EAS Abstracts 2017.indd - Eastern Analytical Symposium [PDF]

2017 EASTERN ANALYTICAL SYMPOSIUM & EXPOSITION CROWNE PLAZA PRINCETON – CONFERENCE CENTER NOVEMBER 13-15, 2017 | PLAINSBORO, NEW JERSEY, USA

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2017 EAS Abstracts

November 2017

2017 EAS Abstracts This volume contains the final abstracts for the oral and poster presentations which take place Monday, November 13, through Wednesday, November 15, 2017. If an abstract is not provided in this volume or the Addendum, then the presenting author did not supply an abstract. For each abstract provided, a complete mailing address for the presenting author is shown. Additional authors are indicated, however, their mailing addresses are not provided.

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Save the Date The 2018 EAS November 12 - 14, 2018 Crowne Plaza Princeton – Conference Center Plainsboro, NJ We want you to be a part of the 57th Eastern Analytical Symposium! 2018 Call for Papers opens March 1

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ABSTRACTS OF TECHNICAL PAPERS 1

Shrinking NMR: From the Laboratory Floor via the Tabletop to the Pocket? Bernhard Blümich, RWTH Aachen University, Worringerweg 2, Aachen, 52056, Germany Compact nuclear magnetic resonance (NMR) instruments operate with permanent magnets at field strengths up to 2 T.[1,2] They are available as tabletop instruments for spectroscopy, imaging and relaxometry and as mobile devices with stray-field magnets for non-destructive testing of materials by relaxometry and 1D imaging. Recent applications with mobile NMR concern studies of tangible cultural heritage such as master violins and antique frescoes. Tabletop relaxometry has extensively been used to study aging of polymers from exposure to temperature and solvents. These phenomena are closely related to aging over centuries of pigment binder in easel paintings, accelerated aging during forgery, and solvent exposure during restoration. NMR spectroscopy with tabletop instruments provides opportunities for chemical analysis of small molecules and for reaction monitoring in the fume hood of the chemical synthesis laboratory, while tabletop tomographs enable engineers to adapt the technology of diagnostic magnetic resonance imaging to control processes and to analyze flow and transport phenomena in chemical reactors. Current advances in methods and hardware focus on further shrinking size and price to convert the current tabletop NMR spectrometers to health trackers that can fingerprint urine metabolites and report on the quality of operation of the human body as a chemical reactor which converts food to energy and life sustaining functions.[3]

tissues. In particular, we highlight the challenges these techniques face in the practical implementation and analysis of data with limited signal-to-noise. We discuss Bayesian approach to understand the uncertainty of the data analysis and the machine learning concept for real-time optimization of the data acquisition to achieve fast and robust measurements.

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Structure and Function in Metal Organic Frameworks are Informed by Portable Magnet Relaxometry, Thanks to Bernhard Blümich Jeffrey A. Reimer, University of California-Berkeley, 201 Gilman Hall, MS: 1462, Berkeley, CA 94720 How do molecules fill and move inside of porous media? Nuclear magnetic resonance (NMR) methods have been developed in recent years to assess these questions; indeed, these methods have been championed by Prof. Blümich and his coworkers in the past decade. I illustrate how relaxometry and diffusometry answers some questions for molecules such as xylenes, carbon dioxide, and methane imbibed into metal-organic frameworks.

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Nanoscale Chemical Imaging with Tip-Enhanced Raman Spectroscopy Richard P. Van Duyne, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208 This lecture discusses our recent research directions in tip-enhanced Raman spectroscopy (TERS). Following a brief introduction to tip-enhanced Raman spectroscopy that highlights the issue of spatial resolution, I focus in on two recent projects in ultrahigh vacuum (UHV) TERS which illustrate the power of this emerging technique. First, new insights into the nature of a dynamic phase boundary involved in the room temperature (RT), UHV-TERS of the Ag tip/ N-N’-bis(2,6-diisopropylphenyl)-1,7-(4’-t-butylphenoxy)perylene-3,4:9,10-bis(dicarboximide) (PPDI) / Ag(100) system are described. We have unraveled the orientation of PPDI molecules at the dynamic molecular domain boundary with unprecedented ~4 nm spatial resolution by UHV-TERS mapping. This topic illustrates that TERS provides access to molecular adsorption geometries in the region with little STM topographical information. Next, unprecedented Angstrom-scale spatial resolution in TERS has been achieved. At room temperature, the strong adsorbate-substrate interaction between the meso-tetrakis-(3,5-di-tertiarybutylphenyl)-porphyrin (H2TBPP) and the underlying Cu(111) substrate leads to the formation of the bowl up/down conformations. Through simultaneous UHV-TERS and STM analysis on the neighboring conformational isomers, we have observed ~15 cm-1 spectral shift in one of the porphyrin-ring Raman modes and analysed the origin of this shift using DFT calculations. Finally, time permitting, I will discuss electrochemical TERS highlighting single molecule electrochemistry and the imaging of electrochemical reactions on the nanometer length scale.

References: [1] B. Blümich, S. Haber-Pohlmeier, W. Zia, Compact NMR, De Gruyter, Berlin, 2014 [2] B. Blümich, E. Pretsch (eds.), Compact NMR, Trends Anal. Chem. 83A, Special Issue, Elsevier, Amsterdam, 2016 [3] B. Blümich, Beyond Compact NMR, Micro. Meso. Mat. (2017), DOI: 10.1016/j. micromeso.2017.04.039

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Compact NMR as a Screening Tool for Diabetes Prevention David P. Cistola, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, MSB1, CoE in Diabetes & Metabolism, 5001 El Paso Dr., El Paso, TX 79905 Thanks to Professor Blümich, compact nuclear magnetic resonance (NMR) technology has gained momentum as a powerful, yet practical tool for solving a wide range of analytical problems. Unlike high-field NMR spectroscopy and imaging, compact NMR has great potential for penetrating new settings, including point-ofcare medical offices. Here we describe how compact NMR is being developed as a screening tool for preventing type 2 diabetes. The diabetes pandemic is threatening the health and well-being of societies worldwide. This situation will not change until improvements are made in early screening and prevention. Current screening tests measure blood glucose or surrogates such as hemoglobin A1c. However, by the time an individual develops impaired glucose tolerance, a 50-70% decline in pancreatic insulin secretion has occurred. There is an unmet need for methods that detect the disease process earlier. Compact NMR relaxometry is filling that need. The approach is based on the ability of NMR to quantify the motional properties of water in blood via the relaxation time constant T2. It exploits the unique properties of water as a molecular surveillance system, as water forms hydrogen bonds with every protein and lipoprotein in the blood. A shift in concentration, as occurs in insulin resistance and inflammation, alters the water binding equilibrium and reduces T2 in a protein-weighted manner. In a study of 72 non-diabetic subjects, low water T2 was independently and additively associated with hyperinsulinemia, dyslipidemia and inflammation. Water T2 offers a surprisingly powerful, yet simple new tool for detecting early insulin resistance syndrome and monitoring cardio-metabolic health.

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Translating SERS into a Robust Detection Platform for Uranium in Complex Matrices Amanda J. Haes, University of Iowa, 205 N. Madison St., 204 IATL, Iowa City, IA 52242 Directly detecting low concentrations of small molecules is often limited by similar molecular structures and function of the targeted species as well as complex sample matrices. When plasmonic nanoparticles synthesized using bottom-up strategies are used to facilitate detection, an additional limitation of signal irreproducibility from changing surface functionality further complicates both qualitative and quantitative detection. Herein, the plasmonic properties of gold nanostars are exploited for understanding how local surface chemistry impact surface-enhanced Raman scattering (SERS) signals using solution-phase gold nanostars for uranyl detection. The engineered roughness of the Au nanostar architectures and carboxylated alkanethiols promote uranyl adsorption. The localized surface plasmon resonance (LSPR) spectral properties are used to quantify alkanethiol layer thickness, which successfully promote uranyl coordination to the Au nanostars in neutral aqueous solutions. Quantitative uranyl detection is demonstrated for various functionalized Au nanostar samples as indicated by enhanced signals and red-shifts in the symmetric U(VI)-O stretch. These results indicate that SERS signals using functionalized Au nanostar substrates can provide quantitative detection of small molecules including the important environmental contaminant uranyl. As such, the development of this novel sensing platform could result in empowering technology to improve the health and safety for those living in regions where uranium contamination of ground water is a concern.

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Porous Media, Magnetic Resonance and Machine Learning Yi-Qiao Song, Schlumberger, 1 Hampshire St., Cambridge, MA 02139 Porous material is ubiquitous in nature and human life. Rocks, soil, concrete, wood, food, and biological tissues are good examples. They are intrinsically multi-phasic, and their microstructure is critical for their functions. In recent years, nuclear magnetic resonance (NMR) / magnetic resonance imaging (MRI) has become an important technique for characterization of a variety of porous media for petroleum exploration, material sciences, and medical imaging. This talk outlines a wide range of techniques used for in-situ measurement of the material porosity, and their physical mechanisms. Examples of NMR/MRI methods and applications are discussed including multi-echo techniques, compressed sensing, 2D methods for diffusion and relaxation to study complex diffusion dynamics in porous media. We also discuss several applications of these techniques in the study of polymer degradation, molecular composition, porosity in sedimentary rocks, food productions and biological

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ers do not always accurately reflect fetal exposure because of the complex portioning of chemicals across the placenta. To address these challenges, the speaker presents a methodology that combines detailed histological and chemical analyses of deciduous and permanent teeth layers corresponding to specific life stages. This approach allows reconstruction of exposure to individual chemicals and chemical mixtures in the second and third trimesters, in early childhood, and also cumulative life-long exposure. Examples are provided from the application of tooth matrix biomarkers to the study of neurodevelopmental trajectories.

Polymer-Enabled Analytical SERS Sensing Christy L. Haynes, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455, Victoria M. Szlag, Seyoung Jung, Theresa M. Reineke, Rebeca Rodriguez This talk focuses on employing polymer-based affinity agents paired with surface-enhanced Raman spectroscopy (SERS) substrates to detect various target analytes within complex matrices. This novel sensing scheme is exemplified through the detection of ricin B-chain (RBC) in water and liquid food matrices using an N-acetyl-galactosamine glycopolymer capture layer on a film over nanospheres SERS substate. The sensing scheme’s detection limit is well below that of the predicted oral exposure limit. Theoretical predictions of the normal Raman spectrum of the glycomonomer give insight into polymer–RBC intermolecular interactions. Preliminary results aimed at other targets, namely agriculturally relevant foodborne toxins, demonstrate the generality of this sensing scheme.

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Volatile Genotoxic Impurity Determination in Oligonucleotide API at Sub-ppm Level Dora Visky, Celgene Corporation, 556 Morris Ave., Summit, NJ 07091 No abstract submitted by the author.

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Novel Approaches to Identify Metabolite-Related Mutagenic Reactions James F. Rusling, University of Connecticut, Dept. of Chemistry, U-3060, 55 North Eagleville Rd., Storrs, CT 06269 Assessing toxicity of drug and impurity metabolites is a key issue in drug development. From the standpoint of chemical toxicity prediction, it is essential to be able to investigate reactions of the metabolites. We have developed a family of genotoxicity prediction assays that first metabolize test compounds, then measure the relative rates of metabolite reactions with DNA. Simple microfluidic technology is applied to produce the metabolites and detect their reactions with DNA. Results predict the possibility of chemical pathways that damage DNA. These arrays feature enzyme/ DNA/metallopolymer films in microwells that form metabolites and facilitate reactions with DNA that are detected by electrochemiluminescence (ECL). Using these principles, we have developed microfluidic reactors by molding PDMS, precision cutting of gaskets, and three-dimensional printing that detect DNA damage from drug metabolites. The ECL detection platform incorporates layered films of metabolic enzymes, DNA and an ECL-emitting ruthenium metallopolymer in the microwells. Liquid samples are introduced into the array, metabolized by the human enzymes, products react with DNA if possible, and DNA damage is detected by ECL with a low-light charge-coupled device (CCD) camera. High-throughput liquid chromatography-mass spectrometry (LC-MS) with DNA/enzyme films on magnetic beads can be used as a follow-up to establish the chemistry of possible genotoxic pathways. These systems can determine possible chemical toxicity profiles for new drugs and chemical compounds. We recently extended this work to employ LC-MS sequencing to detect specific codon damage to p53 tumor suppressor gene, to predict what kind of cancer a metabolite could mediate.

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Imaging Mass Spectrometry on the Nanoscale with Cluster Ion Beams Nicholas Winograd, Penn State University, 209 Chemistry Bldg., University Park, PA 16802 Bombardment of molecular solids with polyatomic projectiles allows interrogation of the sample with reduced chemical damage accumulation. Hence, it is now possible to perform depth profiling experiments with a depth resolution of less than 10 nm. In our hands, the projectile of choice is a gas cluster ion beam (GCIB) consisting of 2000 - 10000 CO2 molecules at a kinetic energy of 70 keV. Under these conditions, the beam can be focused to a submicron spot for chemical imaging purposes. Moreover, erosion of the sample can be performed with minimal chemical damage. With this combination of properties, it is feasible to think about creating three-dimensional molecule-specific images. Here we present GCIB-SIMS (secondary ion mass spectrometry) imaging approaches to elucidate the chemistry of complex biological systems. On a mouse brain tissue section from the traumatic injury model, molecular ions of cardiolipin (CL) and ganglioside up to m/z ~3000 are mapped, demonstrating detailed distributions which have not been reported and lipid loss after the injury at 1-µm lateral resolution. In various mammalian cells (e.g., HeLa), the large intact lipids (e.g., CL) are seen at the single cell level, along with the intact lipids species at m/z 700~1000 and doped pharmaceutically active compounds. This protocol leads to a new level of high resolution molecular imaging, serving as a valuable tool for biological and pharmaceutical studies.

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Capturing Chemical Exposures: The Exposome and Human Health Gary W. Miller, Emory University, 1518 Clifton Rd., Atlanta, GA 30322 The exposome can be defined the cumulative measure of environmental influences and associated biological responses throughout the lifespan, including exposures from the environment, diet, behavior, and endogenous processes. Mapping the exposome is ultimately about understanding the divergence between our genetic predispositions and our biological reality. It requires not just studying environmental chemicals, it is about studying all of the chemicals, endogenous and exogenous alike, that influence human biology. The HERCULES Exposome Research Center is focused on advancing technologies to assess this broad array of chemicals. This includes the development and optimization of assays and platforms for 1) sensitive analysis of environmental chemicals using targeted mass spectrometry, 2) untargeted analysis of exogenous and endogenous chemicals using high resolution metabolomics, and 3) a broad array of data sciences to extract, analyze and interpret data. We continue to expand our analytical capabilities for targeted analysis; however, as we envision the future of healthcare a specific goal of our center has been to develop approaches for comprehensive environmental assessment across all sectors of human health. To this end we have developed and are validating a platform that measures over 200 targeted environmental chemicals and 20,000 known metabolic features for less than $200 per sample. We are testing the new platform across a range of biological systems and matrices including cells (lysates), worms (lysates), animals (blood, tissue), and humans (plasma, CSF, urine, saliva). Such approaches, that allow scaling of environmental assessment to large populations, have the potential to transform precision medicine into population health.

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Improving Metabolite Coverage in Untargeted LC-MS Metabolomics Dajana Vuckovic, Concordia University, Department of Chemistry and Biochemistry, 7141 Sherbrooke St. West, Montreal, ON H4B1R6, Canada, Dmitri Sitnikov, Parsram Ramrup The main objective of global metabolomics is quantitative and qualitative analysis of all small-molecular-weight species (≤1,500 Da) in a given biological sample. The detection of many of these metabolites of interest is complicated by chemical complexity and wide concentration range of metabolome. Recently, we showed that the use of seven extraction methods in parallel only increased metabolite coverage ~30-80% in human plasma depending on liquid chromatography-mass spectrometry (LC-MS) method employed, despite the drastic increase in MS analysis time. The main objective of this work was to develop new sequential extraction method using both solid-phase extraction (SPE) and liquid-liquid extraction (LLE) to improve the metabolome coverage of polar and mid-polar metabolites in blood plasma. The proposed method combines Methyl tert-Butyl Ether (MTBE) removal of lipids with mixed-mode SPE and methanol deproteinization to yield four fractions enriched in neutral, anionic, cationic and zwitterionic compounds. Selectivity analysis showed 81% of standard analytes and 66% of all metabolic features eluted in a single fraction and further improvements are currently under investigation. Median relative standard deviation (RSD) % of signal intensities of all metabolites ranged between 14-20% in SPE fractions as compared to median RSD of 14.1% in methanol extract for n=6 extractions. This indicates suitable repeatability of the method for untargeted metabolomics while achieving significant increase in metabolome coverage in comparison to methanol extract. Additional strategies for further improvement of metabolite coverage, such as use of new extraction materials including ionic liquids and nanomaterials will also be briefly discussed.

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Early Life Exposure to Environmental Chemicals and Health Trajectories Manish Arora, Icahn School of Medicine at Mount Sinai, Box 1057, Dept. of Preventive Medicine, Mount Sinai Hospital, New York, NY 10029 The exposome concept emphasizes the importance of measuring human environmental exposures over the life span. Implicit in this concept is the importance of exposure timing, including exposures during fetal development. Researchers have been limited in their ability to determine critical developmental periods specific to environmental chemicals because such studies invariably require prospective data and exposure assessment tools that may not reflect the correct life stage. This barrier is perhaps most detrimental to the study of fetal environmental exposures, which rely on maternal biomarkers, typically assessed in blood or urine. Maternal biomark-

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Development of Nucleic Acid Preservation and Extraction Methods Jared L. Anderson, Iowa State University, 1605 Gilman Hall, Ames, IA 50011, Kevin D. Clark, Omprakash Nacham, Marcelino Varona Nucleic acids are biopolymers that constitute important diagnostic molecules for a broad range of applications from clinical testing to forensic analysis. A major challenge faced by DNA and RNA analysis techniques is the instability of nucleic acids toward enzymatic degradation by nucleases that are often present in biological sam-

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ples or the surrounding environment. In this talk, hydrophobic magnetic ionic liquids (MILs) will be described as nucleic acid extraction and preservation media. DNA samples stored in various MILs and incubated with deoxyribonuclease I (DNase I) retained their molecular weight for up to 72 h at room temperature, whereas DNA treated with DNase I in aqueous solution underwent complete degradation. Select MILs were also found to enhance the resistance of plasmid DNA (pDNA) toward nuclease degradation. pDNA samples treated with 20 units of DNase I and incubated at room temperature for 72 h were successfully amplified using polymerase chain reaction (PCR). The preservative capabilities of MILs were also investigated for RNA samples treated with the ubiquitous enzyme, ribonuclease A (RNase A). While RNA in aqueous solution was completely degraded by RNase A, storage of RNA within MIL solvent enabled its detection by reverse transcription PCR (RT-PCR).

bination products leads to the great demand for the development of the analytical procedures that are capable to analyze all actives drug substances and their impurities simultaneously. Analytical method development for multicomponent drugs is a complex task that requires a creation of selective, sensitive, accurate, precise and robust methods that are able to control significant number of analytes, which can be from different chemical groups, having different solubility, UV profile and amount constrained by label claim ratio. At the same time these methods have to be practical and efficient in terms of minimal experimental time and resources. Utilization of the new technologies, such as ultra-pressure liquid chromatography (UPLC) can be an alternative to the traditional high-PLC methodology. This presentation discusses various examples from our laboratory experience and particular challenges, limitations, possibilities and solutions for the development of the methods that met above mentioned requirements and developers’ goals.

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Sample Preparation and Precision Medicine Marcel Musteata, Albany College of Pharmacy and Health Sciences, 106 New Scotland Ave., Albany, NY 12208 Prescription drug costs are the fastest growing component of health care spending today. This is not unexpected, since medication is the most relied-upon treatment in health care. However, despite the critical importance of drugs, the current medication-use system is still directed towards the average patient and tends to suffer from problems related to adverse reactions and low drug quality. Successful management of diseases requires individualized therapy to achieve optimal outcomes in treating patients of all age groups. Many drugs have low therapeutic indices and large inter-individual variability in plasma concentration, but also have great therapeutic potential. One of the main impediments to finding good correlations between drug concentration and effect at population levels is the high inter-individual variability in drug distribution between body components and target receptors. Implementation of the precision medicine initiative as it relates to drug therapy requires analytical methods that provide pharmacogenomic, proteomic, and metabolomic data in addition to the usual drug concentration time course; furthermore, this often has to be done for a cocktail of drugs. To reduce cost and patient inconvenience, these data should be provided from a single small biological sample. Therefore, it is crucial to employ sample preparation methods that supply all the needed fractions while also providing information about distribution in the body and reversible binding among sample components. In this regard, processing methods based on microdialysis, microextraction, and ultrafiltration are gaining popularity in clinical laboratories. Such sample preparation methods were successfully applied to investigate proteomics, metabolomics, free concentrations, and normalized concentrations.

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System Suitability Failures Encountered in Pharmaceutical Analysis by UHPLC Xiande (Andy) Wang, Janssen Pharmaceuticals, J&J, 1125 TrentonHarbourton Rd., Titusville, NJ 08560, Jessica Wysocki, Qingjun (Mike) Liu, Rosie Tran, Dave Thomas Ultra-performance liquid chromatography (UHPLC) instrumentation has provided a lot of benefit in terms of high efficiency and short analysis time in pharmaceutical analysis. Therefore, it has become the preferred technical platform in most analytical laboratories. During routine testing of drug products, however, system suitability failures are sometimes encountered after the methods are successfully validated and transferred. A few case studies are presented in this presentation, along with the efforts to investigate the root cause of such failures. The learnings from such events have provided valuable insights on the robustness of the instrumentation, method development and will help the prevention of future problems.

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An Effective Method Development Strategy Using Fused-Core Columns Thomas J. Waeghe, MAC-MOD Analytical, 103 Commons Ct., Chadds Ford, PA 19317, Stephanie A. Schuster, Conner W. McHale The development of a stability-indicating method (related substances method) can be among the most challenging activities in support of pharmaceutical or other ultra-pressure liquid chromatography (UHPLC) method development for complex samples. The objective of such work is to develop a robust and rugged separation of all of the impurities and degradants from a drug substance or formulation, or the analytes from a multi-component mixture. To discover that your stability-indicating method does not separate all of the known and unknown impurities at a later stage can seriously affect product registration. A sensible approach for LC separation development is to screen various selectivity parameters up front, when careful selection of the best combination of stationary phase, organic modifier, mobile phase pH, temperature and other parameters can be made. In this presentation we show examples of how such a method development strategy can be applied using Fused-Core stationary phase selectivities with samples such as a degraded pharmaceutical active ingredient and a double-blind-prepared mixture of acidic, basic and neutral pharmaceuticals.

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What’s the Matter with Sample Prep? Novel Approaches and Solutions Roy Helmy, Merck & Co., MS: RY 80T B173, 126 East Lincoln Ave., Rahway, NJ 07065 An extensive amount of waste is generated by sample preparation methods in analytical laboratories, and as the pharmaceutical market becomes more environmentally cognizant, how can this be justified? Typically more than 99.9% of prepared solutions are disposed of without being used. Manual preparation of samples and standards for analytical methods, such as chromatography, reportedly accounts for up to 82% of solvent usage, 61% of labor time, and 49% of the out-of-specification errors reported. Reducing the quantities prepared has a significant impact on the amount of solvent and substance consumed, as well as the cost of an analytical workflow. In this paper, we describe novel solutions such as gravimetric sample preparation, which also has a positive impact on the quality of results and introduce metrics to measure an analytical method’s environmental impact (AMVI).

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Development of a Robust HPLC Method for Pharmaceutical Analysis Using Quality-by-Design Approach Jinjian Zheng, Merck & Co., MS: RY80T-A150, 125 E. Scott Ave., Rahway, NJ 07065 Method robustness is critical to the successful method validation and transfer, and more importantly, to the routine uses during the lifecycle of the product. However, development of a robust high-performance liquid chromatography (HPLC) separation can be very challenging as the method performance could be impacted by many parameters such as mobile phase pH, organic modifier composition, additive concentration, gradient profile, flow rate, temperature, and system dwell volume etc. In this study, a quality-by-design (QbD) approach to develop a robust HPLC method by leveraging software tools such as ACD/Labs and Fusion AE are presented, which focus on four integrated steps: 1) selection of initial conditions, 2) method optimization, 3) multifactorial robustness evaluation and 4) control strategy.

Size Exclusion Chromatography of Biopolymers and Synthetic Polymers with Common Reversed-Phase and HILIC Columns Joe P. Foley, Drexel University, Dept. of Chemistry, 3141 Chestnut St., Philadelphia, PA 19104, Anna M. Caltabiano Size exclusion chromatography (SEC) is an important separation technique in the polymer industry because of its ability to measure the macromolecular property distributions of synthetic polymers. It is also useful in the life sciences because of its ability to separate proteins and polypeptides of different sizes. As traditionally practiced with cross-linked polymeric stationary phases packed into long, large diameter columns that are often serially-coupled, SEC suffers from a number of column-stationary phase created disadvantages compared to other liquid chromatographic separation modes such as reversed-phase liquid chromatography (RPLC). Following a brief review of recent (> 2011) SEC research, the present study details our investigations of the potential benefits and limitations of using common reversed-phase and hydrophilic interaction liquid chromatography (HILIC) columns for SEC separations of polystyrene, polymethylmethacrylate, and selected proteins. In addition, the accuracy of molar mass determinations by non-aqueous SEC using these columns is reported for both 100% tetrahydrofuran and numerous non-traditional mobile phases for synthetic polymers (i.e., almost any composition other than 100% tetrahydrofuran).

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Analytical Method Development for Multicomponent Drugs, Challenges and Solutions Prasad Panzade, Apotex Inc., 150 Signet Dr., Toronto, ON, M9L 1T9, Canada, Yuliya Yarkho A development of the pharmaceutical products containing multiple active drug substances is the current trend in the pharmaceutical industry. Manufacturing of com-

Titration for Early Drug Discovery and Development Lori Spafford, Metrohm, 6555 Pelican Creek Circle, Riverview, FL 33578 Many assay methods described in the United States and European Pharmacopoeias rely on the accuracy and simplicity of titration; however, these methods usually require several hundred milligrams of the active ingredient, which is expensive and difficult to obtain during discovery and development stages. This analytical chal-

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lenge is addressed by microtitration technology so that sample waste is minimized and reanalysis is avoided. Further optimization can come from selecting the proper solvent, optimizing titration parameters and using alternative techniques such as thermometric titration. Attend this talk to learn how to optimize assay titrations and gain practical insights for developing active pharmaceutical ingredient microtitration methods.

beta-sheet fibrils depending on pH of the solution. And, with advanced AFM imaging modes, enhanced resolution of the fibrils can be achieved further elucidating the hierarchical assembly from sheets to filaments to bundles. Furthermore, attempts and progress at imaging the dissolution process of API crystals in the presence of simulated biological fluids are discussed with the intent to correlate with in-vitro high-performance liquid chromatography (HPLC) dissolution assays. Finally, applications of bimodal AFM techniques to the characterization of controlled release tablet coatings are presented.

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Oil Reservoir Properties at the Nano-Scale: Using AFM in a Bulk Characterization Industry Shannon L. Eichmann, Aramco Research Center-Houston, 16300 Park Row Dr., Houston, TX 77084, David Jacobi, Mohammad H. Haque, Nancy A. Burnham The oil industry relies heavily on new technological advances to locate oil in subsurface reservoirs and improve our ability to recover this oil both economically and efficiently. Even with the newest technological advances, the industry as a whole only recovers up to 50% of the oil contained in subsurface reservoirs. Given the scale of the problem, searching over kilometers of land for the best areas to produce from, the main technologies used for exploration and production are traditionally bulk or large-scale measurements, such as seismic and down-hole logging to provide information about the subsurface. These tools are irreplaceable for providing information at the centimeter to meter length-scales, but at smaller length-scales (i.e., micron and nano), laboratory measurements provide higher resolution information about reservoir rock, fluids, and interfacial phenomena. As such, atomic force microscopy (AFM) has found use in the petroleum industry to provide fundamental understanding of the nanoscale material properties that affect our ability to produce oil, such as the mechanical properties of the organic matter in source rock reservoir and how fluid additives interact with rock surfaces. This presentation provides an overview of AFM in the petroleum industry and shows two examples where we used this nano-scale tool to: 1) provide non-destructive mechanical property measurements for source rocks[1], and 2) demonstrate the effects of salinity on nanomaterial and molecular adhesion in reservoir fluids.[2]

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Atomic Force Microscopy and Nano-IR Characterization of Composites William Haseltine, Solvay, 1937 West Main St., Stamford, CT 06902 Fiber reinforced composites have a heterogeneous structure at multiple length scales. The interface between the fiber and the resin binder is critically important for composite mechanical properties, performance and reliability. In order to engineer the interphase region we would like to be able to measure the interphase thickness, modulus and chemical composition in real industrial composite materials. Sample preparation to expose the interface or interphase region requires cutting the cured composite and polishing to give a smooth cross-section suitable for Atomic Force Microscopy (AFM). Mechanical polishing of composites typically leaves a damaged rough surface on the 10 to 100 nm scale. Ion beam cross-section techniques provide better cross-section samples. Atomic force microscopy and nano-IR have been used to examine broad beam ion milled composite cross-sections. In epoxy/carbon fiber composites we have found the interphase region is about 50 nm thick.

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Learned from the Flint Water Crisis: Communicating Science and Influencing Public Discourse Using Science Ni Zhu, Virginia Tech, 103 Canterbury Ct., Blacksburg, VA 24060, Christina Devine The Flint, Michigan (MI) Water Crisis (2014-present) resulted from a state-appointed emergency manager’s decision to switch to the corrosive Flint River as a drinking water source, and the Michigan Department of Environmental Quality (MDEQ) failing to enforce federally mandated corrosion control treatment of the water. This created a “perfect storm” of drinking water issues resulting in violations for bacteria and disinfection byproducts, caused unprecedented corrosion of iron mains as well as high levels of legionella bacteria and lead in water. For 18 months, about 100,000 Flint residents were exposed to unsafe drinking water whilst city and state officials publicly insisted the water was safe to drink. An “open science” research collaboration between our (Virginia Tech’s) Flint Water Study team and Flint residents in Aug-Sep 2015 revealed citywide lead in water contamination as well as high levels of legionella bacteria in bigger buildings. This was followed by Hurley pediatricians proving how the water switch had doubled instances of childhood lead poisoning in the city. Undaunted by attempts to discredit our work, we fought the MDEQ and the United States Environmental Protection Agency (EPA) to educate the residents about the severe public health risks of lead-tainted water. This endeavor led to declaration of a “Public Health Emergency” by President Barack Obama, garnered hundreds of millions of dollars in relief for Flint residents, and brought much needed attention to glaring issues within the current drinking water legislation and aging water infrastructure (especially, lead pipes and leaded plumbing) plaguing the country fueling a natural debate on “safe” drinking water in America. This talk revisits the events of the Flint Water Crisis.

References: [1] Eichmann, S. L.; Jacobi, D.; Haque, M. H.; Burnham, N. A., Non-Destructive Investigations of Thermal Maturity and Mechanical Properties in Source Rocks. submitted. [2] Eichmann, S. L.; Burnham, N. A., Calcium-Mediated Adhesion of Nanomaterials in Reservoir Fluids. Scientific Reports under review.

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Atomic Force Microscopy of Polymer Systems: From Morphology to Properties to Chemical Imaging and Spectroscopy Gregory F. Meyers, The Dow Chemical Company, Core R&D - Analytical Sciences, Midland, MI 48667, Mark A. Rickard, Carl W. Reinhardt, Jamie J. Stanley Scanned probe microscopy (SPM) has had a long history at The Dow Chemical Company, beginning in the late 1980s when commercial scanning tunneling microscopes were just hitting the market. Since that time Dow has invested in internal and external collaborative efforts to drive and develop atomic force microscopy (AFM) based technologies for property measurements of polymeric materials at nanometer length scales. These capabilities provide both mechanical spectroscopy and mapping. What these techniques lack, however, is chemical specificity. From 2008-2010 Dow worked with Anasys Instruments on the development of an AFM-infrared (IR) capability (commercialized as the NanoIR in 2010). More recently a top-down version of the system was commercialized (NanoIR2 in 2013). The AFM-IR method relies on detection of IR absorption under the AFM tip by rapid photothermal expansion. Such an approach breaks the diffraction limit enabling IR mapping at >LSI>>MALDI) correlating well with ease of desolvation/evaporation of matrix from charged clusters. The production of highly-charged stable ions in

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Influencing the Selectivity of Small Proteins and Peptides on the Raptor™ ARC-18 Thi Do, Restek Corporation, 110 Benner Circle, Bellefonte, PA 16823 With the influx of biotherapeutics in medical research and healthcare, the analysis of small proteins and peptides by liquid chromatography (LC) continues to grow. Many of these analyses utilize acid-modified mobile phases to improve peak shape; however, their effects on selectivity and retention are often not well understood. In

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this presentation we explore the effects of acid type and concentration, temperature, and gradient slope on the selectivity and retention of several peptide probes using the sterically protected superficially porous Raptor™ARC-18 LC column (stable to pH 1).

Expanding the Analytical Toolbox for Protein Aggregation Studies Nicole M. Schiavone, Merck & Co., MS: RY801-A102, 126 E. Lincoln Ave., Rahway, NJ 07065, Erik Guetschow, Alexey Makarov Protein aggregation is related to numerous disease states and can be problematic in biopharmaceutical development by compromising physical stability, production and storage. While it is known that in many cases proteins must unfold prior to aggregation, there remains much to learn regarding the mechanisms of protein aggregation, including the differing pathways to structured (fibrils) vs. unstructured aggregates. Here, we aim to develop new methodology using multiple analytical techniques to allow for a better understanding of how proteins aggregate. Insulin will serve as a model to investigate how the degree of protein unfolding relates to extent of protein aggregation. First, we will monitor the distribution of insulin aggregates over time using capillary electrophoresis-mass spectrometry (CE-MS). CE will separate the aggregates based on their size-to-charge ratio and MS will provide molecular weight information. Then, we will use circular dichroism (CD) and hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to obtain structural information and to measure the degree of protein unfolding at various time points. Through the combination of these powerful techniques, we aim to expand the analytical toolbox for protein aggregation studies.

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Prediction of Long Term Peptide Chemical Stability Jameson R. Bothe, Merck & Co. 126 E. Lincoln Ave., Rahway, NJ 07065, Yuhang Jiang, Yogita Krishnamachari, Paul Walsh, John Lena, Claudia Neri Development of robust peptide and protein solution formulations with desirable chemical and physical stability is a highly challenging task from both a formulation and an analytical standpoint. The prediction of long term stability with high confidence using short term accelerated stability data is not straight forward as the degradation mechanisms can be confounding. The capability to rapidly and accurately predict long term chemical stability of peptides and proteins with confidence would be a powerful tool during formulation development. A major uncertainty when attempting to use limited accelerated stability data to make long term predictions is understanding the ability of the data to make robust predictions. In this study, we investigate the use of Monte Carlo and Bootstrap statistical methods to predict long term experimental peptide chemical stability using short term accelerated stability data. First, we extensively explored the impact of stability study parameters on the quality of long term predictions including number of temperatures, number of time points, and level of degradation. With an understanding of these factors in hand, we carried out an accelerated stability study of two peptide formulations that differ in chemical stability over extended periods of refrigerated storage. Long term stability predictions made from the accelerated stability data coupled with the Monte Carlo and Bootstrap statistical tools reveals several insights and caveats in robustly predicting long term peptide chemical stability.

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Protein Purification and Reconstitution of Biological Clock in Test Tube to Determine Robustness Manpreet Kaur, New Jersey Institute of Technology, 120L Colton Hall, Newark, NJ 07102, Yong I. Kim Cyanobacteria possess the simplest known biological clock consists of three proteins KaiA, KaiB and KaiC that can be reconstituted in-vitro by mixing with adenosine triphosphate (ATP) from E. coli to produce a 24-hour cycle. For in-vitro reconstitution of the clock, protein needs to be highly purified, as ATPase and protease can disrupt in-vitro experiment. The clock can be entrained by applying the alternating light/dark pulse or by altering the biochemical system such as ATP/ADP ratio. Previously by breaking the hydrogen bond network made KaiC phosphorylate without KaiA. We performed mutation at the Glutamate-487 (E487) residue of the A-loop, changing it to Alanine. Glutamate contains a carboxyl group, which contains oxygen atoms capable of hydrogen bonding, whereas Alanine possesses a methyl group, which cannot participate in hydrogen bonding, thus causing a break in the hydrogen bond network. We observed that breaking hydrogen bond and changing the concentration of ATP/ adenosine diphosphate (ADP) ratio caused dephosphorylation of KaiC alone. To achieve high purity protein, we modified the traditional method, i.e., using both glutathione S-transferase (GST) and anion exchange (Q) columns by using only a single glutathione-S-transferase (GST) column with the lengthening of washing time. We study the oscillator mechanism to understand the generation of ~ 24-hour rhythm. If we understand the molecular mechanism of the clock it might help us to uncover the links between our biological clocks and health, we might be able to come up with ways to intervene biological clock disorders such as the risk of jet-lag, cancer, depression.

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Composition Determination of Copolymer of Vinylpyrrolidone (VP) and Poly(ethylene glycol) Methacrylate (PEG-MA): A New Technique for “Gentle” Whitening Zheng Li, Ashland, 1005 Route 202/206, Bridgewater, NJ 08807 This work introduces a simple method to determine the overall composition distribution of VP and PEG-MA as function of molecular weights using size-exclusion chromatography (SEC) combined with UV absorption and refractive index (RI) dual detectors. The signals from UV and RI are linearly proportional to the concentrations of each component, with separation by SEC, the concentrations of each components within each mass fractions can be calculated. This SEC-UV-RI dual detection method is the most contemporary way for analysis of such kind as many copolymer systems have been reported over the years. In this study, PVP is the UV “visible” component due to the strong absorption from pyrrolidone rings and PEG-MA is almost UV “invisible.” VP-PEG-MA is the novel technique being developed for the mouth wash products to provide “gentle” whitening. With both hydrogen bonding and surfactancy properties, stains can be extract and solubilized on the enamel, that the goal of non-abrasive/non-oxidative whitening is achieved. The whitening efficiency of this product is found to be directly related to the chemical homogeneity of the copolymer, and a randomly distributed copolymer is required to provide effective whitening functionality. This study shows the homogeneity of copolymers made through different processes and their according teeth whitening performances.

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Development of a Platform for Peptide Stability Assessment on Solid Substrates Margaret Roeder, Merck & Co., MS: RY80T-B164, 126 E. Lincoln Ave., Rahway, NJ 07065, Jameson Bothe, Yash Kapoor, Paul Walsh, Justin Pennington Microneedle (MN) technologies offer a minimally invasive drug delivery system for peptide and protein molecules, avoiding disadvantages associated with standard injectable routes of administration. Drug-coated MNs penetrate the tough epidermis and deposit drugs within the skin, offering advantages such as ease of administration, targeted drug delivery, and minimal pain associated with drug delivery. The use of intradermal MN systems for drug delivery, including devices and patches, provides a way to overcome common challenges associated with peptide solution formulations, including poor chemical and physical stability even under refrigerated conditions. Key areas of interest involving coated MNs include understanding the coating process, evaluating solid-state stability of the coated formulation, and examining the compatibility of MN surfaces and peptide formulations. Here, we present the development of a platform experimental design to investigate peptide stability on solid substrates. The goal of this study aims to establish a screening tool for new compounds in early stages of development in order to evaluate the compatibility of a typical MN surface with different peptide formulations. To evaluate surface/formulation compatibility, peptide formulations were deposited onto metal disc surfaces and stored under various conditions to assess chemical stability of coated formulations in comparison to solution formulations. A number of variables were tested, including solid substrate material, drying conditions, surface treatment, choice of excipients, and peptide sequence in order to evaluate peptide stability in the solid state. Overall, these studies reveal the complexity and wide array of challenges associated with performing robust compatibility experiments for peptides dried on surfaces.

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Rapid Characterization of Insulin Modifications and Sequence Variations by Proteinase K Digestion and UHPLC-ESI-MS Rong-Sheng Yang, Merck & Co., MS K15-D101, 2015 Galloping Hill Rd., Kenilworth, NJ 07033, Weijuan Tang, Huaming Sheng, Fanyu Meng Discovery of novel insulin analogs as therapeutics has remained an active area of research. Compared to native human insulin, insulin analog molecules normally incorporate either covalent modifications or amino acid sequence variations. From the drug discovery and development perspective, methods for efficient and detailed characterization of these primary structure changes are very important. In this report, we demonstrated that proteinase K digestion coupled with ultra-high-performance liquid chromatography electrospray ionization mass spectrometry (UHPLC-ESI-MS) provides a simple and rapid tool to characterize the modifications or sequence variations of insulin molecules. A commercially available proteinase K digestion kit was used to process recombinant human insulin (RHI), insulin glargine, and fluorescein isothiocynate-labeled recombinant human insulin (FITC-RHI) samples. UHPLC-ESI-MS analysis of the digested samples was used to characterize the chemical modifications and sequence variations. End-to-end experiment and data interpretation was achieved within 60 minutes. This approach is fast and simple, and can be easily implemented in early drug discovery laboratories to facilitate research into more advanced insulin therapeutics.

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Towards a Simultaneous Elemental and Molecular Chemical Imaging Platform via a Combination of Optical and Mass Spectrometries Jacob T. Shelley, Rensselaer Polytechnic Institute, Cogswell Laboratory, 110 8th St., Troy, NY 12180, Sunil P. Badal, Montwaun D. Young, Jessica R. Hellinger Recent advances in analytical instrumentation and data capture/processing have led to the ability to generate comprehensive chemical maps (or “images”) of solid samples. Unambiguous analyte identification within these complex samples necessarily requires multiple analytical approaches performed on the same sample; these methods have been termed ‘multimodal chemical imaging’ approaches. While tandem imaging methods provide a wealth of information, they often suffer from weak sensitivity, poor selectivity, or compromised spatial resolution as dictated by the spectroscopic method employed. Greater success in comprehensive chemical imaging has been achieved with instruments based around mass spectrometry (MS), due to the excellent sensitivity and selectivity; however, MS imaging is inherently destructive in nature. Here, we will present our recent work towards the development of a multimodal chemical imaging apparatus capable of providing simultaneous molecular and elemental information from the exact same spatial location (i.e., each pixel) at high spatial resolution (e.g., better than 100 microns). This capability is achieved through focused laser sampling of the solid material. The aerosol generated from the ablation event is swept to a flowing atmospheric-pressure afterglow (FAPA) molecular ionization source, which desorbs and ionizes molecules from the ablated particles for detection by mass spectrometry. In addition, light from the laser-induced plasma is collected and detected to provide optical emission information on the elements present in the sampled spot. The design of this instrument will be discussed in detail along with preliminary analytical figures-of-merit.

Fragmentation Mechanisms of Protonated Benzoic Acid and Related Compounds: Competitive Generation of Protonated Carbon Dioxide or Protonated Benzene Sihang Xu, Stevens Institute of Technology, Department of Chemistry, Hoboken, NJ 07030, Athula Attygalle, Julius Pavlov The collision-induced dissociation (CID) product-ion spectrum recorded from the mass-isolated m/z 123 ion showed prominent peaks at m/z 45 and m/z 79, in addition to the anticipated peaks for a water loss (m/z 105) and subsequent CO loss (m/z 77). Product-ion CID spectra recorded from protonated (carbonyl-13C) and 18O2-labeled benzoic acids also ascertained that the carbonyl carbon and both oxygen atoms are removed in the CO2 loss, while only one of the oxygen atoms is eliminated in the CO loss. The m/z 79 peak represents the benzenium ion, whereas the m/z 45 peak represents protonated carbon dioxide. Quantum chemical evaluations indicate that the most favorable structure of protonated CO2 bears a proton attached to one of the oxygen atoms. Protonated benzoic acid (m/z 123) upon activation undergoes fragmentation by either eliminating a molecule of benzene to generate protonated CO2 (m/z 45), or ejecting a molecule of carbon dioxide to yield protonated benzene (m/z 79). Preliminary experimental evidence shows that the incipient proton ambulates during the fragmentation processes. For the CO2 or benzene loss, protonated benzoic acid transfers the charge-imparting proton initially to the ortho and then ipso position to generate a transient species which dissociates to form an ion-neutral complex between benzene and protonated CO2. An intramolecular reaction then takes place within the ion-neutral complex, and the protonated CO2 ion acts as an electrophile to transfer a proton to the benzene ring and form the benzenium ion.

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Analysis of Products from the Liquid Phase Reaction of Cinnamaldehyde with Pd/C in the Presence of Lewis Acid Salts Amanda B. Childs, Cedar Crest College, 100 College Dr., Allentown, PA 18104, Lindsey A. Welch Cinnamaldehyde, an ɑ,β-unsaturated aldehyde, is a model compound for studying selective catalysis because of its olefin and aldehyde functionalities. The carbonyl on the aldehyde can be reduced to form an alcohol, but results suggest alternate reaction pathways are possible. Cinnamaldehyde was reacted over palladium on carbon in the presence of Lewis acids (iron (III) chloride hexahydrate, nickel (II) chloride hexahydrate, tin (II) chloride dihydrate, and copper (II) chloride dihydrate) in isopropyl alcohol in reducing and oxidizing conditions. Reducing conditions were investigated by comparing a hydrogen balloon to high-pressure catalytic transfer hydrogenation (CTH). These reactions were carried out under ambient conditions as well as with the use of a microreactor. Oxidizing conditions were investigated via reactions in air assisted by selected oxidizing agents. By varying the Lewis acid, the temperature, and the pressure, the creation of different products was observed. Products were analyzed with gas chromatography mass spectrometry and gas chromatography infrared spectroscopy. Mass spectral data were crucial in the identification products when analytical standards were unavailable. Reaction pathways for the formation of hydrogenation and etherification products are presented. Evidence suggests that activity and selectivity can be tuned with proper selection of reaction conditions.

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Direct, Sensitive Detection of a Broad Range of Analytes from Surfaces with Flowing Atmospheric Pressure Afterglow (FAPA) Mass Spectrometry Sunil P. Badal, Rensselaer Polytechnic Institute, 38 25th St., Troy, NY 12180, Montwaun D. Young, Jessica R. Hellinger, Jacob T. Shelley Plasma-based ambient desorption/ionization (ADI) techniques, which employ a low energy electrical discharge as a means to directly desorb/ionize molecular species from their ambient environment, have received considerable attention due to their simple operation, fast analysis times, and high sensitivity. However, the capabilities of these sources are often limited to small, polar organic molecules. Additionally, reproducible sample introduction and poor spatial resolution in imaging applications are still major challenges. In this presentation, we explore alternative source conditions and unique sampling methodologies to expand the range of detectable analytes with a flowing atmospheric pressure afterglow (FAPA) ADI source. Specifically, it was found that changing the operating parameters of the FAPA source (e.g., discharge current and gas flow rate) provides better ionization efficiency for analytes with low polarity. Addition of small fractions of molecular gases to the helium-based FAPA discharge was found to enhance ion signal for specific analytes as well as produce cleaner mass spectra. To better understand the reasons for the observations, ionization and plasma characteristics for mixed-gas FAPA were determined with optical and mass spectrometries. The issues of reproducible sample introduction and spatial analyses were addressed by combining FAPA with laser-ablation sampling. Aerosolized particles from the ablation event were carried to the FAPA source for desorption/ionization and, subsequently, analyzed by a high-resolution mass spectrometer. By scanning the laser across the sample, followed by appropriate data processing, molecular chemical images of sample surfaces were generated. Analytical figures of merit for the laser ablation-FAPA-MS system are also presented.

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Benefits of ICP-MS with 10 Times Higher Sensitivity and 1/2 of Argon Consumption Iouri Kalinitchenko, Analytik Jena US, Konrad-Zuse-Strasse 1. Jena 07745, Germany, Oliver Buettel Unique specifications like limit of detection (LOD), precision, cost and speed of analysis or productivity in the laboratory make inductively coupled plasma mass spectrometry (ICP-MS) a performance driven technique. When looking at any ICPMS instrument’s technical specifications one of the main parameters – sensitivity – is often under-estimated. While at the same time sensitivity in particular is a feature that directly affects LOD(s), speed and cost of analysis, and overall instrument productivity. Another important parameter is argon consumption. The Analytik Jena ICP-MS runs with half the argon consumption of competitive systems, thus allowing a significant reduction in running cost per sample.

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Using Optimization Algorithms to Determine Isotope Ratios from Tandem Mass Spectrometry William Simon, Princeton University, 4957 Frist Campus Center, Princeton, NJ 08544 Isotope ratios (IRs) play a critical role in the fight against counterfeit pharmaceuticals and crime in general, allowing the determination of the source of a material and demonstration that substandard and falsified drugs or other illicit goods are part of the same supply chain. But current methods for determining IRs are expensive, slow, and limited to only a few elements. Fast isotope ratio mass spectrometry (FIRMS) is a new method under development to determine isotope ratios in a sample of a given compound from tandem mass spectrometry data. FIRMS calculates isotope ratios (IRs) through optimization software that minimizes the sum of absolute percent differences between experimentally observed and theoretically predicted fragment abundances in mass spectra. But the algorithm best suited to FIRMS and optimal method for setting bounds on the IR solution space are unknown. This study seeks to determine an optimization algorithm most likely to accurately calculate IRs when employed by FIRMS and proposes a method for setting bounds on the IR solution space.

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Metals Impurities: Efficient USP 232 Quantification Thomas Rettberg, LGC Standards, 276 Abby Rd., Manchester, NH 03103, Vikas Padhye The implementation of International Council on Harmonization (ICH) Q3D guidelines and United States Pharmacopeia (USP) Chapters 232 and 233 have been ongoing and a range of new instrumentation for measurement and sample preparation are now found in many laboratories performing analyses of pharmaceutical products. Quantification of metals is primarily done using inductively coupled plasma mass spectrometry (ICP-MS) where current instrumentation generally has more than adequate detection capability. Experience has now shown that the most perplexing challenges faced are not instrumental but often relate to factors involving sample handling, including: sample dilutions for stability, contamination avoidance, establishment of calibrations, J-value spiking levels in addition to discretionary aspects of the method (e.g., timings, internal standards, acid or base

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matrices, etc.). For many laboratories, metals testing arose from graphite furnace atomic absorption (GFAA) methods or colorimetry where each metal had unique procedures; however, the great advantage of ICP-MS (and ICP-atomic emission spectroscopy) can be the ability to capture many metals in one run. Achieving this goal requires set-up of procedures suitable for the widest range of individual metals as possible. Work is presented on this poster to show key calibration and sample handling factors for efficient and accurate measurement of pharmaceutical products for metals, including the twenty-four covered elements and others often of interest.

the solute, and it seemed that problems were solved. However, lauric acid is very far from an ideal solvent and the end result was an experiment that does not work consistently. However, this turned out to be a positive in the end, and now we use this experiment to encourage students to examine scientific failures as a learning experience. This is a departure from the standard model of introductory chemistry classes, where all the experiments are successful, all the time. I feel that this is an important educational experience for our students.

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Spin-Lattice Relaxation of Pharmaceutical Polymorphs by HighResolution Solid-State NMR Robbie J. Iuliucci, Washington and Jefferson College, 60 South Lincoln St., Washington, PA 15301, Rosalynn Quiñones-Fernández, Brooke Lininger, Sarah Stuchell, Jordan Hosfelt, Deben Shoup, Grayce Behnke, Taylor Maddox Polymorphs of pharmaceuticals pose a problem due to differing physiochemical properties, such as solubility, stability, and even drug efficacy. In addition to polymorphic characterization being necessary for proper administration of medicine, understanding these physiochemical properties at a molecular level is critical for drug development. High resolution solid-state NMR offers various ways to analyze the crystal structure of drug polymorphs. Because microcrystalline samples are suitable for solid-state nuclear magnetic resonance (NMR), the method is complementary to diffraction techniques especially in cases where growing large single crystals are challenging. Thus, NMR crystallography has evolved into a standard tool to elucidate crystal structures of polymorphs. Spin-lattice relaxation is an NMR phenomenon that potentially can shed light on physiochemical properties. Moreover, spin-lattice relaxation is polymorph dependent, allowing it to correlate crystal structure to drug properties. We will report results of spin-lattice relaxation measurements of polymorphic forms from cimetidine, carbamazepine, erlotinib, acetaminophen, and other drugs. A second aspect of the relaxation study is to demonstrate the ability of growing drug polymorphs that are doped with paramagnetic impurities. Paramagnetic complexes, such as chromium or nickel acetylacetonate, serve as a relaxation center. Microcrystalline polymorphs created with paramagnetic impurities have reduced 1H T1 values that leads to enhanced spectra. The enhancement improves multidimensional NMR as well as acquisition of low abundant nuclei, such as 15N, without altering the crystal structure.

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Acid Number of Crude Oils and Petroleum Products by Catalytic Thermometric Titration Using ASTM D8045 Lori Spafford, Metrohm, 6555 Pelican Creek Circle, Riverview, FL 33578 American Society for Testing and Materials (ASTM) Standard D8045 describes the analysis of acidity in difficult crude oils and petroleum products using thermometric titration. This new technique overcomes solubility and precision challenges encountered with traditional potentiometric measurements. Thermometric titration is more precise, faster and reduces solvent requirements. Through eight years of development work in ASTM and thousands of sample measurements this new standard is shown to be rugged, robust and transportable across many labs and technicians. This poster provides a clear analysis of data acquired with this new method and details the benefits of thermometric titration for safer and more precise acidity analysis.

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The Development of a High-Throughput UHPLC Method for Determination of the Emitted Dose Uniformity (EDU) and Aerodynamic Particle Size Distribution (APSD) by Andersen Cascade Impaction (ACI) for Dry Powder Inhaler (DPI) Jagruti A. Patel, Merck & Co., MS: RY80M -212A, 126 E. Lincoln Ave., Rahway, NJ 07065, Josephine Bermudez An ultra-performance liquid chromatography (UPLC) method was developed to support the early phase development of a DPI program for key performance tests such as APSD by ACI and EDU. As a result of a 4-fold decrease in the predicted human dose midway during product development, the analytical team was quickly challenged with re-developing the UPLC method to increase the sensitivity to accurately and precisely analyze the new low dose. This method was re-developed with the overall goal of increasing sensitivity and efficiency, while decreasing the amount of hazardous waste. The ACI test segregates particles based on their aerodynamic particle size on ACI components and a series of stages/plates and provides insight into drug product aerodynamic properties, at the expense of a significant number of samples for extraction and chromatographic analysis. This UPLC method shows significant improvement in hazardous waste and lessens the turnaround time compared to the previous methods. Overall, several challenges were encountered such as peak detection for ACI plates with less drug and peak interferences for low dose EDU. In the end, the analytical team was able to overcome these challenges to successfully re-develop this green UPLC method that is proven to be sensitive, efficient and validated for Phase I.

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Chemometric Application Development for Benchtop Permanent Magnet NMR Systems Operating at 42, 60, and 80 MHz – Demonstration of Equivalency with Supercon 300 MHz NMR John C. Edwards, Process NMR Associates, LLC, 87A Sand Pit Rd., Danbury, CT 06810 Benchtop high-resolution nuclear magnetic resonance (NMR) systems are available at a number of field strengths and probe configurations. However, beyond the obvious academic instruction market for these instruments, very few applications have been demonstrated across all available platforms which would in turn demonstrate the general applicability of benchtop NMR technology to industrial quality control. We present two chemometric-based applications that have been developed at 4 different field strengths utilizing Varian Mercury 300 MHz, Magritek Spinsolve 42 MHz, Aspect AI 60 MHz, and Thermo Picospin 80 MHz NMR systems. Partial-leastsquares (PLS) regression correlations were obtained on all 4 platforms relating to: 1) Omega-3 fatty acid composition of samples taken from various points in a nutritional supplement manufacturing process. Excellent correlations were obtained on all four NMR instruments proving that NMR technology is applicable to in-lab, at-line, or on-line analysis of fish oil derived omega-3 fatty acid supplements. The 40 second NMR analysis with minimum sample preparation effectively replaces a 60+ minute gas chromatography (GC) analysis that also requires an esterification preparation of the sample. 2) Physical and chemical property determination of diesel fuels where excellent correlations were obtained between 1H NMR variability and parameters such as density, aromatic content by GC, hydrogen content by 1H time-domain (TD)-NMR (American Society for Testing and Materials D7171 method), and sulfur content.

Withdrawn by the author.

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Sustainable Imaging Technology for Thermal Printing Terri Powell, The Dow Chemical Company, 400 Arcola Rd., Collegeville, PA 19426, Brian Einsla, John Roper Traditional thermal printing uses a mechanism that involves a colorless dye and developer, which is activated by the thermal print head. Bisphenol A, which is a material of concern, is often used as the developer. An effort to develop a more environmentally friendly method of printing without Bisphenol A or any other developer resulted in a new type of thermal printing technology exploiting the properties of opaque polymer, which is a hollow, spherical particle. The new printing method was developed in collaboration between the Dow Chemical Company and the Koehler Paper Group. In this method, a colored layer is applied to the base paper, and then a top coat containing the opaque polymer particles is applied. When heat is applied, the hollow particles collapse and become transparent, revealing the colored layer beneath. Scanning electron microscopy was used to characterize and confirm the hypothesized mechanism. The correlation between void size and scattering power and the effect of temperature on the structure will be discussed. This project was the recipient of the 2017 Presidential Green Chemistry Challenge Award for Designing Greener Chemicals.

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H qNMR Analysis of Alcoholic Beverages - Detailed Chemical Fingerprint Information for Quality Control and Process Understanding John C. Edwards, Process NMR Associates, LLC, 87A Sand Pit Rd., Danbury, CT 06810 1 H quantitative nuclear magnetic resonance (qNMR) can be utilized to yield quantitative component distributions of various alcoholic beverages that can then be utilized for product consistency, product authenticity, and mandated regulatory labelling. The qNMR internal standard approach utilized in our laboratory will be detailed and several examples of quality assurance /quality control (QA/QC) or manufacturing process understanding are presented. NMR analysis provides details on ethanol production in fermentation processes as well as information on methanol and fusel alcohols. Various polyols can also be identified and quantified such as glycerol and 1,3-propandiol. Organic acid distributions are readily quantified providing lactic, ace-

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How Not To Do It: (Mis)Adventures in Developing an Environmentally Freezing Point Depression Experiment Jacob M. Newman, Touro College - Lander College for Men, 75-31 150th St., Flushing, NY 11367, At Lander College for Men, we had run a molal freezing point depression experiment using naphthalene, p-dichlorobenzene, and sulfur in our General Chemistry laboratory class. While this was successful, I wanted to replace the foul smelling and toxic materials with more environmentally friendly ones. I turned to published laboratory manuals, found suggestions to use lauric acid as the solvent and benzoic acid as

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A Guide for HPLC Troubleshooting: How to Diagnose and Solve Chromatographic Problems? Imad Haidar Ahmad, Merck & Co., MS: Bldg. 818, Rm. A207, 126 E. Lincoln Ave., Rahway, NJ 07065, He Yu, Hao Luo, Ping Zhuang High-performance liquid chromatography (HPL) is one of the most used analytical techniques in industry. Regardless of whether the HPLC system is in well-maintained condition or not, it is impossible not to run into issues, such as: pressure ripple, artifact peaks, retention time variability, peak shape distortion, etc. Knowledge on how to diagnose, isolate, and fix a HPLC problem is essential for the success of analytical testing. This guide will be useful for analysts who are looking for a summary of the most common problems encountered in HPLC analysis along with troubleshooting clues on how to diagnose and solve these problems. Example cases from our labs and from the literature - are presented to support the decision tree demonstration.

tic, succinic, malic, tartaric and citric concentrations that are of particular interest to sour beer and cider-makers from both a quality and sensory perspective and to other beverage manufacturers in the context of troubleshooting off-flavors. NMR also provides information on the yeast and bacterial uptake of amino acids throughout the fermentation process. It also allows for quantitation of sugars, complex sugars and malt dextrin chemistries throughout the fermentation and ageing processes of various alcoholic beverage types. These chemistry observations are useful in the context of wild fermentations which are becoming quite prevalent and allow the conversion of sugars by different yeast and bacterial mixtures to be followed as well as providing an understanding of the residual carbohydrate chemistry in final products such as beer, mead, wine and fruit containing beverages. NMR also allows for accurate ethanol analysis of sugar and lipid containing liquor drinks that can be difficult to analyze by traditional approaches.

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Polymer Analysis Applications of Thermo Fisher Scientific picoSpin NMR Spectrometers Daniel Frasco, Thermo Fisher Scientific, 1240 E. Columbia Ave., Philadelphia, PA 19125 Nuclear magnetic resonance (NMR) spectroscopy has proven to be an invaluable analytical tool for polymer analysis by elucidating molecular structure, studying reaction dynamics, monitoring reaction progress, and gauging product purity. When analyzing polymers using high-field NMR spectrometers, resonance signals often coalesce as broad peaks due to poor molecular rotation and the marginally different chemical environments the polymer repeating units are situated in. To that end, low-field NMR such as the Thermo Scientific™ picoSpin™ 80 NMR spectrometer readily lends itself as a low-cost alternative to high-field instruments with significant savings on both instrument procurement and upkeep, while still generating similar information to what would be obtained from a high-field instrument. In this work, a picoSpin NMR spectrometer was used to obtain a variety of critical polymer data from a diverse group of applications. Information about polymer structure was accomplished through the molecular weight determination of poly(ethylene glycol) (PEG) acetyl triarm and compositional analysis of a polyol. The picoSpin was also used to qualitatively and quantitatively monitor the polymerization of t-butyl acrylate with a polystyrene reagent by identifying and integrating the resonance signals associated with the t-butyl acrylate monomer.

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Some Hint on How to Make a Standard UHPLC Column with + 300 000 Theoretical Plates/Meter Norikazu Nagae, ChromaNik Technologies Inc., 6-3-1 Namiyoke, Minato-ku, Osaka 552-0001, Japan, Tomoyasu Tsukamoto, Henrik Svennberg The race to achieve the highest number of theoretical plate counts has been going for many years already, even though it could and should be argued that a good separation is mainly governed by good selectivity, a reasonable retention and then high column efficiency. However, the race has still been going, and this presentation shows how + 300 000 theoretical plates/meter can be achieved in a standard column for ultra-high-performance liquid chromatography (UHPLC). The importance of a good bonding and end capping technique is discussed and hints about how this is made is revealed. The importance of base particle choice with a good heat transfer capacity is shown and the importance of small extra column peak broadening is emphasized. The + 300 000 theoretical plates/meter is shown in two different columns lengths with the inner diameter of 2.1 mm, 150 and 50 mm long respectively.

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Investigation into Peak Fronting Observed in Liquid Chromatography Standard Solution during Technical Transfer from HPLC to UPLC Elizabeth Wilson, Amgen, 360 Binney St., Cambridge, MA, 02141, Laura Blue, Kevin Turney, Tawnya Flick As part of the Kyprolis drug substance (carfilzomib) technical transfer to Amgen Singapore Manufacturing, liquid chromatorgaphy (LC) control and release methods were migrated from high performance liquid chromatography (HPLC) instrumentation to ultra-high-performance liquid chromatography (UHPLC) instrumentation that is the quality control network platform. UPLC instruments are capable of running LC methods developed on HPLC instruments, but with smaller tubing diameter and the ability to run at higher pressure, offer the flexibility to run quicker UPLC methods. To facilitate success of the method transfers, a cross-site Attribute Sciences team performed an assessment across all methods. Peak fronting was observed for a component of the standard solution in the Step A LC methods. This peak fronting only occurred for the first of four components in the standard solution to elute, carfilzomib drug substance intermediate PR-026. This poster summarizes the investigation into the root cause for the peak fronting and method characterization.

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Affecting Selectivity and HILIC Retention on a FluoroPhenyl Stationary Phase Shane Stevens, Restek Corporation, 110 Benner Circle, Bellefonte, PA 16823 In this study we have set out to explore the retention mechanisms of the FluoroPhenyl stationary phase. The FluoroPhenyl phase may be described as having mixedmode and/or hydrophilic interaction liquid chromatography (HILIC) retention and selectivity. HILIC and mixed-mode offer retention mechanisms that vary, or are orthogonal to, typical reversed-phase columns like C18. These chemical interactions are generally not well understood or easily demonstrated, which may be frustrating and leave chromatographers not using the phase to the fullest potential. The FluoroPhenyl phase offers unique selectivity by incorporating strongly electronegative fluorine atoms on a phenyl ring. In addition to traditional reversed-phase dispersive interactions, this phase may exhibit polar, cation-exchange, and HILIC retention. Our focus in this presentation is on method changes including temperature, mobile phase composition, and acid strength and concentration and how these changes affect the selectivity of targeted analytes. By demonstrating the influence of method changes on this phase we aim to gather a better understanding of the interactions provided by the FluoroPhenyl phase and its use as a HILIC or mixed-mode phase.

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Development of a Novel Immobilized Polysaccharide Chiral Stationary Phase for Enantiomeric Separations Ernest J. Sobkow, YMC America, Inc., 941 Marcon Blvd., Ste. 201, Allentown, PA 18109, Noritaka Kuroda, Masahide Kobayashi, Toshikazu Adachi, Takehiro Iwadate, Tsuyoshi Watabe Coated and immobilized polysaccharide bonded silicas are known to be useful for separation of a wide range of racemic compounds. Among these two types, immobilized chiral stationary phase (CSP) columns are now common for measuring optical purity, and offer advantages when analyzing and isolating chiral materials. We developed a novel immobilized type CSP (named “CHIRAL ART Cellulose-SJ”), a cellulose tris(4-methylbenzoate) immobilized on silica particles. Cellulose-SJ features durability under acidic and basic conditions and may be used with many organic solvents. A structural feature of the stationary phase features an ester linkage to the selector. This leads to different separation in comparison of other conventional carbamate-linked polysaccharide columns when used with a wider range of organic solvents.

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Unique Chemically Modified Carbohydrate Based Chiral Stationary Phases to Improve Chiral Separations Matthew Przybyciel, ES Industries, 701 South Route 73, Ste. A, West Berlin, NJ 08004, David Kohler The chromatographic separation of chiral compounds is an important tool in the search for new pharmaceutical entities. Both high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) separations of chiral chemicals are important tools for analytical determination and preparative isolation of enantiomeric mixtures. Existing chiral stationary phases can separate a many chiral mixtures. Many of these chiral stationary phases are based on chemically modified carbohydrates. However, even with the existing chemically modified carbohydrates stationary phases there are still many enantiomeric mixtures that are difficult to separate limiting the ability to characterize and purify chemical mixtures containing chiral compounds. In this study we are chemically modifying carbohydrates, such as cellulose, chitin, cyclodextrins and amylose with functional groups that have not been routinely employed. Chemical modifications of the carbohydrates include halogenated, aromatic and hetero-aromatic functional groups. We present information on the chiral separation characteristics and overall separation capabilities for these chemically modified carbohydrate based chiral stationary phases.

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Growing Up With SPME Nicholas H. Snow, Seton Hall University, Department of Chemistry and Biochemistry, 400 South Orange Ave., South Orange, NJ 07079 Solid phase microextraction (SPME), originally invented by today’s honoree and his research group, provides many lessons about chemical analysis applicable to nearly all applications. This presentation presents a historical overview of developments in traditional SPME with a focus on chemistry lessons learned from performing SPME-based experiments. It begins with a fundamental overview; the

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Think Big but Design Small, a Path to Modern Analytical Chemistry Janusz Pawliszyn, University of Waterloo, Department of Chemistry, Waterloo, Ontario, Canada, N2L 3G1 The presentation summaries my involvement in different areas of Analytical Chemistry leading to miniaturized portable devices and on-side applications. My early work as new faculty in 1980’s on application of technologies developed at that time for communication industry including LEDs, laser diodes, imaging semiconductor arrays and optical fibers are discussed. This work led to construction of different microfluidics devices including whole column imaging detection (WCID) system applied in successful “now gold standard” analytical CIEF/WCID instrument widely used in biotech industry for antibody QC and product development. The research on optical fibers lead to development of the SPME technology presently used around globe in number of laboratory and on-site applications. Recent developments of new morphologies of extracting materials and novel sampling configurations as well as approaches compatible with high throughput lab and/or on-site determinations will be outlined. The recent development of matrix compatible SPME coating lead to interesting features experienced during extraction, some of them not anticipated. They are not limited to elimination of fouling and saturation effects during direct SPME of complex samples, but also balance coverage property, enabling “via free form” clean extraction of small molecules widely varying in physical properties leading to some interesting applications. For example, on-site sampling, in-vivo metabolomics, and rapid screening via direct coupling of sample preparation to mass spectrometry were facilitated by this development.

early SPME literature presents one of the finest examples of proper introduction of a new analytical technique. Over the two decades following its introduction, SPME matured and spawned numerous additional techniques, including stir-bar sportive extraction, single drop microextraction and in-tip solid phase extraction. SPME was the founding technique of the sorptive microextraction family. Using examples from forensic and pharmaceutical analysis, key developments and principles of SPME are discussed in the context of broader analytical chemistry principles, including extraction equilibrium and kinetics and sample transfer to the instrument. SMPE has evolved and grown into one of the staple sample preparation techniques in the chromatographer’s toolkit.

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Providing and Transferring Rugged Methodology for Regulated Industries Mary Ellen P. McNally, DuPont Crop Protection, Stine Haskell Research Center, 1090 Elkton Rd., Bldg.S315/2224, Newark, DE 19711, Stephen J. Platz Analytical method validation in a regulated environment is a requirement for submission and acceptance. Exact requirements vary based on country, but include some very standard criteria including linearity, precision and accuracy followed by specificity and ruggedness. Frequently, however, the laboratory where the method development and validation initially take place are not the end users of the method. So the method transfer process after validation can be considered just as critical to the efforts to quantitate an active ingredient and its impurities as the validation itself. Little guidance is available in the literature on how to verify that a method transfer has been effective and stays effective. This presentation gives a high level overview of a validation process and then give details about a successful transfer verification protocol, highlighting successful examples as well as examples where the receiving lab fell short of being able to reproduce the validation parameters.

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Engineering Plasmonic Nanostructures for Ultrasensitive SERS Applications Laura Fabris, Rutgers University, Engn-Materials Science & Engn., 607 Taylor Rd., Piscataway, NJ 08854,Supriya Atta, Ted V. Tsoulos, Manjari Bhamidipati Near field techniques, such as surface enhanced Raman spectroscopy (SERS), rely on the ability of plasmonic nanoparticles to induce localized electromagnetic field enhancements in close proximity to the metallic surface. The possibility of achieving SERS signal enhancements high enough to enable sensitive identification of analytes down to the single molecule level depends on the presence of the so-called “hot spots”, which can be located at the vertices, edges, or crevices in isolated nanoparticles or at narrow junctions between assembled nanoparticles. In turn, the presence of finely tunable hot spots correlates to the possibility of applying SERS as a reliable spectroscopic technique in the analytical and biomedical fields. Herein, I introduce our efforts in engineering nanostructured systems based on gold nanoparticles by focusing on both synthetic and assembly strategies and present examples of applications of these concepts in chemical and biological sensing and in cell imaging. More specifically, I demonstrate how by finely tuning the morphology of gold nanostars synthesized from the bottom up we were able to develop a chemical sensor with limits of detection in the femtomolar regime and a SERS enhancement factor of 109. Finally, I introduce our latest synthesis and characterization work, in which we have identified protocols that enable us to reproducibly synthesize gold nanostars with high sample monodispersity and batch to batch reproducibility and to characterize them to determine those physical and optical parameters that are important for their application in quantitative SERS detection.

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Whole-Column Imaged Capillary Isoelectric Focusing (cIEF): From Academic Idea to Industrial Gold Standard Jiaqi Wu, ProteinSimple, 3001 Orchard Parkway, San Jose, CA 95134 When isoelectric focusing (IEF) was first run in capillary column format (cIEF) in 1985, the potential advantages of cIEF over slab gel IEF were obvious: quantitation and automation. However, until 1992, all commercial capillary electrophoresis (CE) instruments used to run cIEF were equipped with single point detectors. Thus to perform cIEF, a mobilization step had to be used after IEF separation. The mobilization step took significant amount of time compared to the IEF step, and resulted in reduced reproducibility and resolution. In general, cIEF at that time was not easy to use. The advantages of cIEF over slab gel IEF were not realized. The Whole-Column Imaged cIEF proposed in 1992 revolutionized cIEF by eliminating the need for mobilization together with its problems. The Whole-Column Imaged cIEF technology was commercialized by Convergent Bioscience/ProteinSimple. The iCE280 instrument based on UV absorption at 280nm was introduced in 1999 followed by the iCE3 in 2011. Since their introduction, these instruments have quickly become pharmaceutical industrial standard equipment to perform charge characterization for protein therapeutics. 2015 a new generation instrument (Maurice) was launched by ProteinSimple featuring additional native fluorescence whole-column detection. This detection mode led to a 5 X increased sensitivity compared to the original UV absorption detection. This higher sensitivity opens an entire new field for cIEF applications. In this presentation, application examples of the technology are shown and discussed.

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From Research to Routine: Surface-Enhanced Raman Spectroscopy as a Practical Tool in Art Analysis Marco Leona, the Metropolitan Museum of Art, 1000 Fifth Ave., New York, NY 10028 Surface-enhanced Raman scattering (SERS) is an invaluable analytical technique in the study of works of art and archaeological materials for its ability to detect organic colorants with minimal sampling requirements. The spectra of the most important natural dyes and several synthetic ones have been acquired and characterized, digital spectral libraries have been compiled, several notable examples of identification of organic colorants in important works of art have been published, and a variety of analytical methods, plasmonic substrates, and sample treatment options have been described. At the Metropolitan Museum of Art, SERS is now used routinely in the analysis of works presumed to contain organic colorants, as positive results can be obtained from samples as small as 20 micrometer across. Among the examples that are presented, the most recent is a large scale study on the introduction of synthetic colorants in nineteenth century Japan and their subsequent use in the production of woodblock prints. The analysis of over fifty prints issued between 1848 and 1898 allowed us to track the replacement of traditional Japanese colorants such as safflower red, first by the imported dye cochineal, then by naphtol red. Alongside these colorants we followed the introduction of rosaniline, methyl violet, and eosine, developing a timeline for the complex changes in the esthetics of Japanese art in the second half of the nineteenth century based on chemical information.

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Medical and Pharmaceutical Applications of Solid Phase Microextraction Barbara Bojko, Nicolaus Copernicus University in Torun, Dr. A. Jurasza 2, Bydgoszcz 85-089, Poland Solid phase microextraction (SPME) has been well established in environmental and food analysis many years ago mainly for analysis of volatiles. With the development of matrix compatible extraction phases it became widely used in these areas in direct immersion mode. It took longer to introduce SPME to biomedical and pharmaceutical applications, but great flexibility of the technology, including on one hand manual utilization of microdimensional probe and high-throughput automated system based on thin film geometry on the other, showed versatility of applications where SPME can be implemented thus enriching portfolio of currently used conventional methods. In the talk, several examples of drug studies from drug discovery step using cell cultures through in-vivo pharmacokinetics on animal model to therapeutic drug monitoring in patients are shown. This includes monitoring of drugs and drug candidate metabolism, quantitative analysis of the targets as well as impact of the drugs on the metabolome. Also discussed are how unique features of SPME like low invasiveness and sample-free chemical biopsy combined with untargeted metabolic profiling can improve the safety of chemotherapy, help to increase the pool of organ donors for transplantation or diagnostics of brain tumor bridging information provided by histological results and genotyping data. Finally, the future perspectives and directions of SPME, i.e., fast on-site/bed-side analysis with direct SPME-MS coupling or one-tool based translational medicine studies (“from bench to bedside”) are presented.

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Forensic Analysis of Blue Glass Chips by Microspectroscopy and X-Ray Spectroscopy Tiffany J. Millett, CUNY Graduate Center, 467 Ridgewood Ave., Staten Island, NY 10312, Mircea Comanescu Micro-spectrophotometry has long been employed as a technique for adding evidential value to the microscopical and forensic examination of micro transfer evidence, such as glass chips and fibers, as discussed here. A number of commercial units have been available to make transmission, reflectance, and fluorescence measurements on these types of evidence; however, many of these current and past units are quite costly. Here, we describe the use of an Ocean Optics USB2000+ fiber-optic array detector connected to a microscope and a laptop computer to obtain reflectance and transmission visible spectra. Preparation of the samples was minimal and nondestructive, which is of extreme importance in many real-life samples. Color comparison is the logical first step towards inclusion or exclusion of a possible contributing source of glass evidence samples. Color in a single fragment can vary with size and thickness, making direct comparison of two similarly colored glass chips largely subjective to the human observer. Of the forty-six blue glass samples analyzed, similarities and differences between spectra were used to distinguish amongst samples. An X-ray spectrometer, attached to a scanning electron microscope was also utilized to support our conclusions. Throughout the work on this project, surprising facts were revealed about blue glass that are discussed. We aim to improve the technical capabilities of the field of forensic science by making the sorting of glass samples within a single color class simpler and instrumentally supported. Future hopes for this project include branching to other classes of evidence and other instrumental techniques.

The Resonance Raman Spectra of Salmon Oil Fran Adar, Horiba Instruments, 3880 Park Ave., Edison, NJ 08820, Gene S. Hall Wild salmon contain various antioxidants such as the red keto-carotenoid astaxanthin which belongs to a class of compounds called terpenes. These compounds contain multiple double bonds that results in chromophores that have intense absorption bands in the UV-Vis part of the electromagnetic spectrum. Farm raised salmon are sometimes fed artificial astaxanthin that has a different absorbance spectrum compared to natural astaxanthin. Edible salmon oil has a statement of identity as defined by United States Food & Drug Administration (FDA) regulation 21 Code of Federal Regulation (CFR) 161.170 and all foods including dietary supplements must conform to the standard when products are labeled as “salmon oil”. The edible oil is produced from both wild and farmed raised salmon offal using the wet rendering method. The oil is refined and sold as dietary supplements for humans and as a nutrient for use in animal feed including companion pets and has a distinctive red-pink color. Unfortunately, our preliminary data analysis of a random selection of both human and pet salmon oil show products do not contain “salmon oil” and are misbranded. We used a macro and micro Raman spectrometer to characterize salmon oil products. For the micro Raman spectrometer, different excitation wavelengths were evaluated to enhance the forensic investigation of the misbranded products. The resonance Raman effect is very sensitive for the determination of astaxanthin in salmon oil. Detailed Raman spectrum at different excitations, band assignments, data mining, astaxanthin concentrations, and data interpretation are presented.

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The Raman Spectra of Cis and Trans Fatty Acids Gene S. Hall, Rutgers University, Chemistry Department, 610 Taylor Rd., Piscataway, NJ 08854, Fran Adar Partially hydrogenation oils (PHO) have been used extensively in processed foods. However, recent studies have linked these PHO products that have trans fatty acids have been linked to an increase in coronary heart disease (CHD). PHO have been affirmed generally recognized as safe (GRAS) by the United States Food & Drug Administration. However, the GRAS status PHO been recalled and starting June 2018 PHO cannot be marketed in foods in the US. The common method for determination of trans fatty acids in foods requires derivatization to convert the compounds to fatty acid methyl esters. Then, gas chromatography (GC) with special columns are used to separate individual trans methyl esters then either flame ionization detector (FID) or mass spectrometric (MS) detector are used to quantitate FAMEs. Technique is time consuming and may introduce artifacts into the analyses. Fourier transform infrared (FTIR) is also used to determine the total trans fatty acid concentration of a sample but the method is complicated by background signals at 967 cm-1. Raman spectroscopy on the other hand can use other bands (C=C) that are more intense due to Raman selection rules that are different than those for IR. The objective of this presentation is to show that Raman is more sensitive and selective for total trans isomers in foods and dietary supplements than current accepted analytical methods. Gaussian was used to determine theoretical band assignments for the model compounds to assist in the interpretation of the Raman spectra. Detailed sample preparation, real sample analysis, data mining, and data interpretation are presented.

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The Microscopic and Spectroscopic Analysis of Organic Gunshot Residues and Explosives Jennifer Leonard, CUNY Graduate Center, 365 5th Ave., New York, NY 10016 Raman spectroscopy is often employed due to its ability to give a chemical “fingerprint,” or molecularly specific information. It is characterized by high spectral resolution and is quick, non-contact and involves minimal preparation, making it a popular technique for chemists and criminalists alike. Normal Raman (NR) is often classified as having a low intensity and fluorescence interferences at high energy excitations that often overwhelm a spectrum. However, surfaced-enhanced Raman spectroscopy (SERS) may be a solution to these issues. SERS also involves enhancement of the Raman signal. Normal Raman spectroscopy or surface enhanced Raman spectroscopy is made a more powerful tool with the addition of confocal microscopy and results in additional output and sensitivity. This study involves microscopic and SERS analyses via different excitation wavelengths, differing sample preparations, and a variety of samples and substrates. This study involved the use of gold and silver nanoparticle substrates, as well as more novel approaches to sampling analytes of forensic interest for SERS analysis. Of particular interest were standard samples of organic gunshot residue constituents, explosives and simulated “real-life” samples of gunshot residue. These results, and other developments in SERS research are discussed.

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Quantitative and Selective 13C NMR for Determining the Composition of Wood and Cork Klaus Schmidt-Rohr, Brandeis University, Chemistry Department, Waltham, MA 02453, Pu Duan, Xiaoyan Cao Solid-state nuclear magnetic resonance (NMR) can characterize the composition and nanometer-scale structure of complex organic materials in their “native” states and analyze changes due to processing. In wood, the combination of quantitative composite-pulse multiCP 13C NMR and aromatic-signal selection by 1D PASS enables the accurate determination of the amount of native lignin. Four different measures of the syringyl:guaiacyl ratio of lignin are presented, including one based on the aromatics-only spectrum that is insensitive to ether hydrolysis and can therefore also be applied to any extracted lignin. [1] In cork, NMR spectroscopy combined with 1 H spin diffusion has identified three major components that are spatially separated. Most interestingly, the 3.5-nm thick light lamellae seen in electron micrographs have been identified as polymethylene plastic crystals that melt near 75 oC. [2]

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Forensic Science and the Amazing Multicolor Fur Coat: Microscopy of Dyed Beaver Hair Michelle D. Miranda, Farmingdale State College, 2350 Broadhollow Rd., Farmingdale, NY 11735 The results of the microscopy of assorted dyed beaver pelt used for fur apparel is presented. Beaver furs ranging in color from vibrant hues of red, pink, yellow, green and blue to neutral tones of white, beige, brown and grey were obtained for this study. Fibers were examined using polarized light microscopy and microscopy with both reflected and transmitted light. The microscopic characteristics of the hairs are reported. The goal of this research project is to develop a small guide to the microscopy of dyed beaver fur. It is proposed that by adding to the existing knowledge of mammal hairs used in garments, the forensic scientist will be better equipped to identify and compare samples encountered in casework.

References:

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Microscopic Changes in Markings Made by a Tavor Rifle Peter Diaczuk, Penn State University, Forensic Science Program, 329 Whitmore Lab, University Park, PA 16802, Andrew J. Winter The authors were given the opportunity to perform a “torture test” on a new rifle designed by Israeli Weapon Industries (IWI) called the Tavor. This rifle is a gas operated design in 5.56 x 45 mm caliber. We fired just over 2,000 rounds of ammunition through the test rifle in a variety of environmental conditions. Comparison microscopy of collected samples of spent cases and bullets was performed at 200-round intervals to determine whether microscopic changes occurred over time, influencing the ability to determine common origin. The rifle performed flawlessly throughout the experiment. Fired bullets were recovered for microscopy in a steel tube filled with polyaramid fibers to minimize damage and extraneous marks.

[1] Collaboration with Michael Timko et al., Worcester Polytechnic Institute, MA [2] Collaboration with Joe Noel et al., Salk Institute, San Diego, CA

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Electron Decoupling with Frequency Agile Gyrotrons and Fluorescent Polarizing Agents for DNP in Human Cells Alexander Barnes, Washington University-St. Louis, Dept. of Chemistry, One Brookings Dr., St. Louis. MO 63130 Electron decoupling and time domain dynamic nuclear polarization (DNP) show considerable promise to improve DNP nuclear magnetic resonance (NMR) performance. Attenuation of detrimental paramagnetic relaxation effects, increased DNP enhancement at room temperature, and efficient targeted DNP within intact human

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cells are the primary objectives of methods and technology development in our laboratory. Magic angle spinning (MAS) DNP is being applied to study the structure and dynamics of protein kinase C activation in membranes. We demonstrate electron decoupling experiments in conjunction with DNP and MAS NMR spectroscopy. Microwave sweeps through the trityl electron paramagnetic resonance (EPR) line shape are shown as a time domain strategy to significantly improve electron decoupling. For 13C spins on biomolecules frozen in a glassy matrix, electron decoupling reduces linewidths by 11% (47 Hz) and increases intensity by 14% at 90 Kelvin. Custom frequency agile gyrotrons supply >100 W of tunable power at frequencies above 200 GHz and provide the requisite control over EPR spins to implement time domain DNP transfers and also decouple electron-nuclear dipolar interactions. A custom counter flow heat exchanger reduces the liquid nitrogen consumption to 100 L per day for MAS at 82 K. Using helium gas for all spinning and cooling gases enables MAS below 15 K. A novel tri-modal DNP polarizing agent is introduced containing a fluorescent marker, biradical, and targeting moiety. Fluorescent DNP polarizing agents enable sub-cellular targeting and optical localization of DNP enhanced NMR signals. Acknowledgements: This research was supported by the NIH (DP2-GM119131), and NSF-IDBR (CAREER DBI-1553577).

Peak Capacity and Peak Capacity per Unit Time in Capillary and Microchip Electrophoresis Joe P. Foley, Drexel University, Department of Chemistry, 3141 Chestnut St., Philadelphia, PA 19104, Donna M Blackney, Erin J. Ennis Whereas numerous quantitative expressions have been reported for one- and two-dimensional separations, most are focused on chromatographic separations and few, if any, quantitative unbiased expressions have been developed for capillary or microchip electrophoresis. Making the common assumption that longitudinal diffusion is the predominant source of zone broadening in capillary electrophoresis, analytical expressions for the peak capacity are derived, first in terms of migration time, diffusion coefficient, migration distance, and desired resolution and then in terms of the remaining underlying fundamental parameters (electric field, electroosmotic and electrophoretic mobilities) that determine the migration time. The latter expressions clearly illustrate the direct square root dependence of peak capacity on electric field and migration distance and the inverse square root dependence on solute diffusion coefficient. For a given symmetrical range of relative electrophoretic mobilities (µr’s) for co- and counter-electroosmotic species (cations and anions), the peak capacity increases with the square root of the electric field even as the temporal window narrows considerably, resulting in a significant reduction in analysis time. Over a usually desirable broad µr interval an approximately two-fold greater amount of peak capacity can be generated for counter-electroosmotic species although it takes about five-fold longer to do so. The latter result is consistent with the well-known bias in migration time and resolving power for co- and counter-electroosmotic species.

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Multinuclear Quantitative NMR Spectroscopy of Crystalline and Disordered Pharmaceutical Solids Joe Lubach, Genentech, 1 DNA Way, South San Francisco, CA 94080 Quantitative analysis using nuclear magnetic resonance (NMR) spectroscopy has been in practice since the early 1960s, yet precise and accurate results are still surprisingly difficult to obtain in many cases. Quantitative solid-state NMR requires additional consideration in both acquisition and analysis of the data, but is extremely powerful in that pure standards and calibration curves are not necessary. Factors such as longitudinal relaxation, cross polarization (CP) dynamics, crystallographic in equivalence, and chemical shift anisotropy (CSA), among others, all must be considered to yield good results. Solid-state problems warranting quantitation include crystal form mixtures or crystalline/amorphous mixtures, both in drug substance and drug product samples. For pharmaceutical solids, 13C is the most widely studied nucleus and most often used in quantitative studies. It is primarily accessed via CP experiments due to its low natural abundance and long relaxation times. Although not explicitly quantitative as one-dimensional spectra, CP experiments can be treated quantitatively when acquired and analyzed using appropriate measures. Examples of 13C quantitation of crystalline active pharmaceutical ingredient (API) and amorphous solid dispersions using 1H-13C CP experiments are discussed. Fluorine is appearing in many new drug candidates, providing a built-in NMR label. 19 F is 100% naturally abundant and nearly as sensitive as 1H and not found in any pharmaceutical excipients, making it an excellent nucleus for quantitative studies of formulated products. Quantitation of low levels of disordered active ingredient in a formulated product is illustrated. Case studies presented help illustrate the utility of solid-state NMR for quantitative analysis.

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Spiral Design Rotors: A Significant Advance in Countercurrent Chromatography Martha Knight, CC Biotech LLC, 12111 Parklawn Dr., Rockville, MD 20852, Rodrigo A Lazo-Portugal In the last few years, there have been introduced new types of countercurrent chromatography (CCC) rotors or columns based on a spiral arrangement of the flow path or tubing. There are now spiral CCC rotors with capabilities far beyond the early multi-layered tubing spools which were only able to separate small molecules using organic-aqueous bi-phasic solvent systems. The first spiral design was the spiral disk rotor, a stack of eight high density polyethylene plates each with a spiral groove for the solvent flow sandwiched between Teflon sheets. Then in a plate, 4 spirals serially connectd by channels underneath were stacked between gaskets. This served to hold more stationary phase in the rotor which increases the efficiency. More modifications were made in the channels (mixing and settling sections) to retain better the viscous aqueous-aqueous 2-phase solvents like 12.5% polyethylene glycol (MW=1000) and 12.5% K2PO4. The examples of separations of proteins and carbon nanotubes will be presented. By 3 D printing of a nylon material, a circular frame to hold 1.6 mm ID PTFE tubing in layers of 4 spiral loops, was successful in chromatographing small molecules (flavonoids from a South African plant), peptides and proteins. This spiral tubing support (STS) rotor has recently been made with 12 loops. The versatile spiral rotors separate all kinds of molecules because they can use all types of solvent systems. Finally, the new preparative spiral CCC instrument, a planetary centrifuge able to run these rotors will be presented.

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Solid-State NMR Crystallography of Pharmaceuticals Utilizing Proton-Detected and Multidimensional Techniques Xingyu Lu, Merck & Co., MS: WP75B-3813, 770 Sumneytown Pike, West Point, PA 19486, Chengbin Huang, David Hesk, Anthony Leone, Robert T. Williamson, Wei Xu, Yongchao Su Solid-state properties of active pharmaceutical ingredients (APIs) are critical in pharmaceutical development. However, structural investigations at molecular level are of great challenge for crystalline compounds of suboptimal diffraction quality. Characterization of crystalline drug substances within polymer matrix is further complicated for multicomponent solid dosages. Here we present the first example elucidating crystalline structure of posaconazole (NOXAFIL®, an azole antifungal agent) utilizing ultrafast magic angle spinning (MAS) and multidimensional spectroscopy. Recent advances of ultrafast MAS with sample rotation frequencies of 60-110 kHz enables proton detection, which opens a new avenue of characterizing natural abundance pharmaceutics in a multidimensional manner. Our results of homonuclear 1H-1H and 19F-19F and heteronuclear 1H-13C, 1H-15N and 1H-19F experiments of crystalline posaconazole exhibit remarkable improvement of spectral sensitivity and resolution. These two-dimensional correlations facilitate full resonance assignments and reveal the intermolecular interactions and the molecular packing in posaconazole crystals. Intra- and interatomic distances are obtained utilizing spin diffusion and dipolar recoupling techniques for structural determination. Moreover, 1H-13C and 1H-15N dipolar and 1H, 13C and 15N chemical shift anisotropy (CSA) tensors are measured in multidimensional experiments for understanding the structure and interaction mediated site-specific motions at nanosecond to millisecond scales. These approaches presented here exhibit the capability of ultrafast MAS and multidimensional techniques for tackling questions in structural analysis of natural abundance pharmaceuticals.

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Experimental Evaluation of Microfluidic LC Column Performance: Straight vs. Serpentine Channels. Martin Gilar, Waters, 34 Maple St., Milford, MA 01757, Thomas S. McDonald, Fabrice Gritti We prepared capillary liquid chromatography (cLC) and planar titanium microfluidic (µLC) columns, 100 mm long, with 0.15, 0.3 and 0.5 mm column i.d.’s. The columns were packed with 1.8 µm C18 sorbent and tested in isocratic and gradient mode. The efficiency N and peak capacity Pc (gradient elution) of devices were monitored using a micro LC instrument with minimal extra column dispersion. Columns with serpentine channels were found to perform worse than those with straight channels, exhibiting a significant loss of efficiency and peak capacity. This effect was more noticeable for wider i.d. columns, presumably due to the introduction of on-column band broadening from the so-called “race-track” effect. The loss of chromatographic performance was found to be partially mitigated by tapering the turns (reduction in i.d. in the curved region). While good performance was obtained for 0.15 mm i.d. devices even without turn tapering, the performance of 0.3 mm i.d. columns could be brought on par with cLC devices by tapering down to 2/3 of the nominal channel width in the turn regions. The loss of performance was not fully compensated for in 0.5 mm devices even when similar tapering was employed; 10-30% loss in efficiency and peak capacity was still observed. The experimental data were utilized as guidelines for rational µLC column design. The performance of best µLC devices matched of exceeded the performance of conventional 2.1 mm i.d. UPLC columns.

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Superficially Porous Particles with Polar C18 Selectivity Richard A. Henry, Independent Consultant, 983 Greenbriar Dr., State College, PA 16801, Stephanie A. Schuster, Chuping Luo, Conner W. McHale, Robert E. Moran, William L. Johnson Resolution of a critical pair of analytes is usually the most important goal when developing a high-performance liquid chromatography (HPLC) separation. In most cases, the critical pair can be easily resolved with a standard C18 alkyl chain stationary phase or by modifications to the mobile phase. In other cases, a more polar bonded phase such as cyano, phenyl, or a polar-embedded phase offers the best option to resolve the peaks of interest. However, in some cases there may be critical pairs that need a different selectivity than can be obtained with these commonly used phases in order to obtain the desired baseline resolution of solute peaks that co-elute with another analyte. This paper describes superficially porous particles (SPP) with a new polar C18 bonded phase that retains full retention advantages of classic C18 but offers alternate selectivity for analytes that are often unable to be resolved with a C18 phase. The utility of this new selectivity polar C18 phase compared to other bonded phases will be highlighted using various example separations. Other performance measures of this new SPP column material such as use with highly aqueous mobile phases, stability under stressed conditions, and the effect of flow rate on plate count are also presented.

analysis of carcinogenic polycyclic aromatic compounds released into the Gulf of Mexico by the Deepwater Horizon event will be discussed. Acknowledgement: A. D. Campiglia acknowledges financial support from The Gulf of Mexico Research Initiative (Grant 231617-00).

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Optical Characterization of Individual Aerosol Particles for Defense and Environmental Monitoring Vasanthi Sivaprakasam, Naval Research Laboratory, 4555 Overlook Ave. SW, Washington, DC 20375 Our group at the United States Naval Research Laboratory (NRL) conducts experimental research on optical/spectroscopic properties of aerosols for developing new sensors in defense applications, and more recently, for measurement methods of environmental aerosols that could lead to improved understanding of the atmosphere. We have previously investigated several promising spectroscopic techniques, such as two-wavelength excited laser induced fluorescence (266 and 355 nm), and broadband polarized elastic scatter (450 nm to 1600 nm). Currently we are exploring surface enhanced Raman spectroscopy (SERS) on individual aerosol particles. I briefly review these studies and present example results, discussing the strengths and limitations of these techniques as well as possible future directions. I plan to briefly describe our ambient aerosol test facility located in the new Laboratory for Autonomous Sensor Research at NRL, which enables long-term, continuous sampling of local outdoor air for determining false positive rates of developmental detection systems. Our focus on aerosol particles has led to a refined capability for easily introducing, and trapping particles for long-term interrogation. This type of device could be applied to investigate chemical dynamics in materials of interest under controlled conditions of temperature, humidity and concentration of trace gas species. Our current SERS program uses metallic nanoparticles either embedded in, or on the surface of, suspended composite aerosols, and could potentially provide a non-contact means for tracking a changing particle composition. Preliminary data and results, as well as future goals for applying this technique to study atmospheric aerosols, are presented and discussed.

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Evaluating Surfactants Using Potentiometric Titration Kerri-Ann Blake, Metrohm USA, 6555 Pelican Creek Circle, Riverview, FL 33578 The concentration of anionic, cationic and nonionic surfactants is a critical parameter and will often vary across the many formulations of products made for the pharmaceutical, detergent or cosmetic industry. The development of surfactants with improved capabilities leads to more complex matrices that are often difficult to evaluate. Attend this session to hear from expert applications chemist on the unique characteristics of surfactants and the titration techniques that quickly evaluate a surfactant matrix. Hear about the progress that can be made by switching to greener surfactant titration chemistry and discusses the benefits of updating existing United States Pharmacopeia methods.

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Panel Discussion on Monitoring Pollution and Climate Change

No abstract submitted by the author.

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Modeling the Transformation of Inorganic Environmental Pollutants into Microalgal Biomass Frank Vogt, University of Tennessee, Dept. of Chemistry, 552 Buehler Hall, Knoxville, TN 37996, Mohammed F. Hasan Marine phytoplankton acts as a sink of atmospheric CO2 as it sequesters large quantities of this greenhouse gas into biomass. It has been found that the cells’ chemical environment has a major impact on nutrient sequestration capacity. Since cells can only chemically interact with their immediate vicinity, compound sequestration needs to be studied on a microscopic spatial scale. However, experimental analyses are very challenging as cultures contain a large number of microscopic cells; yet it can be efficiently done by means of computer models. Therefore, a novel modeling methodology is presented that describes all steps from atmospheric CO2 to its sequestration by marine phytoplankton for biomass production. It is shown that this model’s predictions are in very good agreement with experimental flow cytometry data. This modeling approach also enables studying impacts of nutrient competition on the biomass production and assessing upper limits of phytoplankton quantities an ecosystem can support.

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Dissolution Testing from Biorelevant to Quality Control Challenges and Gaps Jian-Hwa Han, AbbVie Inc., MS: D-R417, Bldg. AP31-4, 1 N. Waukegan Rd., North Chicago, IL 60064 For dissolution testing, we have heard about the terms of “biorelevant method,” “bio-predictive method,” “clinical-relevant method,” and “QC method” in recent years. No matter what kind of method we are going to use, the expectation is more and more clear from the regulatory agencies that the dissolution results should link to the clinical outcomes. Since biorelevant dissolution and clinically relevant specifications became hot topics, the following question has been posed: When biorelevant methods are established for formulation development, why aren’t they used as the registered quality control (QC) method? To answer this question, both current industry practice and the gap between in-vitro dissolution testing and in-vivo drug performance were evaluated. The biorelevant and QC methods usually live in two different worlds, and have very different purpose (i.e., intended use). Biorelevant methods are often used for early stage formulation screening and therefore not validated and tend to be more generic. The QC method is developed for the later-stage product (after the formulation and process are defined) with appropriate discriminating power for quality control (i.e., “registered method”). Bridging biorelevant to QC dissolution is a destination desired by both industry and regulatory authorities. On the way to this destination there are gaps and challenges to be overcome through both efforts by industry, as well as, the input and agreement from the regulatory authorities. A new working concept and proposed flowchart are presented for discussion. [1] Reference: [1] Dissolution Methodologies from Biorelevant to Quality Control – Bridging the Gaps Authors: Xujin Lu (BMS), Jian-Hwa Han (AbbVie), Danna Mattocks (Therapeutics MD), David Curran (GSK); AAPS Newsmagazine September 2017.

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Exploring the Multidimensionality of High-Resolution Photoluminescence Spectroscopy for the Analysis of Organic Pollutants in the Gulf of Mexico Andres Campiglia, University of Central Florida, Dept. of Chemistry, Physical Sciences Bldg., Rm 211, 4000 Central Florida Blvd., Orlando, FL 32816 This presentation highlights experimental and instrumental developments we have made for the collection of wavelength time matrices (WTMs) and time-resolved excitation-emission matrices (TREEMs) at liquid nitrogen (77 K) and liquid helium (4.2 K) temperatures. Individual elements of our approach had been anticipated and implemented previously, including low-temperature studies with tunable laser excitation, low-temperature measurements, excitation-emission matrix data formats, and the use of fluorescence decays as a mathematically well-behaved way to distinguish emitting components. However, the optimal combination of these features had not been realized and the techniques had generally not been practical enough for straightforward implementation. The main significant aspect of our approach is the practical, smooth way we have integrated all these features together. The complications of traditional low-temperature methodology have been removed by using cryogenic fiber optic probes. We can now routinely perform measurements at liquid nitrogen and helium temperatures; frozen samples are prepared in a matter of seconds. The full dimensionality of photoluminescence is obtained with the aid of a pulsed tunable laser for sample excitation, a spectrograph and an intensified charge-coupled device (ICCD). Because of the spectrograph and the ICCD, WTMs and TREEMs are efficiently recorded in short analysis time. Their application to the

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Linking Dissolution Method Development and Clinical Relevance – When is a Method Appropriately Discriminating? Andre Hermans, Merck & Co., 770 Sumneytown Pike, West Point, PA 19486 A common concern during dissolution method development for solid oral dosage forms for both the innovator and from a regulatory perspective is the evaluation of the discriminating power of the dissolution method towards critical process parameters (CPP) and critical material attributes (CMA). An overly discriminating dissolution method might lead to rejection of batches which could still deliver appropriate bioperformance, while an under discriminating dissolution method could result in the acceptance of tablets with unacceptable bioperformance. Here, we present approaches and case studies on how to develop an appropriately sensitive dissolution

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Mitigating Risk for Oral Solid Dosage (OSD) Extraction Using Design of Experiment (DoE) to Define an Alternate Homogenization Sample Preparation Process Adriene Malsbury, Bristol-Myers Squibb, MS: B109-A216, 1 Squibb Dr., New Brunswick, NJ 08903, Khanh Ha, Jeff Dai, William Fish Robust sample extraction is a cornerstone of reliable analytical methodology. For OSD potency/impurity and content uniformity analysis, manual extraction procedures are often employed. These procedures can be laborious, resource intensive, and time consuming. They can also be error prone and difficult to transfer particularly if the drug product formulation presents unique challenges such as gelling or swelling of the formulation matrix or active pharmaceutical ingredient (API) solubility limitations during the extraction procedure. In these cases, the typical extraction techniques of shaking/sonication may be insufficient to reliably extract the API from the formulation matrix, necessitating additional extraction steps that increase the overall method risk for generating erroneous data. This work explores the application of a homogenizing extraction procedure using a design of experiment (DoE) approach to define the optimal extraction conditions for both a tablet and capsule OSD. The DoE output will demonstrate an alternative sample preparation procedure that significantly reduce risks identified with the manual extraction process. In addition, potential applications of the alternate procedure beyond quality indicating testing are discussed.

method and examine the discriminating power of such method. In some cases, such as non-standard enabled dosage forms, development of discriminating dissolution methods can be challenging. As basis for this approach, a deep mechanistic dissolution understanding needs to be gained during formulation development to link correctly identified CPPs/CMAs with dissolution and ultimately bioperformance leading to a clinically relevant specification strategy.

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Using In-Vitro Dissolution to Support Post Approval Changes Global Regulatory Expectations Xin (Amy) Bu, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903 Dissolution is one of the critical quality attributes for solid oral dosage forms, typically tablets and capsules. In addition as a quality control (QC) test to release commercial products, dissolution is often used as a comparative test to 1) apply biowaiver for lower strength(s) when multiple strengths of one product with the same or similar formulation are marketed, or 2) support post approval changes. In these cases, in-vitro dissolution test is used in place of in-vivo bioequivalence study to establish equivalency between products of different strengths or pre- and post-change. Guidances provided by major regulatory agencies, the United States Food & Drug Administration (US FDA) and European Medicines Evaluation Agency (EU EMEA) are often followed by many countries around the world. However some countries/ regions, such as Australia, Japan, China, Taiwan and Korea have their own country specific guidances. The dissolution requirements by the FDA and EMEA are generally similar, and depend on the type and level of changes as outlined in the relevant guidances. The requirements from other mentioned countries are often significantly different from that of US and EU, and different from each other. For products marketed globally, it’s prudent to understand the differences amongst the different country requirements when applying post approval changes using dissolution to demonstrate equivalency. Several sets of comparative dissolution studies may have to be conducted in order to satisfy all regulatory agencies. This presentation compares differences in dissolution testing requirements among the listed countries and provide examples to illustrate how for conduct studies to comply with the relevant guidance(s).

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Transfer of Manual Sample Preparation for Content Uniformity (CU) Testing for a Capsule Formulation to an Automated Workstation Using Tablet Processing Workstation (TPW) Jasvinder Gaudh, Apotex Inc., 150 Signet Dr., Toronto, ON, M9L 1T9, Canada, Bhupendra Sawant Benefits of automating sample preparation are reducing manual labor, increase safety and improve analytical quality. A manual sample preparation of a capsule formulation has been transferred to an automated workstation using tablet processing workstation (TPW). Every step of automated sample preparation was validated for its precision, Linearity, accuracy, robustness and other critical parameters. TPW results obtained shown to correlate to results obtained with manual method.

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Approaches to the Development of Biorelevant and QC Dissolution Methods Michael D. Likar, Pfizer, MS: 8220-3464, Eastern Point Rd., Groton, CT 06340, Ling Zhang Dissolution and related tests are important tools used during the development and life cycle management of solid oral dosage forms. These tests are used to guide the development of new formulations, monitor the quality and dissolution stability of drug products, and assess the potential impact of scale-up and post-approval changes on product performance. In some cases, these tests can also be used to predict the in-vivo performance of the product and to obtain biowaivers. In this presentation, we show how these tests have been used to guide formulation development and to assess potential biopharmaceutical risks associated with pre-approval changes. We will also provide examples of how traditional methods employing compendial apparatuses and relatively simple dissolution media have been developed that are both biorelevant (i.e., biopredictive) and suitable for quality control purposes.

Biocatalyst Development Using High-Throughput Microfluidics and Mass Spectrometry Shuwen Sun, Merck & Co., MS: RY818-C207, 126 E. Lincoln Ave., Rahway, NJ 07065 Enzyme engineering for drug discovery, development and manufacturing has become a revolutionary technology as enzymatic transformations potentially offer higher yield, selectivity and enantiomeric purity with low cost and green chemistry. Conventional approaches to enzyme evolution by growing cells in microplates, coupled to optical or liquid chromatography mass spectrometry (LC-MS) analysis are robust but hardly meet modern fast-decision-making demands of the pharmaceutical industry. Cell-based protein synthesis (CBPS) takes weeks to obtain a winner enzyme. The relatively large volume of microplates makes the consumption of reagents prohibits large scale screenings. Optical analysis relies on customized surrogate substrates, while LC-MS cannot be faster than 1 min/assay. We propose a system to reduce the material use, eliminate special reagents and improve the overall throughput in enzyme engineering. The three key modules are: cell-free protein synthesis (CFPS), droplet microfluidics and mass spectrometry (MS). CFPS offers the speed advantage by bypassing cell growth and protein harvest, and allows active monitoring, direct manipulation and easy sampling. Droplet microfluidics offers a platform for nanoliter reactions and is adaptive to automation. MS allows completely label-free assays with high sensitivity, high selectivity, multiplexing capability, and fast scanning potential. Merck Enzyme Engineering and High-Throughput Analysis groups will collaborate with the Kennedy Lab at University of Michigan to develop this high-throughput droplet-MS enzyme evolution platform. The project bringing together world-class expertise has been funded by National Science Foundation GOALI program for nearly half a million dollars.

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Automated Forced Degradation Screening for Genotoxic Risk Assessment of Small Molecule Pharmaceutical Candidates Kaitlin M. Grinias, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA 19406, John Campbell, Kenneth Wells Delivering the best possible medicine to the patient is the primary goal of pharmaceutical research and development. Solubility and stability characterization of new target therapies is required to select the most viable compounds to progress into clinical trials. Finding the best molecule and formulation early in the development cycle will reduce the time and effort necessary for that therapy to reach the patient. Due to the large number of molecules in pre-clinical development, the study of any individual molecule is constrained by labor, time, and budgetary resources. Laboratory automation increases the throughput, thus expanding the scope of solubility and stability screening. The Freeslate Core Module 3 (CM3) automates sample preparation through liquid and solid dispensing (with integrated balance) as well as heating, cooling, stirring, and vortexing in a single platform. The system is easily programmable enabling the user to customize each experimental design. The platform interfaces with offline analytical instrumentation (such as high-performance liquid chromatography, X-ray diffraction, and Raman). Using high resolution, robust chromatography techniques and automated data processing also improve the throughput of these experiments. These laboratory automation tools facilitated the design, implementation and validation of an automated forced degradation screen that focuses on understanding the genotoxic risk of therapeutic candidates.

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A New Approach to Analytical Instrumentation and Computerization Scot D. Abbott, Phoenix First Response, 25 Allegheny Ct., Glassport, PA 14540, Ryan Taylor, David W. Faries Computerization in instrumentation design has brought profound advances for experimentation and analytical determinations. Custom, proprietary software and specialized electronics provides real time monitoring, instrument control, sophisticated data analysis and some automation. This design approach serves labs with large budgets and narrowly defined analytical experiments quite well, and has been a boon for instrument vendors. Unfortunately, for labs like ours, with many and changing analytical challenges but a modest budget, a different approach was needed. We needed to do several things: 1) Do many different experiments sporadically, 2) Have a common data file format and a common user interface for all instruments, 3) Do custom automation fix broken instruments, 4) Update good instruments having obsolete software, 5) Fix instruments with broke electronics, and (6) Make our own measurement systems (a.k.a. “instruments”). We have developed a complete

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and generally applicable ensemble of electronics and software to meet all these situations. We have used this new system to automate a number of complete experiments, revive commercial instruments with obsolete software and broken electronics, and also make measurements not heretofore available with commercial instruments. We now have a common data format and user interface for all of our instruments. The open structure of this software (uses Excel as its basis) and a well-engineered interface ensemble makes all this possible and practical for the average lab. Several examples are provided.

able tool for improving peak capacity and selectivity. However, the relatively slow speed of chiral separations has limited the use of chiral stationary phases (CSPs) as the second dimension in 2-D-LC, especially in the comprehensive mode. Realizing that the recent revolution in the field of ultrafast enantioselective chromatography could now provide significantly faster separations, we herein report an investigation into the use of ultrafast chiral chromatography as a second dimension for 2-D chromatographic separations. In this study, excellent selectivity, peak shape, and repeatability were achieved by combining achiral and chiral narrow-bore columns (2.1 mm × 100 mm and 2.1 mm × 150 mm, sub-2 and 3-μm) in the first dimension with 4.6 mm × 30 mm and 4.6 mm × 50 mm columns packed with highly efficient chiral selectors (sub-2-μm fully porous and 2.7-μm fused-core particles) in the second dimension. Multiple achiral × chiral and chiral × chiral 2-D-LC examples (single and multiple heart-cutting, high-resolution sampling, and comprehensive) using ultrafast chiral chromatography in the second dimension are successfully applied to the separation and analysis of complex mixtures of closely related pharmaceuticals and synthetic intermediates, including chiral and achiral drugs and metabolites, constitutional isomers, stereoisomers, and organohalogenated species.

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SPE is LC: Using Automation and Chromatographic Principles to Achieve High Performance SPE (SmartSPE) Mark Hayward, ITSP Solutions, 212 Northlake Dr., Hartwell, GA 30643, Jonathan Ho, Tom Moran, Kim Gamble Despite >40 yr of solid-phase extraction (SPE) using liquid chromatography (LC) sorbents, LC principles have been ignored. This is due to the lack of flow control in SPE devices. Variable flow results in variation in results. Internal standards are used to achieve meaningful results. Measuring absolute recovery against external standards to demonstrate absence of matrix effect (gold standard) isn’t done. With invention of SmartSPE, all above is changed. SmartSPE cartridges are designed around a syringe in order to achieve both automation (cartridge transport) and accurate flow (performance like LC columns). The result of its use with a CTC-PAL-MPS autosampler is total automation of SPE and LC-tandem mass spectrometry (MSMS) (or gas chromatography-MS-MS) measurement in a single parallel workflow. SPE method development is a matter of systematically applying chromatographic principles. van Deemter curves can be measured for SPE cartridges and SPE performed at optimal flow rate to achieve >99% absolute recovery versus external standard. Key operational needs are addressed such as increasing speed using smaller particles, performing mixed mode ion exchange load/elute steps at optimum flow for recovery while using different optimum flow for solvent wash for maximized (>99%) matrix removal. In addition, since SmartSPE is a micro SPE device, sample dry down is no longer needed to achieve catch/release sample enrichment as high as 200x. Finally, SPE can be performed with single use devices efficiently, economically, and with a performance level that matches all chromatographic knowledge gained in the last 50 years. Compelling examples of clinical and forensics measurements using reverse phase and ion exchange SPE will be provided.

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Innovative Approaches in High-Throughput Chromatographic Analysis in Support of Pharmaceutical Development Research Kerstin Zawatzky, Merck & Co., MS: RY800-C367, 126 E. Lincoln Ave., Rahway, NJ 07065, Christopher J. Welch High-throughput experimentation is increasingly important in modern pharmaceutical development research, with a growing need for the analysis and interpretation of hundreds or even thousands of samples in a single day. High-throughput analysis techniques have been evolving in recent years to keep pace with these demands. Recent innovations in the use of multiple injection techniques, short columns, fast autosamplers and other techniques for pushing the speed limit of high-throughput chromatographic analysis are presented. Examples are provided illustrating how such methods can be routinely developed and used to support enantioselective synthesis investigations and enzyme screening. In addition, strategies for improving the quantification of minor components and a new approach for kinetic profiling of organic reactions are also presented.

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Pharmaceuticals, Separations and Separation Science Christopher J. Welch, Welch Innovation, LLC, 29 Washington Dr., Cranbury, NJ 08512 Separation science is a key enabling technology for pharmaceutical discovery, development and manufacturing, where there is a never-ending need for the invention and application of new separation techniques and strategies. In this presentation several case histories illustrating how innovation in the field of separation science has enabled important developments in pharmaceutical research are presented along with thoughts on current emerging trends and the future progression of the field.

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Practice and Consequences of Ultra-Fast LC and SFC Daniel W. Armstrong, University of Texas at Arlington, 700 Planetarium Pl., Arlington, TX 76109 There are a number of reasons and strategies for performing ultra-fast and high efficiency liquid chromatography (LC) and supercritical fluid chromatography (SFC) separations (~0.1 to 40 sec range). These approaches are particularly important in the 2nd dimension of comprehensive LC, high-throughput analysis and in increasing the peak capacity of complex single dimension separations. However, there are consequences for doing so. One must play close attention to efficiency trends, detector limitations, secondary thermal effects, selectivity and; of course, well-known extra column effects. Some of these parameters and the factors that affect and control them can differ among instruments and particularly between high-performance liquid chromatography (HPLC) and SFC. Sometimes the chromatogram produced is not indicative of the actual peak elution profile that exits the column. The use of the sub-2µ particles, superficially porous particles (SPPs) and short columns will be taken as logical points of departure for these discussions. The “ultra-fast effects” are demonstrated and discussed using the separation of enantiomers, peptides, fluoro and desfluoro drugs, etc., as representative analytes that also require high selectivity.[1-5] Various “peak sharpening” or “resolution enhancement” data treatments also are shown to be invaluable.[5, 6]

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Approaches to Modify Silica Particles for HPLC Luis A. Colon, University at Buffalo, The State University of New York, Department of Chemistry, NS Complex, Buffalo, NY 14260, Joseph R. Ezzo, Amaris C. Borges-Muñoz, Josmely Vélez-Gonzålez We report on the surface modification of silica particles to create a thin polymeric layer on the silica surface baring amine functionalities. The modified particles have been characterized by means of thermogravimetric analysis, infrared (IR) spectroscopy, elemental analysis, and gas adsorption measurements. The amount of material on the silica particles is controlled by manipulating the reaction conditions. After characterization, the particles were packed into chromatographic columns and tested under liquid chromatographic conditions. The packed column showed to be stable under the chromatographic testing used. The particles exhibited reversed phase adsorptive characteristics while providing additional interaction with compounds having polar moieties. In this presentation, we discuss the synthetic approach used to produce the aminated layer on the surface of superficially porous silica particles as well as its characterization. In addition, we present the initial chromatographic characterization of the modified silica particles under liquid chromatographic conditions.

References: [1] Anal. Chem. 87 (2015) 9137-9148 [2] Anal. Chim. Acta 898 (2015) 128-137 [3] J. Chromatogr. A 1426 (2015) 241-247 [4] Anal. Chim. Acta 907 (2016) 31-44 [5] Anal. Chem. 88 (2016) 8821-8826

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Multidimensional Gas Chromatography (GC): Are More Dimensions Always Better? Nicholas H. Snow, Seton Hall University, Department of Chemistry and Biochemistry, South Orange, NJ 07979 Gas chromatography has evolved into a multidimensional technique; modern methods often include multiple separation chemistries that can assist or hinder analysis. New column technologies such as GCxGC and ionic liquid columns, selective sample preparation and multidimensional detectors can generate up to six separation dimensions. These additional dimensions place an additional burden on the analyst to fully understand their chemistry, for both the benefit and the possible detriment to the analysis. A solid-phase microextraction (SPME)-GCxGC-time-of-flight-mass spectrometry (TOF-MS) analysis may include up to six dimensions; each has the

[6] J. Chromatogr. (2017) http://dx.doi.org/doi:10.1016/j.chroma.2017.06.031

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High-Speed Enantioselective Chromatography as the Second Dimension in Multiple Heart-Cutting and Comprehensive 2D-RPLC Analysis Erik L. Regalado, Merck & Co., MS: RY818-B218, 126E. Lincoln Ave. / PO Box 2000, Rahway, NJ 07045, Chandan L. Barhate, Daniel W. Armstrong, Christopher J. Welch Chromatographic separation and analysis of complex mixtures of closely related species is one of the most challenging tasks in modern pharmaceutical analysis. In recent years, two-dimensional liquid chromatography (2-D-LC) has become a valu-

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potential to either aid or disrupt the separation: sampling, extraction, injection, two dimensions of GC and MS detection. Using examples from forensic, organic contaminant and pharmaceutical analysis, multidimensional GC is examined for both beneficial and non-beneficial selectivity and separation power. The addition of a new separation dimension, such as a second column or a more selective detector may allow the analyst to re-think or reduce the selectivity of other steps in the method, especially simplifying sample preparation, or it may allow improved analytical performance. In gas chromatography, additional dimensions of separation provide both more capability and more challenge: more dimensions may not always be better.

electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS). The preliminary results have shown that the particles fall at considerably lower rates than expected. The results found may provide investigators with more accurate information regarding the potential contamination of those entering the crime scene following the discharge of a firearm.

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An Investigation of the Effect of Varying Impact Force on the Formation of Three-Dimensional Fabric Impressions in Automotive Finishes Jessica E. Hovingh, The Pennsylvania State University, 329 Whitmore Lab, University Park, PA 16802, Ralph R. Ristenbatt III, Rachel E. Downey, Ted Schwartz In vehicle-pedestrian impacts with sufficient force, fabric imprint patterns may be formed in automotive finishes on vehicle surfaces. Currently, a crime lab’s focus while investigating hit-and-runs is on the individualization of paint deposited by the vehicle onto the pedestrian or into the surrounding area. In the absence of paint evidence, or biological material on the vehicle, it is often difficult to provide an evidentiary link between the vehicle and victim. The aim of this study is the elucidation of pattern production mechanics—type of fabric, surface coating, angle of impact, and impact force of the vehicle—and the eventual individualization of imprint patterns to specific fabrics. This initial study currently utilizes a pendulum. A fabric-covered dome-shaped weld cap is mounted to the pendulum arm, simulating a clothed human kneecap. The pendulum impacts a section of either a door or hood, mounted in a frame. To vary the impact force, the pendulum arm is raised or lowered. With the fabric type, surface coating, and impact angle held consistent, preliminary results show repeatable fabric imprint pattern formation, provided a narrow range of pendulum heights is utilized. Precise impact force will be calculated via analysis of highspeed video. This will allow for impact force determination and study of pattern variability with changing fabric type, surface coating, or impact angle. Initial evaluation of the patterns has been conducting using DSLR photography, optical microscopy, and micro-level terrain mapping. Other variables, including angle of impact, surface coating, and fabric type, will be examined in future studies.

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Microfluidic High-Throughput Screening Consumables that Leverage Existing Laboratory Tools Vincent T. Remcho, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331 Plate readers are to biology and biochemistry what gas chromatographs are to chemistry and petrochemistry: reliable, indispensable high-throughput sources of critical data. Plate readers have permeated the high throughput screening market as inkjet printers permeated the printing market; both are precision tools that handle small fluid volumes, both have modest acquisition cost, and both are heavily dependent on consumables. Likewise, the quality of their output is dependent on the quality of the consumables. We have explored a space that is common to plate readers, inkjet printers and bioassays by designing, building and deploying microfluidic paper-based analytical devices (umPADS) using a materials palette that leverages the best qualities and capabilities of each of the materials and methods selected. The materials include microfibrous media, polycaprolactone, and semiconductor nanocrystals, and the methods include thermal inkjet deposition, lamination, paper chromatography and electrophoresis, and plate reader-based optical spectroscopy. These selections enable high-throughput screening and analysis with minimal reagent handling and at low cost. Minimization of reagent handling leads to increased precision and accuracy, contributing to data quality and diagnostic utility. Hybrid microfluidic devices with open and wicking channels were modeled, designed, prepared and applied for colorimetric diagnostic assays for clinical biomarkers such as glucose, bilirubin, and creatinine as well as targets of forensic analysis such as cyanide and heavy metal ions, in environmental and biological samples. Assay results were analyzed using commercial UV-Vis and fluorescence plate readers for detection using custom-built devices of a form factor readily accommodated in unmodified standard plate readers.

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Evaluation and Preservation of Urine in Forensic Toxicology Meaghan M. Ringel, Arcadia University, 450 S. Easton Rd., Glenside, PA 19038, Karen S. Scott, Gail A.A. Cooper, Shanan S. Tobe Urine is a commonly encountered matrix when screening for illicit substances in driving under the influence of drugs (DUID) cases within forensic laboratories and clinical testing within hospital laboratories. Upon receiving samples, after a period of storage, samples generally undergo an extraction procedure to remove any matrix interferences prior to instrumental analysis. Solid phase extraction techniques require pretreatment of samples to achieve an appropriate pH so the analyte of interest is in the appropriate form, generally through use of a buffer. During method development for synthetic cathinones, it was determined that urine pH was changing over time and affecting these processes. It was discovered that as the time a sample remained at room temperature increased, so did the urinary pH. This was hypothesized to be due to derive from the breakdown of urea and creatinine into ammoniated compounds. This hypothesis was tested using Nessler’s reagent and the Jaffe test, which test for urea and creatinine respectively. The addition of an appropriate preservative or buffer to stabilize urinary pH was investigated to help decrease the number of failed extractions and increase drug stability in urine over time. Sodium fluoride at 0.2 % w/v was added to urine samples and pH was monitored to see if this was appropriate. Buffers of varying molarity were also evaluated. Samples were monitored in duplicate at 21 °C and 5 °C over a period of 150 days, and it was found that sodium fluoride helped maintain the pH within the necessary pH range required for successful extraction.

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Carbon Nanotube Based Chromatography, Sample Preparation and Membrane Separations Somenath Mitra, New Jersey Institute of Technology, Dept. of Chemistry, Newark, NJ 07102 The research findings highlight the applications of carbon nanotubes (CNTs) and their functionalized analogs as chromatographic stationary phases, sorbents for sample preconcentration and as fillers in membranes. Chemical vapor deposition (CVD) is an excellent method for the self-assembly of CNTs on a variety of substrates. Unique self-assembly processes are presented for both single and multiwalled CNT self-assembly. Self-assembled CNTs have been used to form gas chromatography (GC) columns in open tubular format and also as sorbents for micro- solid-phase extraction (SPE). Uniqueness of sorption characteristics of these nanocarbons is seen from the chromatographic separation of compounds with diverse volatility and polarity. A complimentary approach is the combination of CNTs with polymeric materials to develop membranes that exhibit greater permeation rates and higher selectivity. For example the incorporation of CNTs in a membrane offers several advantages because there can be several alternate mechanisms of solute/analyte transport. The high aspect ratio and nano structuring dramatically increases the active surface area and mass transfer. The CNTs serve as molecular transporters and increase partition coefficients. Together these contribute to enhanced permeation. The incorporation of CNTs in the pores of membranes is presented for different types of separations that range from analytical scale extraction to sea water desalination. In extraction of organic molecules, the CNTs serve as nano sorbents that facilitate solute exchange while in desalination via membrane distillation they provide selective sorption and transport of water vapor.

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Using SPME-GC-MS to Detect Volatile Compounds Remaining from the Storage of Dead Mice Angelica D. Wilz, Cedar Crest College, 4183 Ford Dr., Emmaus, PA 18049, Thomas A. Brettell, Thomas Pritchett One of the main objectives in forensic science is to analyze evidence so that a linkage can be made. One possibility is linking a body to a certain area based on the volatile organic compounds (VOCs) detected in the surrounding area. The goal of this study is to determine if VOCs can be detected and identified after a body has been removed and if so, how long they persist. In these studies, the headspace above decaying mice inside arson cans was analyzed by utilizing solid-phase microextraction (SPME) with gas chromatography-mass spectrometry (GC/MS). First, three different solid-phase microextraction fibers were compared. One was chosen as the optimal fiber and used for the rest of the studies. The studies examined which VOCs were detectable after the body was removed and for how long they remained. The effects of a matrix was also investigated to see how it affected the detection. It was found that as long as decomposition fluids remained in the can, the decomposition VOCs could be detected. Also, if the fluids were absorbed on a matrix (like carpet) and the matrix was moved to a location where there was no decaying body, it is still possible to detect and identify VOCs. This is significant because detecting

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Determination of Gunshot Residue Settling Velocity Cassidy Schultheis, Duquesne University, 1420 Centre Ave., Apt. 1413, Pittsburgh, PA 15219, Stephanie Wetzel, Allison Laneve, Stephanie Horner Gunshot residue (GSR) is a valuable type of trace evidence that is could be found at crime scenes where a firearm was discharged. GSR can provide investigators information about the individuals present at the scene. Once a firearm is discharged GSR particles are released from various openings of the firearm; this research will study the rate at which GSR particles settle to the ground once the firearm has been discharged. This was performed by using air suction filters placed at different heights; at each height, the three filters will be turned on at various time intervals to collect the GSR from the air. The filter samples were analyzed using scanning

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and identifying VOCs can help identify a clandestine grave or link a body to a certain area like a secondary crime scene. These linkages help law enforcement officers reconstruct the events that took place during a crime, which aides their investigation.

ssNMR parameters. In this presentation, the ssNMR crystallographic investigation of Form I of atorvastatin calcium (ATC-I) is given. As a best-selling drug, the crystal structure of ATC-I is unknown, partly due to difficulties with flexible chains involving with a large number of degree of freedom in the molecule. Multinuclear solid-state NMR and NMR crystallography, thus, become an ideal tool of studying crystal structures of ATC-I. With multinuclear 13C, 19F, and 15N ssNMR, we have focused on structural investigation of atorvastatin ligands. Recently obtained 43Ca ssNMR data of ATC-I at two fields (11.7 and 19.7 T) along with extensive ab initio calculations allowed us to gain insights into the detailed structures of calcium site. The 43Ca NMR crystallography method presented here is applicable to the structure determination of pharmaceutical APIs of calcium salts or to calcium-containing solids in general.

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Application of Gold and Silica Nanoparticles for Explosives Detection Alexandra P. Sterner, West Chester University of Pennsylvania, Schumcker Science South, Dept. of Chemistry, 750 S. Church St., West Chester, PA 19383, Monica Joshi, Gaea Lawton Nanoparticles are ultrafine particles with sizes between 1-100 nm. The physical and optical properties of nanoparticles are dependent on their composition, size and shape. Due to their large surface area, nanoparticles can be used for sensitive detection in solution or assembled as monolayers on surfaces. This presentation discusses the synthesis and application of gold and silica nanoparticles for the detection of trace amounts of explosives such as dynamite (TNT), 2,4-Dinitrotoluene (DNT), Pentaerythritol tetranitrate (PETN), and Octogen (HMX). Trace explosives detection has many challenges, one of them being the interferences due to the matrix. Nanoparticles end-capped with functional groups that give a color change in the presence of an explosive improves the specificity and sensitivity even in challenging matrices such as soil. When gold nanoparticles are in the presence of explosive particles, a color change from a wine-red to various shades of blue and purple (depending on the type of explosive) are observed. Likewise, when silica particles are in the presence of explosives, a color change from green to red is seen. The use of scanning electron microscopy (SEM), as well as optical microscopy techniques can be used to measure the size of these end-capped nanoparticles while in the presence of miniscule amounts of explosive material. This color changing solution can be used as a low-cost, sensitive, and easy to use sensor for forensic scientists to detect trace amounts of explosives in the field.

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Moving Towards Fast Characterization of Polymorphic Drugs by Solid-State NMR Spectroscopy Rosalynn Quiñones, Marshall University, Chemistry Dept., 1 John Marshall Dr., Huntington, WV 25755, Robbie J. Iuliucci Solid-state nuclear magnetic resonance (ssNMR) spectroscopy has become a common technique to study polymorphism in pharmaceutical solids at high-resolution. However, high-throughput application of high resolution ssNMR spectroscopy is severely limited by the long 1H spin-lattice relaxation (T1) that is common to solid phase compounds. Here, we demonstrate the use of paramagnetic relaxation reagents such as chromium (III) acetylacetonate (Cr(acac)3) and nickel (II) acetylacetonate (Ni(acac)2) for fast data acquisition by significantly reducing the T1 value for carbamazepine Forms I, II, III, and dihydrate, cimetidine Forms A and B, nabumetone Form I, and acetaminophen Form I polymorphs. High resolution 13C cross-polarization and magic angle spinning were used to measure T1 values for each polymorph. In order to confirm the absence of polymorphic transitions during ssNMR experiments, powder x-ray diffraction was implemented. The amount of chromium ions incorporated by the recrystallization process was quantified by using inductively couple plasma optical emission spectroscopy. Our results suggest that the paramagnetic ions added to the polymorphs do not affect the polymorphic transformation or the quality of NMR spectra. We believe that this successful demonstration of fast data collection will enable high-throughput utilization of ssNMR techniques to study polymorphic solids and could set the groundwork for NMR crystallography studies.

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Characterization of Solid Pharmaceutical Compounds and Their Dosage Forms Using Solid-State NMR of Quadrupolar Nuclei and Plane-Wave DFT Calculations Robert Schurko, University of Windsor, 401 Sunset Ave., Windsor, ON N9B3P4, Canada, David A. Hirsh, Sean T. Holmes, Austin A. Peach The screening of solid forms of active pharmaceutical ingredients (APIs) for the presence of polymorphs, impurities, and new phases is of great importance to the pharmaceutical industry. Traditional screening methods include X-ray diffraction (XRD), 13C solid-state nuclear magnetic resonance (SSNMR), and calorimetric techniques. While these are generally good for probing the bulk forms of APIs, they often have limited use for dosage forms (i.e., tablets and capsules), due to interfering signals from the excipient matrix and numerous ambiguities in interpretation of their data. We have demonstrated that both 35Cl and 14N SSNMR spectroscopy are excellent methods for characterizing APIs. 35Cl SSNMR finds utility for the study of HCl salts of APIs, since the 35Cl electric field gradient (EFG) tensors are extremely sensitive to the local chloride ion environments.[1,2] 14N EFG tensors are also dependent on the covalent and hydrogen bonding environments of nitrogen atoms in APIs.[4] These NMR handles show promise for characterization of APIs, due to recent developments in plane-wave densityfunctional-theory (DFT) calculations that continue to make NMR crystallography a reality.[5] I discuss our recent 35Cl and 14N SSNMR studies of APIs and their cocrystals, along with complementary DFT-D2 computations of EFG tensor parameters,[6] which feature a reparameterized twobody dispersion force field. This combination of techniques shows much promise for structural refinements using NMR crystallographic methods.

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Developing Accurate Crystallography without Diffraction James Harper, University of Central Florida, Dep .of Chemistry, 4111 Libra Dr., Orlando, FL 32816 This presentation explores the development of nuclear magnetic resonance (NMR) and modeling methods designed to provide crystal structures for materials that are not suitable for conventional diffraction methods. One area of focus is a highly promising non-diffraction alternative known as crystal structure prediction (CSP) - the accurate computational prediction of solid-state structure solely from the knowledge of atomic connectivity. The promise of CSP arises from its complete independence from diffraction data, eliminating difficulties associated with diffraction (e.g., poor X-ray diffraction from lighter elements). Despite over three decades of intense research, however, theoretical prediction of crystal structure has remained an elusive goal. Recent work in our laboratory demonstrates that when solid-state NMR data from powders are included in the CSP process, the correct structure can consistently be identified from among dozens of candidates. Presently, most CSP structures are of low quality and even when a correct structure is identified, a refinement step is needed to “clean up” the structure. Thus, a second area of emphasis is the theoretical refinement of CSP structures using densityfunctional-theory (DFT) methods that include lattice effects. Solid-state NMR chemical shift tensors (13C and 15N) are remarkably sensitive to these refinements and are used in our lab to monitor refinements. This NMR work suggests that such refinements have the potential to create ultra-high resolution structures that rival the accuracy of single crystal neutron diffraction coordinates. These structural improvements are realized with both conventional diffraction data and CSP structures.

References: [1] Hamaed, H. et al. J. Am. Chem. Soc. 2008, 130, 11056. [2] Hildebrand, M. et al. CrystEngComm 2014, 16, 7334. [3] Veinberg, S. L. et al. Phys. Chem. Chem. Phys. 2016, 18, 17713. [4] NMR Crystallography; Harris, R. K. et al. Eds.; John Wiley & Sons, Ltd.: Chichester, U.K., 2009.

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Root Cause Investigation of Contaminants in Raw Materials and Active Pharmaceutical Ingredients Olga Laskina, rapID Inc., 11 Deer Park Dr., Ste. 201, Monmouth Junction, NJ 08852, Kathryn Lee, Oliver Valet, Markus Lankers Raw materials and active pharmaceutical ingredients (API) need to be of a certain quality with respect to physical and chemical composition. They also need to have no contaminants, including no contaminants in the form of particulates. Otherwise, the raw materials are not pure enough to make a good quality product. The presence of particles in API can be considered product adulteration. Particle visibility is due to color, size, or shape characteristics that are different from those of the raw or API materials. When particles are found, it is important to identify them to determine the effect on quality and to find the source in order to improve the product. Sources of materials include, but are not limited to the environment, process equipment, processing materials, and packaging. Microscope versions of Raman, laser-induced breakdown (LIB), and infrared (IR) spectroscopies are excellent tools for identifying

[5] Holmes, S. T. et al. J. Chem. Phys. 2017, 146, 064201.

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Multinuclear Solid-State NMR Crystallographic Studies of Atorvastatin Calcium Steve Bai, University of Delaware, Dept. of Chemistry and Biochemistry, 033 Brown Laboratory, Newark, DE 19716, Sean Holmes Characterization of subtle structural differences among polymorphs of active pharmaceutical ingredients (API) is critical for the interpretation of stability, quality, safety, and efficacy of pharmaceutical products. The most common tools of characterizing API’s are single crystal X-ray diffraction (scXRD) and powder X-ray diffraction (pXRD). Solid-state nuclear magnetic resonance (ssNMR) crystallography is an increasingly popular tool for deriving structures of solids. When ssNMR parameters such as chemical-shift (CS) tensors and quadrupolar-coupling (QC) tensors are combined with ab initio calculations, the validity of predicted and/or refined structures can be assessed through comparison between experimental and calculated

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particles in materials because they are fast and accurate techniques requiring minimal sample preparation that can provide chemical and elemental composition as well as images that can be used for identification. The micro analysis capabilities allow for easy analysis of different portions of samples so that multiple components can be identified and sample preparation can be eliminated or reduced. The complementarity of the techniques provides the advantage of identifying various chemical components, as well as elemental and image analyses.

Validation of ATR Correction and Reverse ATR Correction Algorithms, Improved by Optimized Corrections Gregory M. Banik, Bio-Rad Laboratories, Informatics Division, 2000 Market St., Ste. 1460, Philadelphia, PA 19103, Michelle Dsouza, Keith Kunitsky, Robin O’Connor To address wavelength-dependent differences in intensity between attenuated total reflectance (ATR) and transmission Fourier transform infrared spectroscopy (FTIR) spectra, a mathematical ATR correction can be applied to an ATR spectrum to provide a better match when searching against a transmission FTIR reference database. Conversely, the reverse ATR correction can also be applied mathematically to a transmission FTIR spectrum to provide a better match when searching against an ATR reference database. An original study validated the ATR correction and the reverse ATR correction algorithm in Bio-Rad’s KnowItAll Informatics System spectroscopy software. This study updates and compares search results from the original study to search results using a patent pending Optimized Corrections technology recently added to the software that iteratively optimizes the query and reference spectra for each comparison by automatically applying multiple corrections to compensate for differences between spectra caused by the variability of different instruments and accessories as well as other factors, including human error. This technology solves many of the issues surrounding spectral search that cannot be adequately addressed by traditional algorithms and manual methods. Corrections include: baseline correction, clipping, horizontal shift, vertical shift, intensity distortion, and ATR correction.

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In-Vivo, Glucose Detection Using Mid-Infrared Laser Spectroscopy and Multivariate Analysis Alexandra Werth, Princeton University, Dept. of Electrical Engineering, Engineering Quadrangle, Princeton, NJ 08544, Sabbir Liakat, Claire Gmachl Diabetes affects over 387 million people worldwide, a number which grows every year. The most common method of measuring blood glucose concentration involves a finger prick which for some can be a debilitating process. Therefore, a portable, accurate, noninvasive glucose sensor can significantly improve the quality of life for many of these diabetics who draw blood multiple times a day to monitor their glucose levels. Recently, we have implemented a noninvasive, mobile glucose sensor based on this technology. Our system has three main components: a quantum cascade lasers (QCLs), an integrating sphere, and a thermal-electrically cooled mercury cadmium telluride (MCT) detector. The QC laser is swept over 2 µm in the mid-infrared (from 8-10 µm). The mid-infrared region, often called the fingerprint region, has strong molecular absorption features for a multitude of biomarkers including glucose. The light penetrates into the dermis layer of the skin where it is absorbed by the glucose molecules in the interstitial fluid. The backscattered signal is spatially averaged by the integrating sphere then detected by the MCT detector. Backscattered spectra were measured from multiple subjects every 5-10 minutes after eating along with a corresponding blood glucose measurement using a common electrochemical meter. Glucose has a particularly strong absorption feature due to the C-O stretching mode at 9.26 µm; this absorption peak as well as two other known glucose absorption peaks at 9.07 µm and 8.70 µm correlate strongly with the principal component (PC) of the spectra collected using the QC laser system.

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CLS & PCR Analysis of Liquids, Elucidating the Connection between Physics and Chemometrics Howard Mark, Mark Electronics, 69 Jamie Court, Suffern, NY 10901 Modern near-infrared (NIR) analysis is subject to a near-universal, but widely ignored, problem. Chemometrics has covered up the problem and enabled calibration models that “worked.” But while valuable for enabling practical use of the technology, this is game-playing, not science. Experienced practitioners of NIR sense that something is “wrong,” and more broadly, the methodology is sensed as not being part of the rest of the universe of science. For many years the underlying cause of this situation eluded detection. The cause was ultimately determined to be non-linearity in the reference values, due to the use of incorrect units to specify the concentrations of the analytes, as was presented at the 2011 EAS. A minimal algorithm applied to a very small set of specialized data supported the hypothesis. This confirmed the hypothesis, but did not demonstrate that the proper units would correct the calibration problems. What was still needed was a large enough spectral dataset to support conventional algorithms. A suitable dataset was found, and I present the results of applying CLS & PCR to that data. It looks promising to be able to close the gap and finally connect the physics of NIR spectroscopy to the rest of the scientific universe.

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Integrating Instrument Standardization Methods into Data Preprocessing Schemes Barry M. Wise, Eigenvector Research, Inc., 196 Hyacinth Rd., Manson, WA 98831, Robert T. Roginski, Benjamin Kehimkar Multivariate calibration, classification and fault detection are ubiquitous in the monitoring and control of chemical and pharmaceutical processes. Model maintenance can be defined as the on-going servicing of these multivariate models in order to preserve their predictive capabilities. It is required because of changes to either the sample matrices or the instrument response. The goal of model maintenance is to sustain or improve models over time and changing conditions with the least amount of cost and effort. Instrument standardization methods are an important element in the model maintenance toolbox. The goal of instrument standardization is to map the response of a separate instrument, or the same instrument at a later time, onto the response of the master/standard instrument. Many methods have been developed for instrument standardization, including Direction Standardization (DS), many variations on Piecewise Direct Standardization (PDS), and Spectral Subspace Transformation (SST). Multivariate calibration models often include a number of preprocessing steps before the actual regression, classification or other model is applied. But how should the standardization method be integrated with the preprocessing scheme? Should standardization be done before or after preprocessing? Or even between preprocessing steps? Our experience suggests that this question does not have a universal answer, and the optimal approach is case-specific. Based on this we have developed a framework for standardization that allows insertion into calibration models before, after, or in between preprocessing steps. This preprocessing/standardization framework is presented in this talk, and several representative cases are demonstrated.

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Quantification of Oleic Acid in Biologics Solutions Enables Early Detection of Host Cell Protein Mediated Polysorbate 80 Degradation Meng Xu, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08902, Zhihua Liu, Dilusha Dalpathado, Joseph Valente, Mark Bolgar Polysorbate 80 (PS80) is a non-ionic surfactant widely used in biologics product formulations to stabilize proteins against aggregation, particle formation and adsorption to surfaces. PS80 contains a polyethoxylated (PEG) moiety and an oleic acid moiety linked via an ester bond. Recently, it was found that PS80 in biologics product formulations can undergo ester hydrolysis into oleic acid and PEG moiety, due to the presence of lipase(s)/hydrolase(s) originating from host cells used for manufacturing. Current literature reported assays for monitoring this enzymatic activity rely on measuring a decrease in signal from intact PS80 which can take weeks to manifest. Direct measurement of lipases/hydrolases is impractical due to the low abundance and diverse specificities of these enzymes. We therefore envisioned an assay to monitor the accumulation of oleic acid should enable significantly more sensitive and encompassing detection of enzymatic activity. A sensitive liquid chromatography mass spectrometry (LC-MS) method was developed to monitor the accumulation of oleic acid in biologics solutions. The quantitation of oleic acid proved challenging due to its poor solubility, low molecular weight and matrix effect from PS80. A mixed mode column with a step gradient was used to separate oleic acid from protein and other abundant non-polar solution components. A quadrupole mass spectrometer with electrospray ionization source was used for mass analysis under the negative ionization mode. The assay enables rapid and confident detection of enzymatic hydrolysis to PS80 within as little as 24 hours. It can be widely used to monitor lipase activity across different stages of biologics manufacturing and formulation development.

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Applications of FT-NIR in HME Process Monitoring Herman He, Thermo Fisher Scientific, 4410 Lottsford Vista Rd., Lanham, MD 20706, Anh Vo, Jiaxiang Zhang, Michael Repka, Scott Martin Hot melt extrusion (HME), as one of continuous pharmaceutical manufacturing processes is of increased industrial interest because it allows formulation scientists to design pharmaceutical products with unique active pharmaceutical ingredient (API) delivering profiles. A HME process development requires multivariate process data from different operation variables to be aligned and explored within the quality-by-design (QbD) context. This presentation describes an application of Fourier transform near-infrared (FT-NIR) spectroscopy technique to monitor real-time API concentration during a HME process and the use of principal component analysis (PCA) to investigate extrudate spectral responses to HME operation variables, such as extrusion temperature, screw speed, and feed rate.

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Effective Determination of Pharmaceutical Impurities by TwoDimensional Liquid Chromatography (2DLC) Zhimin Li, Waters, 34 Maple St., Milford, MA 01702, Paula Hong, Patricia McConville, Chris Desjardins Analysis of many pharmaceutical compounds often involves evaluation and quantification of impurities that co-elute with active pharmaceutical ingredient (API). Impurity peak co-eluting with API is a major challenge, particularly when other related compounds are also analyzed. To address these challenges, a minimum of two methods are typically employed: a primary method to assess majority related compounds and a secondary method to address the impurities that co-elute with API in the primary method. Two-dimensional liquid chromatography (2-DLC) offers a solution to effectively analyze all mentioned impurities in one chromatography system setup, saving analysis time and increasing productivity. In this work, a targeted 2-DLC platform is evaluated for effective impurity analysis to address impurity peak co-elution issue. In the first dimension, a reversed phase (RP) method is used to separate related compounds from the main compound. The main peak is then transferred to a second dimension to separate an additional impurity that co-elutes in the first dimension. The approach uses a heartcut process combined with trap and elute. To dilute the organic composition of the effluent prior to the second dimension, an additional pump is used for at-column-dilution (ACD). Hence, the targeted 2-DLC platform, “Heartcut-ACD-Trap-Elute,” is shown. The work flow and strategy for the targeted 2-DLC platform is described including the rationale of adding both ACD and a trap column. The impact of the targeted approach on the data quality in the second dimension is explained. Using the proposed targeted 2-DLC platform, we demonstrated a strategy to quantify low-level impurities in pharmaceutical drug substances, including geometric (cis/trans) isomers.

Differentiation of Isomeric Metabolites of Diclofenac Using Electron Induced Dissociation Mass Spectrometry Zhidan Liang, Merck & Co., 126 E. Lincoln Ave., Rahway, NJ 07065, Wendy Zhong 4’-OH-diclofenac and 5-OH-diclofenac are two major metabolites of diclofenac in humans. The structural distinction of this pair of isomers has relied heavily on nuclear magnetic resonance and synthetic standards. In this study, a 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer was used for the structural elucidation of 4’-OH-diclofenac and 5-OH-diclofenac. Collisional induced dissociation (CID) and electron induced dissociation (EID) experiments were both performed for diclofenac, 4’-OH-diclofenac and 5-OH-diclofenac in order to compare the fragmentation behavior of EID to that of CID and determine if EID could generate diagnostic fragments for the differentiation of the two isomers. CID fragmentation of all three compounds exhibited the same fragmentation behavior, generating three major fragments, which corresponded to the same bond cleavages at identical locations across three compounds. EID fragmentation of these diclofenac compounds was particularly efficient, generating about 20 extra fragments over CID. The resulting fragments provided detailed structural information to locate the hydroxyl groups on different rings, thus allowing differentiation of the pair. EID demonstrated superior capability over CID in differentiation of isomeric compounds in this study.

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Fast Isotope Ratio Mass Spectrometry (FIRMS): A Tandem Mass Spectrometry Technique for the Rapid and Semi-Comprehensive Evaluation of Isotope Ratios Fredrick M. Ochieng, South Dakota State University, Department of Chemistry and Biochemistry, 1055 Campanile Ave. SAV 131, Brookings, SD 57006, Brian A. Logue, Paul J. Hinker Isotope ratios (IRs) are a measure of the variation in abundance of light isotopes versus heavy isotopes of an element, e.g., 1H/2H,12C/13C, and 14N/15N. Because IRs change as a molecule undergoes certain chemical and physical processes, they represent unique fingerprints that can be used to trace the source of a chemical compound. Current techniques for determination of IRs each have major limitations, such as loss of structural information, vulnerability to contamination, high cost, large samples size requirements, low precision, limited applicability, and lengthy analysis. Fast isotope ratio mass spectrometry (FIRMS) was developed as a next-generation analytical technique for robust measurement of IRs using tandem mass spectrometric data. Based on the FIRMS nonlinear mathematical model, the difference between predicted and experimental tandem mass spectrometric data was minimized by modifying isotopic fractional abundances of the atoms involved. FIRMS was used to calculate the isotope abundances of molecules, including diisopropyl methylphosphonic acid (DIMPA), and acetylsalicylic acid from multiple sources. FIRMS produced excellent precision, with IRs standard deviations of less than 0.1‰, for carbon and oxygen, and 0.01‰, for hydrogen isotopes, for all atoms involved when compared with IRMS in a fraction of the time. FIRMS also produced delta values within 350‰ and 2450‰ for acetylsalicylic acid and DIMPA, respectively, compared to IRMS. FIRMS offers several advantages over other methods of IR determination, and may ultimately become the preferred method for determination of isotope ratios.

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Sol-Gel Capillary Microextraction with Niobia-, Tantala-, and Zirconia-Based Sorbents Providing Selective Enrichment of Phosphopeptides and Neurotransmitters for On-Line HPLC Analysis Abdul Malik, University of South Florida, 4202 E. Fowler Ave., CHE 205, Tampa, FL 33620, Sheshanka Kesani, MinhPhuong Tran, Abdullah Alhendal, Mohanraja Kumar In current analytical practice, extraction media are predominantly silica-based. A notable shortcoming of silica-based materials is their poor pH stability. However, in many important biomedical, pharmaceutical, and environmental applications pH stability of separation/extraction media is often a pre-requisite. As a result, significant research initiatives have been undertaken toward developing alternative sorbent materials. In an effort to address the pH stability problem, our group has developed titania- [1, 2], zirconia- [3, 4], and germania-based [5, 6] sol-gel microextraction media in the form of surface-bonded coatings. In this presentation, we highlight our recent work on the synthesis of niobia-, tantala-, and zirconia-based sol-gel sorbents and their applications in chemically modified electrode (CME) for on-line preconcentration of phosphopeptides and catecholamine neurotransmitters. We present experimental data demonstrating remarkable selectivity of niobia-based sorbents toward phosphorylated peptides. Examples of on-line CME-high-performance liquid chromatography (HPLC) analysis of underivatized catecholamine neurotransmitters and related compounds are presented highlighting microextraction performance and pH stability of the newly developed zirconia- and tantala-based sol-gel sorbents.

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Application of Pre-Filtering Human Serum for the Determination of Perfluoroalkyl and Polyfluoroalkyl Substances (PFASs) by HighPerformance Liquid Chromatography-Online Solid Phase Extraction-Tandem Mass Spectrometry Chang Ho Yu, New Jersey Department of Health, 3 Schwarzkopf Dr., Ewing, NJ 08628, Bhupendra R. Patel, Zhihua (Tina) Fan, Bahman Parsa This study presents an optimization of previous methods for the measurement of PFASs in human serum, utilizing an online solid phase extraction (SPE) unit for sample processing. Direct injection of human serum into the liquid chromatography mass spectrometry (LC-MS) instrument causes operational and analytical challenges to arise – namely, column clogging. Biological macromolecules (e.g., proteins) present in serum are too large to pass through the column (3.5 µm) unhindered, which clogs the column, elevates column pressure, and results in system shutdown. In order to reduce column clogging and therefore enable uninterrupted instrumental analysis, a system was developed to pre-filter human sera prior to autosampler injection. Three filter types (Polyethersulfone (PES), Polyvinylidene difluoride (PVDF), and nylon) were tested to determine the optimal filtering media, giving special consideration to each filter’s ability to bind proteins while minimizing analyte loss. The evaluation was conducted on five bovine serum replicates spiked with 12 analytes and internal standards. Un-filtered sera were analyzed as control samples to evaluate analyte loss during filtering and resultant concentration deviations. PES filters recovered most internal standards (recovery range 90-119%), except Et-PFOSA-AcOH and PFOSA (67% and 36% recoveries, respectively). Lower recoveries (mean ± SD [range]) were observed in PVDF (53±19% [28-82%]) and nylon filters (59±28% [13-107%]). Analyte recoveries in PES filters [90-105%] compared most closely to the un-filtered samples, followed by PVDF [76-124%] and nylon filters [89-121%]. Paired comparison for human sera (n = 40), un-filtered and pre-filtered

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Advantages of Cold Electron Ionization LC-MS for Structure Elucidation Ryan D. Cohen, Merck & Co., MS: RY800-D133, 126 E. Lincoln Ave., Rahway, NJ 07065, Christine Fisher, Renee Dermenjian Combining electron ionization mass spectrometry (EI-MS) with liquid chromatography (LC) has a long history predating the development of electrospray ionization. However, unlike in gas chromatography, EI-LC-MS has not seen widespread use due to the dearth of robust and commercially available instruments. In this study, we evaluated the first commercially available “cold EI-LC-MS” interface developed by Aviv Analytical, which was paired with a single quadrupole mass spectrometer from Agilent Technologies and controlled by Chemstation software. LC effluent is sprayed via a nebulizer into a vaporization chamber at up to 0.15 mL/min. Solvent and analyte are rapidly vaporized at 250 °C, transferred via a heated capillary through a specially designed nozzle into vacuum, and finally ionized by a custom tungsten filament. This process generates a supersonic molecular beam leading to cold electron ionization. This approach has the advantages of reduced ion tailing, increased molecular ion abundance (M*+), and extended mass range up to 1000 Da. We found the technology to be robust over a year of operation with minimal downtime. Performance metrics (i.e., sensitivity, precision, and linearity) and several case studies highlighting the advantages of this technique over other LC-MS ionization modes will be discussed: a) ionization of non-polar compounds including those of high MW, b) extended fragmentation for regioisomer determination, and c) rapid structure elucidation from National Institute of Standards and Technology library searching.

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with PES filters, must be conducted to further evaluate the efficacy of pre-filtering serum in this method.

Study of VOC Exposure at Fuel Stations with a Portable GC System Anika Poli, Defiant Technologies, Inc., 6814 Academy Parkway West NE, Ste. A, Albuquerque, NM 87109, Douglas R. Adkins, Patrick R. Lewis Defiant Technologies performed a survey of potential hydrocarbon exposure at refueling stations using the TOCAM™ portable gas chromatography photoionization detector (GC-PID) system. TOCAM™ is a portable GC-PID that performs air monitoring for volatile organic compounds (VOCs). The system has a micro-GC column to separate the components of a sample, and a micro-preconcentrator (PC) to collect and inject VOC samples into the column. A 10.6 eV photo-ionization detector at the exhaust of the GC measures the elution time and signal level of the VOC components as they emerge from the column. This information is used to identify the component and determine the concentration. The TOCAM™ is also equipped with a second PID at the inlet of the system to provide real-time feed-back on the presence of VOCs in the air. This screening PID was found to be useful in correlating specific refueling activities with VOC exposure. This presentation covers the results of this study and discusses other testing procedures that could be useful in VOC studies such as vapor intrusion characterization or industrial hygiene surveys.

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Chemometric Approach to Purity Analysis and Quality Assurance of Pure and Adulterated Natural and Essential Oils Sayo O. Fakayode, University of Arkansas Fort Smith, Dept. of Physical Sciences, Box 3649, 5210 Grand Ave., Fort Smith, AR 72913, Brianda Elzey, David Pollard, Carol M. Babyak Natural oils (NOs) and essential oils (EOs) are widely being used in the food, cosmetic, and agrochemical industries. However, NOs & EOs with high market values are often counterfeited and adulterated with cheaper, lesser quality oils, with serious economic implications for industries, and potential health implications for unsuspecting consumers. This study explored the possible use of ordinary Fourier transform-infrared spectroscopy (FTIR) in conjunction with partial-least-squares (PLS) multivariate regression analysis for the purity determination of selected adulterated NOs (neem oil and flaxseed oil) and EOs (wintergreen oil, tea tree oil, rosemary oil, and lemon eucalyptus oil). The FTIR spectra of the calibration sample sets of known compositions of adulterated NOs & EOs at a concentration range of 1 to 99 wt/wt% were measured and subjected to PLS regression. The figures-of-merit of the PLS regression models were excellent with good linearity (R2≥0.999039). The PLS regression models accurately determined the % compositions of the validation samples of adulterated NOs & EOs with errors of prediction. Microwave assisted acid digestion and inductively coupled plasma-optical emission spectroscopy were used to further determine element concentrations in NOs & EOs. The element levels were oil dependent and varied considerably in the samples. Element levels were subjected to principal component analysis (PCA) for pattern recognition in NOs & EOs. The first two principal components explained 100% of the variability in the element compositions. The PCA were found capable of accurately distinguishing NOs & EOs based on the element composition and physical properties of oils

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High-Throughput Implementation of Gas Chromatography with Automated Sample Preparation and Universal Carbon Response Calibration Marcelo Filgueira, Dow Chemical Company, 400 Arcola Rd., Collegeville, PA 19426, Reetam Chakrabarti, Marie Devlin The state of the chemical industry is rapidly changing with more complex processes and products. With the development of modern laboratory equipment and software, leading companies and research organizations are heavily investing in high-throughput screening capabilities. Accurate measurement of analytical process parameters is key for an efficient and successful product development and has traditionally been very time consuming due to the need for sample preparation and calibration of multiple components. Therefore, generating more data with limited workforce and tighter timelines presents a challenge. In this presentation we will describe an implementation that combines automated sample preparation, gas chromatography and flame ionization detection with universal carbon response. This implementation requires minimal calibration and also enables accurate quantification even when the reference materials are not available or are difficult to obtain.

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Field-Deployable Applications of Raman Spectroscopy for Screening of Unapproved and Counterfeit Drugs Jason D. Rodriguez, United States Food & Drug Administration, 645 S. Newstead Ave., St. Louis, MO 63110 The availability of safe and effective drugs is crucial in the event of a national emergency such as a pandemic. Currently, chromatographic techniques are the prevailing methods used to assess drug quality. While highly accurate, these methods are time consuming and require destruction of the sample. This presentation focuses on the United States Food & Drug Administration’s (FDA) efforts to expand the use of portable instrumentation to screen antibiotics. Portable spectroscopic instruments are capable of screening samples at a much higher throughput than can be achieved by laboratory analysis. These tests can be carried out in the field and can typically be conducted in less than a minute without destroying the sample, which is important should further analysis be necessary. Raman spectroscopy is one of the techniques that may be used to screen for counterfeit or substandard drug products. The seminar focuses on FDA’s progress in the development of qualitative and quantitative models that can be used with portable Raman instruments. Overall, the results obtained for identity, dissolution rate and assay by Raman spectroscopy compare favorably with those obtained by traditional laboratory methods. This study indicates the potential for implementation of portable Raman-based approaches into the suite of analytical techniques for pharmaceutical analysis.

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Fast Gas Chromatography of Residual Solvents in Pharmaceutical Excipients Using a Novel Vacuum Ultraviolet Spectroscopy Detector Lindsey Shear-Laude, VUV Analytics, 715 Discovery Blvd., Ste. 502, Cedar Park, TX 78613, Jack Cochran Residual solvents used in pharmaceutical manufacturing have no therapeutic value and are regulated to various levels based on their toxicity. Analysis of these organic volatile impurities in excipients is often done with static headspace – gas chromatography (GC) – flame ionization detection. Given that the flame ionization detector is non-specific, long GC runs may be necessary to avoid coelutions of the analytes of interest with each other or matrix-evolved compounds. This inhibits analytical throughput and could lead to false positives without a confirmatory technique. A new, fast acquiring, sensitive gas chromatography detector based on vacuum ultraviolet (VUV) spectroscopy is available that collects absorbance spectra to unequivocally identify compounds of interest even when they directly co-elute. Spectral deconvolution can even be accomplished for isomeric species with VUV spectra, unlike in electron ionization mass spectrometry. The detector is also not carrier gas flow-rate limited like a mass spectrometer. These VUV detector features allow for chromatographic compression to decrease run times and increase throughput. This presentation demonstrates the capabilities of the VUV detector when used with static headspace – GC for residual solvents analysis. In addition to analysis of Class 1 (e.g., benzene and carbon tetrachloride) and Class 2 solvents in model excipients, GC and VUV detection of water and formaldehyde, compounds relevant to pharmaceuticals analysis but not amenable to flame ionization detection (FID), are shown.

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Portable Raman Spectroscopy for Rapid Identification of Unknown Precious Gemstones Kristen A. Frano, B&W Tek, 19 Shea Way, Newark, DE 19713, Dawn Yang Gemstones of questionable origins have recently flooded the jewel market, making the identification and authentication of unknown precious stones an extremely important task. Often, even experienced jewelers may require additional analytical resources when determining the identity of an unknown gemstone. Here, we develop a solution using a portable Raman spectrometer to create libraries of Gemological Institute of America-certified gemstones, including quartz, sapphire, ruby, tourmaline, and cubic zirconia. An identification software was used to match Raman spectra obtained from unknown gems to the gemstone libraries via a correlation coefficient algorithm. Fourteen unknown gemstones were compared against the library and correctly identified based on their correlation coefficients with high separating power. This rapid identification enables jewelers as well as customs and border patrol agencies to identify unknown stones.

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Towards Understanding the Basis of Oddy Test Failures via Volatile Organics and Other Analytical Analyses Eric B. Monroe, Library of Congress, 101 Independence Ave. SE, Washington, DC 20540, Kelli Stoneburner, Cynthia Connelly Ryan, Fenella France As part of a secure storage vault renovation which is designed to have a relatively low air turnover, we have been examining and testing a range of building and construction materials for their suitability and level of risk to the collection. The vast majority of tested materials were found to fail the Oddy metal coupon test, often in a rather unusual or spectacular manner. We have since been examining the compounds emitted by these materials and how they interact with the metal coupons. Through the use of thermal desorption gas chromatography mass spectrometry, we are able to identify and quantify the compounds emitted from a material. In addition,

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Biotherapeutics Counterfeit Determination Ishan Barman, Johns Hopkins University, Dept. of Chemistry, Baltimore, MD 21218 No abstract submitted by an author.

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other analytical tools are being utilized to examine what compounds are depositing or have reacted at the surface of the Oddy test coupons during exposure to elevated temperatures and humidity. Coordinating and comparing these chemical analyses with the results from the Oddy test are improving our ability to understand the mechanism(s) behind the failure of the Oddy test and, in turn, guide future product selection. While the goal of this screening is to select materials that produce a minimal risk to the collection, this risk often cannot be entirely removed by product choice alone (i.e., all tested materials damage a metal coupon to some extent). As a means of mitigating the residual risk from volatiles, we are also examining and characterizing commercially available sorbent materials for their selectivity, capacity, functionality and adsorption/desorption characteristics.

plan as illustrated by recent work that was successfully completed at the Philadelphia Museum of Art.

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Vibration Risk Assessment for Immovable Artworks in Churches during a Tunneling Work Anna Henningsson, Disent AB, Coldinutrappan 3, Stockholm, 11228, Sweden From 2007 to 2014 a 6 km long railway tunnel was constructed under the city of Stockholm, Sweden. Because the city is built on granite, blasting techniques had to be used. The tunnel construction project took place under or very close to five historic churches. In these churches, extensive ensembles of immovable artworks and liturgical furnishing such as large-scale altarpieces, pulpits, wall paintings and stucco are present. In Sweden, risk assessment related to tunnel construction and blasting mainly focusses on risks associated with the building. As a consequence, risk mitigation is geared towards structural aspects of the historic building rather than protection of immovable works of art and architectural surfaces integrated into the building. The limited research and hands-on experience with vibrations related to tunnel blasting and its possible effects on immovable artworks led to the development and application of a risk assessment model customized for large-scale immovable artworks. The basis for the model was continuous monitoring during the tunnelling work (blasting). This monitoring not only consisted of measuring the levels of vibration and deformation of the building structure. It also focussed on continuous visual monitoring of the works of art are architectural features to detect any visual damage which could be related to the measured vibration/deformation data. This model also served as a communication platform during construction, providing project stakeholders with information about the associated risks. It also provided insights into how different stakeholders and professionals perceive value and risk related to vibrations and historic immovable art.

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Guidelines on Selecting, Determining, and Interpreting Analytical Detection Limits Ephraim M. Govere, Pennsylvania State University, 116 ASI, Dept. Ecosystem Science & Management, University Park, PA 16802 All analytical instruments have limits of detection which indicate their functional sensitivity to specified measurands or analytes. Several terms are used to refer to these limits including: limit of blank, lower range limit, upper range limit, minimum detectable value, limit of detection, instrument detection limit, method detection limit, reliable detection limit, limit of quantification, estimated quantitation limit, practical quantitation limit, program required quantitation limit, and decision limit. While these detection limit terms may sound familiar to chemists, the guidelines on selecting, determining, and interpreting them are not necessarily easy or consistent. For example, which term should one use and for what purpose? How should one instrumentally measure the selected limit and what method should be used to calculate that limit? At what point would one consider using empirical approaches to estimating the selected limit? What confidence level should one choose and why? And what number of analyses are required to characterize the selected limit at the chosen confidence level? The objective of this presentation is to answer these and many more questions on limits of detection to enable chemists make informed decisions that guarantee the production of valid, reliable, and meaningful analytical data.

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Vibration Protocols to Protect Museum Collections during Major Demolition Works: Experiences from Liverpool’s Library Project Siobhan Watts, UK National Trust, Erddig, Wrexham, Wales, LL13 0YT, Great Britain, David Crombie, Tracey Seddon Major demolition and construction works to Liverpool’s Central Library in 2011-12 presented a potential vibration risk to collections at Liverpool’s Walker Art Gallery and World Museum, neighboring museums that are part of National Museums Liverpool. The Library shared party walls with galleries containing sensitive collections: Renaissance paintings on the galleries sharing walls to the east, and Egyptian galleries, with painted coffins displayed against party walls, to the west. National Museums Liverpool negotiated a vibration protocol with the contractor for the Library works, which involved continuous monitoring and two thresholds – a warning level at 1.5 mm/s peak particle velocity and a stop level at 3 mm/s peak particle velocity. This paper considers how successful this approach was in protecting collections, and in enabling business continuity during 18 months of intensive demolition and construction works. It also discusses how vibration data and the experience gained from this project contribute to our understanding of the risks to collections from vibration.

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Considerations of the Potential for Damage to Individual Works of Art caused by Vibration during Construction/Demolition Projects Andrew Lins, (retired) Philadelphia Museum of Art, 22 West Southampton Ave., Philadelphia, PA, 19118 Construction and demolition are activities that take place with surprising frequency in the operation of museums, historical sites, and organizations involved in the preservation of cultural heritage around the globe. Such activities often put art at risk of damage from vibrations generated by heavy mechanical equipment. Similarly, a critical part of the daily operations of museums is the movement of works of art into and out of exhibits, and their packing and shipping for exhibition or loan, where minimizing the potential for damage from excessive vibration during installation or transportation must be carefully managed. While human error due to mishandling is the commonest source of damage in moving artwork, misunderstanding the fragility of individual works of art and the levels of vibration to which they are exposed is a close second. On the other hand, sometimes these considerations indicate that the risk of damage is less than anticipated when objectively considered. This paper illustrates some of these points with practical examples from the Philadelphia Museum of Art over the past 25 years, in particular the protection of Marcel Duchamp’s Large Glass, a problematic masterwork of the twentieth century.

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Infrared Spectroscopy Based Approach to Assess Metabolic Profile and Damage in Cardiac Tissue Saumya Tiwari, University of Illinois at Urbana-Champaign, 405 N. Mathews Ave., Urbana, IL, 61801, Rohit Bhargava, Jai Raman Cardiovascular diseases are the leading cause of mortality in the USA, accounting for 1 in 4 deaths. Heart failure (HF) is the most common cause of hospital admissions in North America with over 5 million patients and $18 billion in costs in the US alone. Differentiating between cardiac conditions that are reversible and those that are irreversible is crucial since it determines therapy options. It is known that myocardial metabolism is altered in many irreversible cardiovascular diseases, and is linked with differential outcome.[1] In this project, we sought to identify spectral signatures associated with metabolism in cardiac tissue by utilizing animal models that expressed experimentally regulated levels of glycogen and lactose. Frozen samples were embedded in optimal cutting temperature (OCT) compound and sectioned in 10μm slices onto LowE (Kevley) slides. Using analysis of variance (ANOVA) we identified spectroscopic features that were independent of the source animal and experimental variance. Machine learning approaches were used to further separate samples with differential metabolism levels. This is the first demonstration of utilizing metabolic profiling using infrared spectroscopy in cardiology. Cardiovascular disease management could greatly benefit from infrared imaging based molecular profiling since additional information is critically needed to determine the best possible intervention.

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A Blueprint for Managing Construction Vibration Risk Near Sensitive Structures Douglas Rudenko, Vibra-Tech Engineers, 109 E. 1st St., Hazleton, PA 18201, Mohamad Sharifinassab Building and renovation projects for museums often bring both joy and anxiety for the staff responsible for the display and protection of exhibit pieces. Vibration – that perceptible yet mysteriously invisible force brings justifiable concern. To manage vibration risk, you must understand what the risk is and develop a plan to manage that risk. A proper protection plan should contain some key elements: 1) A walk through the exhibit space with the construction team, conservation staff, and vibration consultant to understand the means and methods to be used during each phase of construction, and to identify the location of vibration sensitive exhibits relative to construction activities. 2) Performance of vibration testing in each exhibit space identified to determine vibration characteristics such as vibration transmissibility, natural frequency, and attenuation. 3) Establishment of vibration criteria based upon vibration testing results. 4) Prediction of vibration levels induced by construction equipment and defined setback distances for each piece of equipment relative to the defined vibration criteria. 5) Vibration testing on each piece of equipment to be utilized to assure the actual vibration levels are similar to the predicted vibration levels. Once a piece of equipment is qualified it can operate within the defined setback distance. 6) Develop a monitoring plan for each phase of the construction sequence. The plan should include alarm levels for notifications and protocols to cease/resume vibration producing activity. This lecture discusses elements of this

Reference: [1] Ussher JR, Elmariah S, Gerszten RE, Dyck JRB. The Emerging Role of Metabolomics in the Diagnosis and Prognosis of Cardiovascular Disease. J Am Coll Cardiol 2016;68:2850–70. doi:10.1016/j.jacc.2016.09.972.

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oligosaccharide analysis. Solid-state nanopores with the capability to tailor-make the pore sizes, holds promise for polysaccharide analysis. For the first time, we demonstrate the ability of silicon nitride nanopores to detect polysaccharides and enzyme-digested products and ability to differentiate them by a fingerprint profile. Moreover, we demonstrate the nanopore fingerprint capability in detecting contaminants in polysaccharide based drugs by analyzing a heparin sample deliberately contaminated with over-sulfated chondroitin sulfate.

A Quantitative Mass Spectrometry Imaging Workflow Using IRMALDESI and MSiReader Mark T. Bokhart, North Carolina State University, 2620 Yarbrough Dr., Raleigh, NC, 27695, Ken Garrard, Elias Rosen, Corbin Thompson, Craig Sykes, Angela DM Kashuba, Jeffrey Manni, David C. Muddiman Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid ionization technique combining laser desorption and electrospray ionization. IR-MALDESI is well-suited for mass spectrometry imaging (MSI) where the sample is analyzed in a spatially resolved manner. The spatial distribution and ion abundance can then be represented as a two-dimensional (2D) ion map. The field of MSI has routinely provided important qualitative distributions, however, obtaining quantitative MSI data has proven to be much more challenging. IR-MALDESI has several advantages over other MSI platforms, such as complete ablation of the sample at each position and ionization by electrospray, that produce quantitative data. A quantitative IR-MALDESI MSI method using normalization to a structural analogue and incorporation of a spatial calibration curve has been developed for thin tissue sections. This method was applied to study the spatial distribution of antiretroviral drugs in a multi-organ, dosed-animal and human studies. The MSiQuantification tool was developed and incorporated into MSiReader, an open-source MSI software to simplify quantification data analysis. This tool allows the user to define calibration spots and regions to be quantified on the displayed MS image, with linear regression and quantification being performed by the software. Finally, the spatial resolution of IR-MALDESI MSI was improved through the incorporation of a multi-element optical system. An adjustable iris, 3.75x beam expander, and aspheric focusing lens were used to decrease the IR laser ablation diameter from 150 μm to 50 μm for a tissue imaging experiment.

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Investigation of Photoanodic Water Oxidation Surface Species on Hematite Using SI-SECM Mihail R. Krumov, University of Illinois at Urbana-Champaign, Roger Adams Laboratory 60, Urbana, IL 61801, Burton H. Simpson, Joaquin Rodriguez-Lopez Hematite is a promising photoanode for solar driven water splitting because of its narrow band gap, chemical stability, and elemental abundance. However, high overpotentials are required to overcome sluggish water oxidation kinetics which curtails its efficiency and prevents practical application.[1] To offer insight on the best strategies for improving hematite anodes, we use a toolbox of electroanalytical techniques based around scanning electrochemical microscopy (SECM). SECM is unique in its ability to directly quantify local coverage and reactivity of surface species across the broad range of spatial and temporal scales relevant to lifetimes of adsorbed species during reaction.[2] Here, we use the surface interrogation mode (SI-SECM) to probe in-situ the reactive oxygen species (ROS) that are responsible for hematite’s slow kinetics. Satisfactory fitting of the SI-SECM signal obtained on hematite photoanodes required the existence of two populations of surface species reacting with different rate constants. We observed that while the surface coverage of both species was found to increase as a function of applied bias, the rate constants did not change appreciably, showing that the identity of the species doesn’t change with increasing activation. The characteristic lifetimes of ROS were studied by varying the time spend undisturbed before titration. Kinetic analysis of these results showed that the decay of ROS happens through a second order process. Using pulsed substrate generation/ tip collection mode, we detected hydrogen peroxide as a product of this decay.

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An Integrated Platform of Three LC-MS-MS Methods for the Quantification of Urinary Metabolites Differentially Expressed in Respiratory Illnesses Mona M. Khamis (Hamada), University of Saskatchewan, College of Pharmacy and Nutrition, Health Sciences Building, Saskatoon S7N 5E5, Canada, Hanan Awad, Kevin Allen, Darryl J. Adamko, Anas El-Aneed Asthma and chronic obstructive pulmonary disease (COPD) are chronic conditions of the respiratory airways. Their differential diagnosis can be hampered by their overlapping clinical presentations. There is a need for better objective tests in primary clinical settings. Metabolomics has demonstrated promising potential in biomarker discovery, and we have been studying its diagnostic use for respiratory diseases. Our 1H-nuclear magnetic resonance (NMR) data has suggested 50 highly polar, low molecular-weight urinary metabolites as candidate biomarkers for the differentiation between asthma and COPD. The metabolites were divided into 4 subgroups, based on chemical nature/concentration. Groups 1 and 2 contain amine and carboxylic acid-bearing metabolites. Their quantification was achieved using differential isotope labeling (DIL) with dansyl chloride and dimethylaminophenacyl bromide reagents, respectively. Group 3 contains 7 metabolites of diverse chemical properties rendering them unquantifiable through DIL. Accordingly; we developed a hydrophilic interaction liquid chromatography tandem mass spectrometry (MS-MS) method using stable-isotope internal standards for matrix effects correction. Group 4 contains 3 trace-level metabolites and their quantification method is currently under development. We have fully validated groups 1 and 2 high-pressure liquid chromatography-MS-MS methods. The recommended validation criteria were sometimes insufficient for some metabolites, but we have made important procedural modifications in order to confirm methods’ robustness. Preliminary data from patients’ samples were analyzed for groups 1 and 2 metabolites, and we see good diagnostic potential. Method 3 is currently being validated according to the United States Food & Drug Administration and European Medicines Agency guidelines, and we expect patient data to be added to the statistical model in the near future. The involved biochemical pathways will be revealed upon the completion of sample analysis.

References: [1] Iandolo, B.; Wickman, B.; Zorić, I; Hellman, A. J. Mater. Chem. A 2015, 3, 16896– 16912. [2] Simpson, B.H.; Rodríguez-López, J. Electrochimica Acta 2015, 179, 74–83.

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Efficient Method for the Identification of Common Herbicides in Rain Water and from Air Filters by UPLC-MS/MS Steven L. Kolakowski, University of Connecticut, Chemistry Department, 55 North Eagleville Rd., Storrs, CT 06269, James D. Stuart, Christopher R. Perkins, Anthony A. Provatas This investigation presents a straightforward method for the quantification of four commercially abundant herbicides: 3,6-dichloro-2-methozybenxoic acid (DICAMBA), 2,4-dichlorophenoxyacetic acid (2,4-D), 3,5,6-trichloro-2-pyridinyloxyacetic acid (Triclopyr), and N-(phosphonomethyl)glycine (Glyphosate) and its degradation product, aminomethylphosphonic acid (AMPA) in rain water and extracted from air filters for all of these acidic herbicides. Acidic herbicides are used primarily to combat broadleaf weeds and pose a significant risk to the environment and human health due to their widespread use and potency at minimal concentrations. It is suggested that the herbicide 2,4-D, a known endocrine-disrupting compound, is a cause of endocrine related cancers. It has been reported that exposure of male mallards to Roundup, of which glyphosate is a primary constituent, produces harmful effects to their reproductive systems and hormone production. Previous studies conducted by our research group reported a highly sensitive method for the direct analysis of glyphosate and AMPA without derivatization utilizing ultra-pressure liquid chromatography tandem mass spectrometry (UPLC-MS-MS) in single ion mode (SIR) with a UPLC amide column. This report expands upon this work, applying a simple sample preparation for the four herbicides and using two chromatographic methods utilizing a Waters UPLC-MS-MS system for the quantification of the targeted compounds. Due to the contrasting polarities of the compounds, a hydrophilic interaction liquid chromatography (HILIC) column was used for the separation of glyphosate and AMPA, while a C18 column was utilized for DICAMBA, Triclopyr, and 2,4-D. The presented method is significantly faster than those in the current literature, eliminating complications from analyte derivatization. It overcomes difficulties stemming from the polarity of the targeted chemicals, allowing for a more comprehensive analysis of the commonly applied herbicides.

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A Sweet Promise from Solid-State Nanopores: PolysaccharideSugar Analysis Challenge Buddini Iroshika Karawdeniya, University of Rhode Island, Department of Chemistry, 140 Flagg Rd., Kingston RI, 02881, Y.M. Nuwan D.Y. Bandara, Jonathan W. Nichols, Robert B. Chevalier, Jason R. Dwyer Polysaccharides are a complex class of biomolecules that play a vital role in energy storage, structure, cellular communication, and even as drugs, e.g., heparin. Compared to other biomolecules like DNA or proteins, polysaccharide analysis is significantly complicated with over 100 naturally occurring monomer units that build polysaccharides of wide variety of linkages, chain lengths, isomeric forms and branching. Recent contamination of heparin - a widely used anticoagulant drug caused adverse effects and over hundred deaths in the United States and it took a combination of classical analytical techniques (only after the deaths) to pinpoint the contaminant - over-sulfated chondroitin sulfate - another polysaccharide molecule structurally close to heparin. Nanopore single molecule sensors have shown immense promise for DNA and protein analysis and have promise to do the same for polysaccharides. So far, nanopores have been limited to monosaccharide and

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Comparison of GC Column Conditions Using the Mahalanobis Distance Alexandra Clifford, The College of the Holy Cross, Department of Chemistry, Worchester, MA 01610, Edward J. Soares, Amber M. Hupp Due to environmental concerns, the use and study of biodiesel fuels has increased as a form of clean, renewable energy in the field of green energy. Biodiesels can be

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synthesized from a variety of plant oils and animal fats. Biodiesels produced with methanol can be characterized by their unique fatty acid methyl ester (FAME) profiles, as each feedstock contains a unique profile of resulting FAMEs. In this study, FAME content was analyzed for various biodiesel feedstocks using gas chromatography coupled with mass spectrometry (GC-MS) to produce a unique fingerprint for each feedstock of origin. Biodiesel samples were analyzed using two GC columns of differing column lengths and stationary phase thicknesses. Gradient temperature programs were optimized for each column individually. Chromatograms were baseline corrected, aligned via correlation optimized warping (COW), area normalized, and analyzed with principal components analysis. Using the Mahalanobis distance to compare corresponding sets of PC scores from the two columns, we demonstrated that certain combinations of COW alignment parameters could be found so that there is no statistical significance between the data from the two columns. Thus, the column with the shorter length (and therefore shorter analysis time) can be used to acquire comparable peak resolution and overall separation.

0.32 mm x 1.8 µm) and Elite-BAC2 columns (30 m x 0.32 mm x 1.2 µm). Helium carrier gas at a flow rate of 12.30 mL/min was utilized, and the column temperature was set to 70 °C. Calibration curves were created by analyzing eight standard ethanol calibrator solutions (10 mg/dL to 500 mg/dL) in duplicate over five days, and pooling the data; r2 values exceeded 0.999 for both columns, showing good linearity. Intraday and interday variation was assessed using aqueous solutions, bovine blood, human blood, and human oral fluid. A controlled dosing study was performed utilizing 12 female subjects who consumed a pre-determined amount of wine (11.5%) in order to reach a target BAC of 0.05 g/dL. Blood and oral fluid samples were collected and analyzed. Results showed the ethanol concentration profiles correlated well between blood and oral fluid; Pearson correlation values were calculated to be 0.92-0.97.

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Development of a Spectroscopic Method for the Forensic Analysis of Fingernail Polishes and Gels Alyssa Smale, Lebanon Valley College, 140 Dickison Ln., Stroudsburg, PA 18360, Donald Dahlberg, Nicole Bois, Brooke Kammrath Fragments of fingernails are a common type of trace evidence found at crime scenes or on suspects, especially in cases where the victim utilized self-defense. Five replicate Fourier transform infrared (FTIR) spectra of 98 different shades of pink and red fingernail polishes and gels were recorded using three different infrared spectrometers. Several classification methods and data pretreatments were used to classify the 490 samples in a leave-one-out cross validation. KNN coupled with Multiplicative Signal Correction and Generalized Least Squares Weighting was able to correctly classify 98% of the samples. In no case was more than one replicate misclassified. No two instruments of the same type produce exactly the same spectra, which would require the construction of a k nearest neighbor (KNN) model on each instrument throughout the country that is to be used to identify the polishes and gels. Spectral transfer methods require the spectra of a small number of samples taken on the “parent” and each “daughter” spectrometer. The Piecewise Direct Standardization algorithm is being used to create spectral transfer matrices that corrects each spectrum taken on the daughter spectrometers. This allows the use of the parent model on spectra taken on daughter instruments. Blind tests of the model are presently being performed to further evaluate the model and to determine if the transfer model is working correctly. Spectra of new bottles of polish and samples exposed to various environmental conditions such as light, water and soil will also be used to evaluate the model.

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Electrochemical Characteristics of a Class of Pyrenequinones Rebecca Kubena, Cornell University, Department of Chemistry and Chemical Biology, Baker Lab, Ithaca, NY 14853, Luxi Shen, Héctor D. Abruña As the demand for high-performing batteries is growing dramatically, there has been an increasing interest in developing environmentally friendly and abundant organic battery materials. Here, we report the electrochemical assessment of a group of organic, aromatic compounds known as quinones. Because of their carbonyl groups, the quinone redox-active moiety are ideal candidates for the storing of electrons and protons. We aim to learn more about the redox properties of these organic compounds by comparing a class of quinones containing carbonyls in different positions: 1,6 pyrenequinone, 1,8 pyrenequinone, 4,5 pyrenequinone, and 4,5,9,10 pyrenequinone. We used cyclic voltammetry to investigate the electron transfer between electrolyte dissolved in solution and the quinones. We uncovered interesting results that depended on both the placement and number of carbonyl groups. The 1,4 and 1,8 pyrenequinones showed a two-step electron transfer whereas the 4,5 and 4,5,9,10 showed a three-step process. Using electrochemical methods such as cyclic voltammetry and rotating disk electrode voltammetry, we were able to calculate diffusion coefficients for each quinone. It was found that all three quinones with two carbonyl groups had a diffusion coefficient within the 10-6 cm2 s-1magnitude while the quinone with four carbonyl groups had a slower diffusion rate with a magnitude of 10-7 cm2 s-1.

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A Novel Solution for EtG/EtS Analysis in Human Urine by LC-MSMS Connor Flannery, Restek Corporation, 110 Benner Circle, Bellefonte, PA 16823 Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are unique biomarkers of alcohol use. The detection of these metabolites has proven advantageous for zero tolerance treatment programs and abstinence enforcement where information regarding recent alcohol consumption is required. The analysis of EtG and EtS offers many advantages for abstinence monitoring including the detection window (~3 days), stability in stored specimens (non-volatile), and specificity. EtG and EtS are both polar which makes them difficult to retain via reversed-phase chromatography. Both compounds are also very sensitive to matrix interferences which can result in being unable to achieve low limits of detection. Isobaric interferences can also make quantitation impossible. In this study, a simple dilute and shoot method was developed, validated, and applied to patient samples for the analysis of EtG and EtS in human urine by liquid chromatography tandem mass spectrometry (LC-MS-MS) enabled by the use of the novel Raptor EtG/EtS column.

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Implementation of a Novel Parallel Solid-Phase Microextraction with DART Technology for High-Throughput Screening of Drugs Brittany Laramee, IonSense, Inc., 999 Broadway, Ste. 403, Saugus, MA 01906, Brian Musselman, Joseph Tice, Steve Shrader, Frederick Li Ambient ionization was proposed to reduce sample preparation and facilitate rapid determination of drugs in biological fluids. We enabled several early attempts at direct thermal desorption of drugs from plasma and urine using direct analysis in real time – mass spectrometry (DART-MS); however, matrix interferences limited the utility of the method. Eventually the use of solid phase micro extraction (SPME) fibers and sorbent coated screens were demonstrated to remove those matrix related interferences. We report here the most recent implementation of this DART-SPME technology. Direct thermal desorption of samples from the surface of novel solid phase micro extraction-transmission mode (SPME-TM) devices has been completed using a DART-MS system. These devices consist of a mesh coated with a thin layer of polymeric particles that enables the extraction of analytes from a matrix. We demonstrate here the utility of these SPME-TM devices for reducing matrix interference by isolating the drugs from the matrix. The system is utilized for semi-automated collection of 12 samples per experiment, thus facilitating sample collection of up to 96-separate samples in less than 30 minutes. In order to permit rapid, reliable determination of drugs in biological samples a novel data program that permits the creation and search of DART-MS library databases has been developed. Here we searched the acquired mass spectral information for drugs that were extracted using the SPME-TM devices against an internally developed database consisting of a wide array of drugs and controlled substances, which includes designer drugs, opioids, stimulants, hallucinogens and novel psychoactive substances.

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Analysis of Plasma Free Metanephrine, Normetanephrine, and 3-Methoxytyramine by Hydrophilic Interaction Liquid Chromatography Connor Flannery, Restek Corporation, 110 Benner Circle, Bellefonte, PA 16823 Metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) are methylated metabolites of epinephrine, norepinephrine, and dopamine, respectively. These catecholamine metabolites are released from the adrenal medulla and sympathetic nervous cells and are maintained at very low concentrations in the bloodstream. Measurement of these metabolites in plasma is highly sensitive for the diagnosis of pheochromocytoma and paraganglioma as observed by elevated levels in circulation. Analysis of these polar compounds using typical reversed-phase LC is difficult due to limited chromatographic retention and lack of sensitivity. As a solution, an liquid chromatography tandem mass spectrometry (LC-MS-MS) method was developed based on hydrophilic interaction liquid chromatography using a Raptor HILIC-Si column. Combined with a simple and fast solid-phase extraction (SPE) procedure, an accurate and precise analysis of plasma free metanephrine, normetanephrine and 3-methoxytyramine can be achieved and applied to high-throughput clinical assays.

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Utilization of Headspace Gas Chromatography for the Analysis of Ethanol in Blood and Oral Fluid Samples from Dosed Individuals Emily R. Parchuke, Cedar Crest College, 100 College Dr., Allentown, PA 18104, Matthew Wood, Marianne Staretz, Thomas A. Brettell Oral fluid has the potential to provide a better estimation of blood alcohol concentration (BAC) when driving while impaired is suspected. A method to analyze ethanol in both blood and oral fluid was developed in order to assess utility of headspace gas chromatography for this purpose and to correlate ethanol levels in both matrices. In this study, a Perkin Elmer HS-Clarus 580 headspace gas chromatograph with two flame ionization detectors and a TurboMatrix™ 40 autosampler was utilized. A single headspace injection was split between two columns, Elite-BAC1 (30 m x

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Automated Online Desorption and Analysis of DNPH Derivatives of Airborne Aldehydes and Ketones Using a New Robotic Autosampler Fred D. Foster, GERSTEL, Inc., 701 Digital Dr., Ste. J, Linthicum, MD 21090, Jackie Whitecavage, Kurt Thaxton, John Stuff The analysis of airborne aldehydes and ketones first involves collection of the analytes by passing air through a cartridge containing 2,4-dinitrophenylhydrazine (DNPH). As the air passes through the cartridge, the analytes react with the DNPH to form hydrazones which are immobilized on the cartridge. The cartridges are then eluted with solvent and the DNPH derivatives can be analyzed using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The new GERSTEL MPS roboticPRO sampler with a special tray to hold DNPH cartridges enables the entire process of desorbing the analytes and injection of the samples into the LC-UV system to be easily controlled. Automating the desorption of these cartridges can result in significant improvement in accuracy and reproducibility as well as reducing potential experimental errors by the operator. An integrated Balance Option allows weight data to be automatically collected following the desorption of the cartridges to further improve the accuracy of the reported results. The intuitive software includes tools that allow the desorption of a cartridge to take place during the chromatographic separation of a previously injected sample to ensure maximum sample throughput. In this report, the automation of the online desorption and analysis of airborne DNPH derivatives by the robotic autosampler is discussed. Examination and calibration for a variety of aldehyde- and ketone-DNPH derivatives is described. Finally, DNPH cartridges collected from representative matrices are desorbed and analyzed online and the resulting precision data are provided.

amide-forming coupling reactions, which are of great importance to the pharmaceuticals industry.

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Determination of Chemical Composition of Copolymers by SizeExclusion Chromatography with Dual Detection Yejia Li, Ashland, 1005 Route 202/206, Bridgewater, NJ 08807 A new method with size-exclusion chromatography-ultraviolet-refractive index (SEC-UV-RI) dual detection was developed to quantify the copolymers along the molecular weight. Traditional SEC-UV/RI method requires homopolymers to determine the response factors in both detectors. However, some of the homopolymers are not available and some homopolymers may not be soluble in the solvent used for the analysis of copolymer, which limit the application of the traditional method. The new method created a calibration curve with copolymers of various copolymer composition and enabled the analysis without homopolymers. The results revealed that different copolymers with the same comonomer ratio in average contained different profile of chemical composition as a function of molecular weight, result in different performance. This method provides an efficient and cost effective way to characterize copolymers.

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LC-MS Method Development for the Detection of Phosphate Lipids Junling Gao, Merck & Co., MS: RY-818-A106, 126 E. Lincoln Ave., Rahway, NJ 07065, Pei Huo, Wendy Zhao, Yan Lin Support for vaccine research and a method to monitor the synthesized phosphate lipids (Plds) prompted the development of a fast and sensitive liquid chromatography mass spectrometry (LC-MS) method. Retention of Plds was obtained using a Waters X-Bridge C18 column eluted with a mobile phase of A (H2O/20 mM ammonium carbonate) and B (ACN). Mass detection was achieved by a Waters QDa Mini-MS with both electrospray in both the positive and negative ion modes. Commercially obtained Plds, (dihexadecyl phosphate, L-alpha-phosphatidyl-DL-glycerol and phosphatidylcholine) and Merck synthesized Plds were evaluated, and both molecular ions, [M+H]+ and [M-H]- were detected with the reasonable sensitivity. The X-Bridge C18 column trifunctionally bonded and packed with BEH (bridged ethylene hybrid) particles provided good peak shape, excellent stability for basic mobile phase, and high retention or separation efficiency based on the lipid acyl chain length. Advances in LC-MS allowed low abundance Plds to be more readily detected. The method presented highlights the application of Pld analysis and offers a reliable approach for the detection of Plds.

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Method Development on a Combined Reverse Phase and SFC HPLC System Peter C. Ratsep, Shimadzu Scientific Instruments, 19 Schoolhouse Rd., Ste. 107, Somerset, NJ 08873, John R. D’Alessio Traditionally, standard reverse-phase high-performance liquid chromatography (RPHPLC) and super critical fluid chromatographic (SFC) systems have had dedicated hardware for each application. With the Shimadzu UC (Unified Chromatography) system, not only can a user switch between RP and SFC modes on the system, but it can be accomplished in a single batch using either manual methods, or using Shimadzu MethodScouting software. The MethodScouting software was used to illustrate the optimization process on both the HPLC and SFC modes. The MethodScouting software is also capable of Blending different solvents, and combinations of different pH buffers. For testing the SFC to HPLC switching capabilities, switching was done on a single C18 column and separate columns for each mode. Results are discussed.

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Simultaneous Determination of M12 and M17, Unique Human Metabolites of a BTK Inhibitor (BMS-986142) Using LC-MS-MS: Assay Development, Qualification, and Case Studies Yulia A. Kim, Bristol-Myers Squibb, Rt. 206 and Province Line Rd., Princeton, NJ 08540, Ang Liu, Jian Wang, Weiping Zhao, Ihab Girgis, Wenying Li Identification and characterization of major circulating metabolites in humans are required at the early stage of drug development. However, prediction of human metabolite profile is challenging from in-vitro systems (microsomes, hepatocytes) or studies in pre-clinical species. BMS-986142, a potent and reversible inhibitor of Bruton’s Tyrosine Kinase (BTK), is currently at clinical development stage. The pharmacologically active N-desmethyl metabolite M11 was the major metabolite in rat and dog and was considered to have high probability of being a major circulating metabolite in humans. However metabolite profiling of FIH samples identified two unique human metabolites M12 and M17, while M11 was determined to be minor in human plasma. A combined liquid chromatography tandem mass spectrometry (LCMS-MS) assay was developed to quantitatively evaluate the exposure of M12 and M17 simultaneously in human plasma from MAD study. The plasma samples were prepared by protein precipitation and the separation of the analytes was achieved by gradient elution on an Acquity UPLC BEH C18 analytical column. Selected reaction monitoring (SRM) of m/z 589.3 > 553.2 was used for the detection of both M12 and M17. The assay was quantified over the concentration range from 2 ng/mL to 2000 ng/mL. Assay development, fit-for-purpose assay qualification, and clinical case studies are discussed in this presentation.

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Resolving the Issues in Challenging Analysis of a Compound without a Chromophore and a Compound with a Reactive Primary Amino Group Van Truong, Merck & Co., MS: RY818-B221, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ 0065, Hao Luo, Bing Ma Compounds without a chromophore and compounds with a reactive primary amino group are particularly challenging in analytical method development to meet the strict requirements for regulatory validation, method transfer, and release. In this presentation, we used two common building blocks in drug synthesis, Hydroxy pipecolic acid (HPA, compound without a chromophore) and Boc-piperidine amine (BPA, compound with a reactive primary amino group), as examples to demonstrate how to solve the problems and successfully validate and transfer the methods for GMP release testing. HPA and its related impurities have no UV chromophore. Thus, a corona aerosol detector (CAD) was used for assay of impurities. However, the CAD detector provided very low sensitivity, non-linear response, and inconsistent impurity profiles, which made the method validation and transfer difficult. In this poster, we demonstrate how high quality solvents/reagents, and a stainless steel nebulizer can improve the sensitivity; how the ion pairing between the volatiles and mobile phase reagents can introduce a ghost peak detectable by CAD; and how to quantitate impurities with the quadratic response from CAD. BPA is a primary amine that reacts spontaneously with CO2 from the air to form carbamic acid. The CO2 intake posed great analytical challenges on conventional compound assay by HPLC and identification by IR. To reduce the complication from the CO2 reaction, a reference-free assay by titration and infrared analysis with special sample preparation were developed to ensure consistent and accurate analytical results.

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Comparison between 13C and Deuterated Dansyl Chloride for Differential Isotope Labelling Targeted Metabolomics Teagan J. Holt, College of Pharmacy and Nutrition, University of Saskatchewan, 104 Clinic Pl., Saskatoon, SK S7N 2Z4, Mona M. Hamada (Khamis), Anas El-Aneed, Darryl J. Adamko Targeted metabolomics involves the accurate quantification of preselected metabolites in biological samples. A differential isotope labelling (DIL) approach was adopted for 17 amino acid metabolites using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The amino and/or phenol moieties of the metabolites were derivatized with dansyl chloride (DNS-Cl). DIL uses light and heavy isotopologues of a derivatizing reagent to produce light and heavy-derivatized metabolites, respectively. Allowing for the creation of an internal standard (IS) for each target metabolite. Two forms of heavy labeled dansyl chloride are 13C2- DNS-Cl and d6-

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Analysis and Derivatization of Water-Sensitive Activated Carboxylic Acids Using Reversed-Phase Liquid Chromatography Maor F. Baruch, Hovione, 40 Lake Dr., East Windsor, NJ 08520, Michael Puppolo, James Choi, Niharika Chaganti, Chen Zhou Herein, we report a facile method for the derivatization and analysis of water-sensitive carbonyldiimidazole (CDI) activated carboxylic acids using reversed-phase liquid chromatography. The method is shown to be viable for a variety of carboxylic acids, and a number of derivatization agents are compared. The reported protocol can be applied to the determination of the reaction progress (in-process control) of

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DNS-Cl. The 2 Da mass unit difference between 13C2-DNS-Cl and the 12C analyte is undesirable in MS due to the natural isotopic contribution from 12C. The deuterated form, however, will not demonstrate natural isotopic contribution due to the 6 mass unit difference. In this work, both forms of DNS-Cl were used to derivatize solutions of the 17 metabolites. Two calibration solutions were prepared for each IS and analyzed using LC-MS/MS. The aim is to evaluate the performance of d6DNS-Cl compared to 13C2- DNS-Cl. Our results demonstrate significant deuterium effects resulting in large variations in the retention times between the d6 and 12C dansylated analytes. In contrast, the 12C labeled analyte co-eluted with its analytes for all metabolites. However, this work observed significant contribution of the 12C labeled analyte on the lower IS concentrations. In Conclusion, deuterated ISs are not suitable for DIL-based MS assays and a high concentration of 13C ISs is needed to minimize the effects of isotopic contribution from the 12C target analytes.

and optimizing the acid/ base concentration used to reach a final pH where the formulation was a solution up to 50 mg/mL for each test article. The three challenging aspects of the method was establishing a short analytical method to separate the individual test articles, detection of the individual test articles down to 1 µg/mL with reproducible response, and ensuring no interference from the vehicle components, specifically HPMC. The assay was successfully validated with three sessions and the formulations were shown to be solutions at 50 mg/mL. Stability of the formulations was established for both test articles for up to 15 days of refrigerated storage. This study shows strategies and general approaches for formulation optimization and analytical chemistry method development for multi component formulations.

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Sensitive UV-HPLC Method for the Analysis of the Docusate and Related Compounds. Michael Breslav, Johnson & Johnson Consumer Inc., 7050 Camp Hill Rd., Fort Washington, PA 19034, Rajesh Darji, Gail Reed Docusate [dioctyl sulfosuccinate], is an anionic surfactant that is used to decrease surface tension between water and organic phases due to its amphiphilic properties. Because of its ability to breaking uniform organic phase into smaller particles, Docusate is used in both environmental and medical applications. In the environmental field, Docusate sodium (DOSS) is used to treat ocean water contaminated with oil, such as during the 2010 oil spill in the Gulf of Mexico. In the medical field, both the sodium and potassium salts of the Docusate are used as actives in the rectal enema preparations. Because of its lack of good chromophores and its ion-pairing nature, Docusate remains a challenge for chromatographic methods. Thus, a review of the literature reveals that studies of Docusate stability are typically performed using liquid chromatography-mass spectrometry techniques. We have developed a firstin-class sensitive ultraviolet high-performance liquid chromatography (UV-HPLC) method that is being used to analyze docusate and docusate related compounds in an over-the-counter (OTC) formulation. This methodology has been demonstrated to be useful in the understanding of the Docusate degradation pathways.

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Rapid and Sensitive Quantification of Desmosine in Body Fluids Using Stable-Isotope Labeling and MALDI-MS2 Pratikkumar N. Rathod, York College-City University of New York Graduate Center, 94-20 Guy R Brewer Blvd., Jamaica, NY 11451, Manjeet Kaur, Hsin-Pin Ho, Basant Dhital, Marissa Louis, Kevin J. Mark, Gregory Boutis, Jong-III Lee, Emmanuel Chang The pyridinium-based amino acids, desmosine (Des) and its structural isomer isodesmosine (Isodes) stemming from the condensation of four lysine amino acid residues, serve as a cross-linking network of elastin. The elastin protein contributes to the structural foundation of lung tissue, skin and blood vessels, and provides elasticity so that tissues can maintain their shape and normal physiological functions. Quantification of elastin degradation products, desmosine and isodesmosine may be used as a biomarker for various pathological conditions including chronic obstructive pulmonary disease (COPD). The current study presents a novel approach to quantify desmosine and isodesmosine using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2). MALDI-MS2 analysis of desmosine and isodesmosine are performed using stable-isotope labeled desmosine as an internal standard in different biological fluids such as urine and calf serum. The important analytical parameters such as, linearity, detection limits and reproducibility have been evaluated in this report to assess the applicability, sensitivity and efficacy of MALDI-MS2 quantification method. Quantification of Des degradation upon UV radiation (250nm) at different time points is studied using MALDI-MS2 method.

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Data Integrity for Analytical Instrument Benchtops Leticia Quinones, Bristol-Myers Squibb, One Squibb Dr., New Brunswick, NJ 08903, George Bouziotis, Robert Falana, Robert Reba Data integrity in pharmaceutical testing release laboratories provides unique challenges to the scientists generating and managing the data. Scientists use a variety of bench-top computer-controlled instruments with different software and file structures and must ensure that their installation and up- keep sustains integrity of the data through the instrument’s life cycle. In many cases the software was not design-compliant with today’s expectations on United States Food & Drug Administration (FDA) Part 11/EMEA Annex 11 and vendors do not always have the expertise to support their customers on data integrity. Scientists need to partner with their IT and quality assurance to devise installation and maintenance practices that minimize the risk brought by electronic record security gaps of their systems. These practices need to be as clear and simple as possible to facilitate onboarding of new technology and compliance through the lifecycle of the instrument. This poster provides the lessons learned by an analytical laboratory on its journey to Part 11/data integrity compliance of bench top instruments addressing vendor collaboration, data owner responsibility and solutions for compliant data management.

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Mass Spec Profiling and Antimicrobial Efficacy of Asparagus Officinalis, Bombax Malabarica and Moringa Oleifera Plant Extracts Lindsey Bodnar, NJCSTM, Kean University, 1075 Morris Ave., Union, NJ 07083, John Mikhail, Yassel Hernandez, Rachana Bhatt, Anima Ghosal, Dil Ramanathan As more and more strains of bacteria are becoming resistant to available antibiotics, drug choices are starting to become limited and more expensive. The current situation demonstrates the increased need for the development of new antibiotic therapies. This study is intended to identify chemical compounds from three medicinally important plants and analyze their antimicrobial potential. Asparagus officinalis, Bombax malabarica, and Moringa oleifera are the three plants of the study’s focus. All three plants are known to have anticancer, antioxidant and antimicrobial effects. The extracts of these plants were obtained by using direct and serial extractions techniques with solvents of different polarity. The chemical constituents obtained after extractions were further analyzed using Perkin-Elmer GC Clarus 680 and MS clarus SQ 8T. Profiling was further done using the National Institute of Standards and Technology (NIST) library and the United States National Library of Medicines PubChem database for establishing the medicinal properties of the constituents from extracts of all three plants. Further antibacterial efficacy of these extracts was checked using agar well diffusion technique against E. coli and S. aureus. By using the agar technique, B. malabarica showed slight inhibition towards solvent types hexane and ethyl acetate on S. aureus. Further testing will be conducted to confirm these results.

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Finding the Solution to Analytical Sample Preparation: An Automation Story Alexandra Andrews, Merck & Co., MS: RY80T-B164, 126 E. Lincoln Ave., Rahway, NJ 07065, Margaret Roeder, Edward Mularz, Jameson Bothe, Elizabeth Pierson Automation in the early development space is not very common for drug product finished goods due to challenges such as lower sample testing volumes or limited sample availability. However, analytical teams within Merck Pharmaceutical Sciences are embracing automation solutions as the first route for new projects. Some of the first projects challenged with this new mindset were two programs with limited samples due to difficult active pharmaceutical ingredient (API) chemistry. Ultimately, the project teams realized that utilizing the Tablet Processing Workstation (TPW) to perform sample preparation could significantly save time and solvent while also providing more accurate and reliable results. The manual preparation was performed in large volumetric glassware with one or two dilution steps depending on concentration. When moving to the automated method, the dilutions were performed gravimetrically in a test tube resulting in a drastic decrease in diluent consumption. Also, when considering a project transferring to late stage development and manufacturing, utilization of the TPW would be critical to preparing large amounts of batch samples quickly with minimal solvent usage. Overall, development of an automated method has led to improvements in assay reproducibility (or spread between replicates), decreased sample preparation time, and significantly reduced solvent consumption.

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Analytical Validation, Separation and Stability Study of Multi Compound Formulations – Challenges and Approach Shiladitya Sen, Charles River Laboratories, 1407 George Rd., Ashland, OH 44805 Multi-component formulations are useful as introducing multiple test article complement the effects and side-effects of one another and recently has shown to be an effective strategy1, but often presents analytical and formulation challenges. Optimization, validation and stability assessment of a two component unique formulation was carried out in a vehicle ensuring the formulations to be solutions for an IV route of dosing. A single analytical method was developed to quantitate two different test articles without any interference from the vehicle components. The test article G1 and G2 were formulated in a formulation containing 17mM NaHC03+ 0.3% HPMC (Hydroxypropyl Methylcellulose) in DI Water, pH 10±0.2. The most significant challenge was to optimize the order in which these formulation components were added

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Mapping Polyethylene Reactor and Product Space Using Multivariable Analysis of Digital Distributions. Paul J. DesLauriers, Chevron Phillips Chemical Company, Bartlesville Research & Technology Center, Hwys 60 & 123, Bartlesville, OK 74003, Jeff S. Fodor One approach that aids in efficient polyolefin catalyst and product design is to apply mathematical models linking polymerization kinetics with a given catalyst to the microstructure of the polymer they produce, and polymer microstructure to polymer physical properties (mechanical, rheological, and optical). The goal of such models is to provide a simulation tool that reasonably predicts polyolefin properties from experimental or digitally generated polymer microstructural data, and accounts for influential factors in an integrated way. That is, given a set of catalyst and reactor variables, as well as the resulting resin properties, correlations (forward models) between the two can be established using various chemometric methods. The catalyst and reaction conditions needed to produce polyethylene resins with specific properties can also be determined using a reverse model. During the development of these models, a correlated output from an analytical method to a measured property is very often not enough to understand the physical underpinnings of the developed correlation. To achieve this, digital data sets that are tied to physical realities need to be used to probe the developed correlation and “model the predictive model.” Furthermore, if the digital data is in the form of a fixed distribution, several additional benefits are found. In this presentation, we illustrate the use of digital data coupled with chemometric methods to better understand and further develop modeled reactor space as well as resin properties.

Analysis of the Moisture Content in Lyophilized Proteins by NearInfrared Spectroscopy Ewa Kowalczyk, Seton Hall University, 400 South Orange Ave, South Orange, NJ 07206, Mary Krause, Ming Huang, Robert Wethman, John Wasylyk, Bristol-Myers Squibb In the biopharmaceutical industry, protein therapeutics can be lyophilized in order to generate a stable bioactive product which can be easily stored and transported to the end user. Typically, the protein is prepared as a buffered solution which may contain one or more additives such as amino acids, carbohydrates and/or polymers, which are designed to maintain the integrity of the protein throughout the drying, reconstitution and administering processes. The typical procedure for determining the moisture level is by Karl Fischer (KF) analysis. Preparation for KF analysis involves opening the lyophilized vial in either a containment unit such as a purged glovebox or on a benchtop, exposing the dried material to the atmosphere, grinding of the material, weighing a specific amount, and finally analyzing by either a coulometric or volumetric KF method. Analysis time varies based on the techniques and may take up to 45 minutes per sample. In order to enhance productivity and limit sample handling, we utilized Fourier transform near-infrared (FT-NIR) spectroscopy as an alternative approach to KF analysis for moisture determination. Lyophilized samples were collected, primarily during secondary drying, and analyzed by KF and FT-NIR. A general chemometric model was developed, based on the combination and overtone of vibrational water bands from collected samples encompassing three different proteins in two different aqueous platforms. The FT-NIR technique is non-invasive and the analysis time is under two minutes. We present statistical data on the general chemometric model as well as models generated from the individual lyophilized proteins used in this study.

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Stacking the Deck in Calibration: Better Models and Better Transfers with Stacked Calibration Methods Steven D. Brown, University of Delaware, Dept. of Chemistry and Biochemistry, 163 The Green, Newark, DE 19716, Dominic Poerio Multivariate calibration is routine in spectroscopic applications, but problems with dealing with application of a calibration model to data from a secondary instrument remain, as does the need to deal with uncalibrated effects in spectral data used for prediction. Both the transfer of a calibration and the compensation of uncalibrated interferents often require correction of spectral responses for localized effects, either in wavelength or in frequency. Stacking, a way to build and combine local models on data, offers an attractive way to compensate for localized effects produced from an interferent or from a transfer of calibration. This presentation demonstrates stacking of orthogonalized, wavelet-transformed responses for transfer of calibration. We show that a calibration model corrected for use on a secondary instrument can also be used to predict data from other, similar, but unseen instruments as well as if the calibration was performed on these. We also briefly report the use of stacked, sparse PLS modeling for calibration, where only part of a spectrum need be used to calibrate effectively, as a first step toward isolation and elimination of some spectral interferences.

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Applying USP 921 to your Moisture Determination Lab and Karl Fischer Titrations Bruce Herzig, MilliporeSigma, 2909 Highland Ave., Norwood, OH 45212 Many Pharmacopeia materials contain water as hydrates or in adsorbed form. Therefore the determination of the water content is important to maintaining compliance with the United States Pharmacopeia guidelines. Generally one of the three methods are given in the monographs, Method I (Titrimetric), Method II (Azeotropic), or Method III (Gravimetric). This poster discusses the application of these methods to moisture determinations focusing on Karl Fischer techniques.

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Development and Implementation of Novel Automated MicroTitration Instrument for Physical Characterization in Early Stage of Pharmaceutical Development Steve Wang, Merck & Co., 126 E. Lincoln Ave., Rahway, NJ 07065 Over the years, miniaturization of solubility measurements have afforded the generation of significant amounts of data at very small scales. However, pH solubility titrations for compounds exhibiting complex phase behavior remains a challenge to both titrate and characterize the excess solids that would be representative of bulk material. Robust, standard manual titrations can require a prohibitive amount of active pharmaceutical ingredients (API), where definitive experiments must be deferred by several weeks. Furthermore, these experiments are fairly labor intensive as they require much time for equilibration. Recently, a microliter titrant delivery system from Mettler Toledo was introduced to the market. However, the available, large titration vessels precluded use at the required miniaturized scale. To address this gap, three-dimensional (3D) printing technology was leveraged to design and construct a compatible Merck customized small volume titration vessel. Adaption of the 3D printed titration vessels enables the successful development of automatic microtitration methodology to support physical characterization work in early stage of pharmaceutical development. In this study, we demonstrated the feasibility of this automatic microtitrator to support early phase development. pH dependent solubility was measured in 3 mL selected vehicle (a 6 times reduction of API and vehicle usage). The automated small volume titration results were consistent with manual and large volume titration. Good method precision was also demonstrated for automated small volume titration (RSD < 1% for 5 replicates). Moreover, application of this novel miniaturized automatic titration instrumentation enabled quick support for buffer selection with significant improvement of operation efficiency and minimal API consumption.

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Investigation of Meteor and Meteor Impact Samples by Raman Spectroscopy and Multivariate Curve Resolution Karl Booksh, University of Delaware, Dept. of Chemistry, 163 The Green, Newark, DE 19716 No abstract submitted by the author.

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The 48 Siblings of Ibuprofen Jens Boertz, LGC Standards, Mercatorstr. 51, Wesel 46485, Germany, Omar Mneimne During the processing, manufacturing and storage of Ibuprofen and many of its various drug products, numerous impurities and degradation products are created and must be tested and identified. The development, validation and implementation of analytical methods to correctly identify and quantify these compounds are key and required to fulfill Guidelines Q3A and Q3B of the International Council on Harmonization (ICH). This poster demonstrates how the use of proper analytical reference standards can significantly facilitate the work required to identify and quantify the 48 siblings of Ibuprofen.

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Multivariate Curve Resolution, Genetic Algorithms, and Cross Correlation Library Searching Applied to the Forensic Examination of Automotive Paints Barry K. Lavine, Oklahoma State University, Department of Chemistry, 107 Physical Science, Stillwater, OK 74078, Undugodage Perera, Matthew D. Allen, Francis Kwofie, Collin White In the forensic examination of automotive paint, each layer of paint is hand sectioned and then analyzed by a Fourier transform infrared spectroscopy (FTIR) spectrometer, which is time consuming. Sampling too close to the boundary between adjacent layers is also a problem as it produces an IR spectrum that is a mixture of two layers. Not having a “pure” spectrum for each layer prevents a scientist, who is searching an automotive paint database, from developing an accurate hit list of potential suspects. We have addressed these problems through collecting concatenated IR data from all paint layers in a single analysis by scanning across the cross-sectioned layers of the paint sample using an FTIR imaging microscope fitted with an attenuated total reflection accessory and equipped with an array detector. Decatenation of concatenated IR data is achieved using multivariate curve resolution techniques to obtain a “pure” IR spectrum of each paint layer. This approach, not only eliminates the need to analyze each layer separately, but also ensures that the final spectrum of each layer is “pure” and not a mixture. By integrating the imaging experiment including the use of multivariate curve resolution techniques to decatenate spectral data with a prototype pattern recognition IR library searching system that consists of search prefilters to reduce the size of the library and a cross correlation library searching algorithm to assess the quality of an IR spectral match, we show that the forensic examination of automotive paints is facilitated using this approach to paint analysis.

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of clandestine methamphetamine laboratories as well as the challenges and evolution of the chemists’ approach to processing these laboratories.

High-Throughput Analysis in Support of HTE in Process Development Wes Schafer, Merck & Co., MS: RY818-B224, 126 E. Lincoln Ave., Rahway, NJ 07065 High-throughput experimentation (HTE) is an important tool in process development. Our lab has focused on the unique analytical challenges in supporting HTE from method development to high-throughput data acquisition and finally high-throughput data processing and knowledge management. Standardized method development platforms and state-of-the-art high-throughput analysis (HTA) techniques are discussed with an emphasis on chromatographic techniques and stereochemical analyses. We also look forward to promising new analytical techniques and automation to further increase the efficiency of HTA in support of process development.

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Investigating One Pot Methamphetamine Clandestine Laboratories Jarrad Wagner, Oklahoma State University CHS, 1111 W 17th St., Tulsa, OK 74107 One Pot Methamphetamine Clandestine Laboratories are a prevalent form of illicit drug manufacture, but they are very hazardous. The specific mechanisms of the One Pot are presented, along with safety issues associated with this modified Birch production. Hazard mitigation, field screening and characterization of these clan labs are discussed, including current and future research efforts.

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Drug and Explosive Overlap in Clandestine Laboratories Jarrad Wagner, Oklahoma State University CHS, 1111 W 17th St., Tulsa, OK 74107 Clandestine laboratories (clan labs) that produce illicit drugs are commonly encountered, and recently there have been an increase in clan labs where improvised explosives were produced. Energetic materials are hazardous and unpredictable, so recognition is a critical aspect of scene response. Common drug clan lab characteristics are presented and contrasted with explosives labs. Limited material characterization can take place with field screening tools, and several key techniques are discussed, including flame tests, spectroscopy (IR and Raman), and color tests. Factors that can affect field screening test response as well as attempts to mitigate through neutralization are discussed.

Reactivity-Based High-Throughput Analysis of Heavy Metals 188 Kazunori Koide, University of Pittsburgh, 219 Parkman Ave., Pittsburgh, PA 15260 Trace metal analysis in the pharmaceutical industry requires rapid turnaround so that analysis will not slow down pharmaceutical production. Currently used techniques, such as inductively-coupled plasma mass spectrometry (ICP-MS), have proven to be accurate but do not always meet the turnaround criteria. My laboratory has been developing reactivity-based high-throughput analysis of heavy metals. In collaboration with Merck Research Laboratories, we have developed and implemented a colorimetric method to quantify palladium. This method is based on palladium catalysis that cleaves an allylic carbon-oxygen bond to unmask resorufin. Catalysis-based assays can suffer from signal saturation over time, which we overcame by developing a stop-and-go technique to initiate and stop the assays. The palladium detection method exploits a Tsuji-Trost reaction. Typically, chemosensors for specific metals are designed and synthesized on the basis of hypotheses that are derived from the inherent reactivity of metals. We wished to develop a different approach that does not rely on such known reactivity. Rather, combinations of metals, ligands, and fluorescence-masking groups were screened, leading to a novel copper detection method. We briefly describe this unpublished and highly selective and robust method.

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Laboratory Analysis of Clandestine Labs Noel Vadell, United States Drug Enforcement Administration, 99 10th Ave., Ste. 721, New York, NY 10011 To understanding synthetic routes of illicit methamphetamine evidence from clandestine laboratories, an analysis would include methods of separation of naturally occurring or synthetic by-products in a drug, often by chromatographic methods, to form a ‘fingerprint’ that may be characteristic of its origin or manufacturing route. The majority of the methamphetamine clandestine laboratories in the United States synthesize it by one of two different routes being the Hydroiodic Acid/Red Phosphorus reduction method and the Lithium-Ammonia reduction method. This presentation discusses the reaction mechanisms of each method, explain by-products formation, and apply the findings in a chemical analysis.

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Asymmetric Reaction Screening with Chiroptical Sensors Christian Wolf, Georgetown University, Chemistry Department, 37th and O Streets, Washington, DC 20057 Chirality plays an essential role in nature and in the chemical sciences. The high demand for high-throughput screening of chiral compounds in the pharmaceutical arena has established a strong impetus for developing molecular probes and sensing assays that are compatible with automated experimentation equipment. Chiroptical chemosensors hold unique promise to address the need for fast stereochemical analysis of small analyte amounts. The design and applications of a variety of chirality sensors that can be used for time-efficient determination of the absolute configuration, enantiomeric excess and amount of chiral compounds are discussed. Particular emphasis is given on crude reaction analysis of the asymmetric Sharpless dihydroxylation and enantioselective ketone and imine reductions.

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Unequivocal Determination of Complex Molecular Structures with Anisotropic NMR Measurements Yizhou Liu, Merck & Co., MS: RY800-C163, 126 E. Lincoln Ave., Rahway, NJ 07065, Josep Saurí, Emily Mevers, Mark W. Peczuh, Henk Hiemstra, Jon Clardy, Gary E. Martin, R. Thomas Williamson Assignment of complex molecular structures from nuclear magnetic resonance (NMR) data can be prone to interpretational mistakes. Anisotropic NMR data, such as residual dipolar couplings (RDC) and residual chemical shift anisotropy (RCSA), provide a spatial view of the relative orientations between bonds (RDCs) and chemical shielding tensors (RCSAs), regardless of the distance separation between these bonds and atoms. Hence, these data are sensitive reporters of global structural validity. Anisotropic data can be used directly to evaluate investigator-proposed structures, as well as candidates generated by a computer-assisted structure elucidation (CASE) program for simultaneous structural proposal and validation. Structural evaluation is based on the agreement between experimentally measured data and theoretical values calculated for the corresponding three-dimensional densityfunctional-theory (DFT) models. The combination of CASE, DFT, and anisotropic NMR data represents an orthogonal approach to conventional NMR data interpretation that is not subject to the interpretational biases of human investigators and, as such, mitigates the risk of incorrect structure assignments.

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Enabling Modern Catalysis in Drug Discovery and Development with High-Throughput Experimentation Chemistry and (Ultra!) Fast Analysis Spencer Dreher, Merck & Co., 2000 Galloping Hill Rd., Kenilworth, NJ 07033 Drug discovery and development are increasingly utilizing high-throughput experimentation chemistry tools to increase the rate and quality of chemistry in pharmaceutical research. Parallel arrays of chemistry conditions can greatly improve the chances of making a new molecules within tight business timelines and parallel substrate combinations (parallel synthesis) allows chemists to make more compounds per unit time in search of optimal structures. In the best chemistry applications, both conditions and substrates are scanned simultaneously to produce the biggest library of compounds with the most compounds successfully prepared. These approaches have evolved to the point that a single chemist can run thousands of miniaturized experiments in a single day, creating a new need for analysis techniques that can keep up with chemistry demand. Furthermore, pro-active chemistry evaluation to create data that can be used to create predictive models promises to further increase our ability to make new molecules, but also further increases our needs for fast analytics. To this end, in the best case, chemistry samples can be analyzed in as little as 0.3 seconds per sample using fast mass-spec techniques.

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Towards Unbiased and more Efficient NMR Based Structure Elucidation: A Powerful Combination of CASE Algorithms and DFT Calculations Alexei V. Buevich, Merck & Co., MS: K15-LL-0450, 2015 Galloping Hill Rd., Kenilworth, NJ 07033, Mikhail E. Elyashberg Computer-assisted structure elucidation (CASE) expert systems were developed for structure elucidation of new organic compounds and natural products. These systems are capable of generating a set of all possible structures which satisfy the molecular formula and basic two-dimensional nuclear magnetic resonance (2D NMR) data (correlation spectroscopy, heteronuclear single quantum coherence, heteronuclear multiple bond coherence). Then the selection of the most probable structure is done from the analysis of carbon chemical shifts predicted with empirical methods based on hierarchical organization of spherical environments (HOSE) code, neural networks and additive rules. However, in some cases, these methods are unable to distinguish the correct structure when average deviations of chemical shifts are too large or when top-ranked structures have acceptable but very similar deviations. Herein, we show that the combination of CASE and density functional theory (DFT)

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Clandestine Labs: A Walk Though Time Edward J. Kovacs III, US Drug Enforcement Administration, 99 Tenth Ave., New York, NY 10011, James DiSarno Clandestine laboratories have a long, storied history dating back to early explosives and alcohol. With regard to controlled substances and the synthetic routes of methamphetamine in particular, the clandestine laboratories have evolved with time and challenges from the legal system. This presentation covers the history and evolution

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of NMR data predictions allows determination of correct structures even in these challenging situations. We believe that this synergistic approach can serve as an unbiased, reliable, and efficient structure elucidation method and can be applied to those difficult situations when molecular systems are proton deficient, chiral or conformationally flexible. The proposed method has been tested on three challenging structure elucidation problems of aquatolide, coniothyrione and epoxyroussoenone.

od. While conventional NMR methods that rely on scalar (J) couplings and through space (nOe/rOe) interactions are essentially local in nature, anisotropic NMR data are indifferent to separation. Hence, anisotropic NMR data provide an orthogonal and unequivocal global assessment of the correctness of given structure and its configuration that is free from any investigator bias.

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Broadband 19F TOCSY with Spin Lock Effected by BURBOP Pulses Alexander A. Marchione, Chemours, Experimental Station, 200 Powder Mill Rd., Wilmington, DE 19803 For the first time, a spin lock sufficient for liquid-phase total correlation spectroscopy (TOCSY) correlations has been effected across a spectral window appropriate for typical fluorocarbon analyses by 19F nuclear magnetic resonance ( NMR) (ca. 50 kHz). The spin lock is based on a series of broadband universal rotation by optimized pulses (BURBOP) broadband inversion pulses; in contrast to adiabatic pulses such as CHIRP, the BURBOP retains phase uniformity across the spectral window. The moderate power requirements of the BURBOP, gammaB1 = 15 kHz over a spin lock duration of 80-128 ms, are tolerated by standard liquids probes with samples of relatively low dielectric loss. A selective one-dimensional variant is also successfully applied. This discovery renders the TOCSY experiment, long useful in 1H NMR spectroscopy of complex mixtures, applicable to similar problems in 19F NMR.

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NMR Characterization of Complex Natural Products: Opportunities and Challenges in Structure Elucidation Kirk R. Gustafson, National Cancer Institute, NCI-Frederick, Bldg. 562, Rm. 201, Frederick, MD 21702, Kentaro Takada, Naoya Oku, Yizhou Liu, Josep Saurí, R. Thomas Williamson, Gary Martin Nuclear magnetic resonance (NMR) provides powerful structural elucidation tools that are particularly well suited for natural products studies. Comprehensive spectroscopic characterization of a native metabolite may be sufficient to fully assign a planar structure. However assignment of the relative and absolute configuration of a molecule when there are multiple stereogenic centers often requires the development and application of additional experimental strategies. Many successful approaches in this regard rely on the formation of appropriate derivatives for more detailed NMR study. Since natural products are often obtained in very limited quantities, micro-scale chemical manipulations and the ability to analyze the structure of the resulting products is often key to the complete structural and configurational assignment of complex metabolites. Anisotropic NMR parameters such as residual dipolar coupling (RDC) and residual chemical shift anisotropy (RCSA) provide a powerful and complementary means to help assign and verify structures deduced from conventional NMR analyses. Application of a wide spectrum of NMR techniques and methodologies will be described in the structural elucidation of a new phosphomacrolide marine natural product with 8 stereogenic centers.

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Recent Advances in Raman Microscopy for Pharmaceutical and Life Science Applications Alexander Rzhevskii, Thermo Fisher Scientific, 2 Radcliff Rd., Tewksbury, MA 01876 Raman microscopy is now well established as one of the most powerful instrumental techniques for a diverse range of applications in both research and analytical laboratories. In this presentation, I consider the recent technological advancements in Raman microscopy and spectral imaging that brings additional analytical possibilities in analyzing two and three-dimensional spatial distributions of materials on a sub-micrometer scale with an information-reach content while maintaining a reasonable image acquisition time. I demonstrate that Raman imaging microscopy is a valuable technique for hyperchemical characterization of pharmaceutical and biological materials. The essential advantages of Raman microscopy and spectral imaging are being illustrated with the examples of end-product content uniformity, stability and drug-excipients compatibility testing in the pharmaceutical industry, biological and life-science applications.

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Discovery of Unique Hydrogen Bonding Motif in Nucleosides Leads to a Novel Protecting-Group Free Selective 3’ Nucleoside Functionalization Chemistry Mikhail Reibarkh, Merck & Co., MS: RY800-D231, 126 E. Lincoln Ave, Rahway, NJ 07065 Direct chemoselective 3’-functionalization of nucleosides has not been described in a literature. Because 3’-OH is less reactive than 5’-OH, the only means of achieving selective 3’-functionalization synthetically was a tedious protection-deprotection approach. In the process of developing a synthetic route for therapeutic nucleoside analogues we discovered that the presence of a strong base leads to increased selectivity for 3’-phosphorylamidation. We explored the mechanism of this rare selectivity through a combination of nuclear magnetic resonance (NMR) spectroscopy and computational studies. The NMR and computational findings demonstrated that a unique hydrogen bond between 5’-OH and a nucleic base is responsible for observed 3’-selectivity. Based on these findings, we developed a predictive computational model that accurately assesses the potential for 3’-functionalization for a broad range of nucleosides and nucleoside mimetics. The synthetic utility of this model was demonstrated experimentally on a broad scope of nucleosides and electrophiles. Potential implications of the discovered hydrogen bonding motif in nucleosides are discussed.

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Applications of Nanoscale IR Spectroscopy and Imaging in Pharmaceutical Science Curtis Marcott, Light Light Solutions, PO Box 81486, Athens, GA 30608, Eoghan Dillon, Kevin Kjoller, Craig Prater Many pharmaceutical ingredients have sub-micron particle sizes. The chemical nature of the interactions between active pharmaceutical ingredients (API) and excipient components plays an important role in both the formulation and end use performance of pharmaceutical products. Ingredient distributions, molecular level interactions, as well as tablet coating materials all affect the timing and effectiveness of the release of the API into the patient. Better nanoscale chemical characterization techniques should enable the development of improved formulations and provide a better understanding of the finished product composition. Nanoscale infrared spectroscopy has been successfully demonstrated in an expanding range of applications in recent years due to significant increases in capability. One method of nanoscale infrared spectroscopy, atomic force microscope based infrared spectroscopy (AFMIR) directly detects IR radiation absorbed by the sample using the AFM probe tip to sense thermal expansion. This thermal expansion depends primarily on the absorption coefficient of the sample and is largely independent of other optical properties of the AFM tip and the sample. The use of a quantum cascade laser (QCL) as the excitation source has dramatically improved the sensitivity of AFM-IR and enabled spectra and absorbance images to be collected much more rapidly. Example applications of AFM-IR and other new nanoscale spectroscopy and chemical imaging capabilities in pharmaceuticals are presented.

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Applications of New Pulse Sequences and Unequivocal Determination of Complex Molecular Structures Using Anisotropic NMR Measurements Gary E. Martin, Merck & Co., MS: RY-800 D-133, 126 E. Lincoln Ave., Rahway, NJ 07065, Yizhou Liu, Josep Sauri, Alexei V. Buevich, Mikhail Reibarkh, R. Thomas Williamson The inherent limitation of heteronuclear multiple bond coherence (HMBC) to primarily 2JCH and 3JCH with only occasional 4JCH correlations has been supplemented by the long range heteronuclear single quantum multiple bond correlation (LR-HSQMBC) experiment that routinely provides 4JCH to 6JCH correlations. Ambiguities in the “length” of correlation pathways (2JCH vs. 3JCH) in HMBC spectra was addressed by the development of a series of ADEQUATE experiments that exploit 1JCC coupling pathways at natural abundance. The low sensitivity of the ADEQUATE experiments benefits greatly from the incorporation of pure shift in conjunction with non-uniform sampling (NUS) data acquisition. The utilization of anisotropic nuclear magnetic resonance (NMR) data, first residual dipolar couplings (RDC) and, beginning in mid-2016, residual chemical shift anisotropy (RCSA) has also been exploited as a powerful compliment to heteronuclear shift correlation methods to assign complex structures and configuration. As an example, the elucidation of the structure of the natural product homodimericin-A stymied conventional attempts to elucidate its structure even when computer-assisted structure elucidation (CASE) methods were applied with the data. It was only with the utilization of advanced NMR methods that the unprecedented hexacyclic 20 carbon skeleton of the molecule, which incorporates 14 non-protonated carbons, could be elucidated. In parallel, the utilization anisotropic NMR data was explored as an adjunctive meth-

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Confocal Raman Microscopy Characterization of Waterborne Coatings Dana Garcia, Arkema Inc, 900 First Ave., King of Prussia, PA 19406, Wenjun Wu Confocal Raman microscopy (CRM) integrates the high molecular specificity associated with Raman spectroscopy with the opportunity of spatial resolution characteristic of microscopy. X,Y and Z (depth) mapping can yield, based on the Raman spectra, a compositional or structural image for multi-component systems. An interesting application for CRM is the study of coating films comprising polymer, pigment and additives. CRM was effectively used to evaluate species distribution and surfactant enrichment at the surface of the coating films. Coating film Raman depth profiles in the first 25 - 50 microns showed that blends of acrylic latex and model water-soluble species are compositionally uniform. Additionally the Raman exper-

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iments provided corroborative spectral evidence for surfactant surface enrichment in acrylic latex coatings.

repulsive interactions between neighboring amines that affect the integrity of the polymer coating, and the availability of amine groups to interact with the membrane.

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Mapping the Surface Concentration of Coatings on Metal and Glass Surfaces Using FTIR Reflectance Spectroscopy Mary Thomson, Remspec Corp., 512 Leadmine Rd., Sturbridge, MA 01566, Peter Melling, Robert Kertayasa A reliable, convenient method for checking the thickness and uniformity of lubricant coatings on various surfaces, including metals and glass, is a long-standing concern in several industries. Performing the measurement non-destructively, and preferably without any surface contact, is also extremely important when coating integrity is a concern. We are reporting an approach that uses an out-of-compartment grazing-angle Fourier transform infrared spectroscopy (FTIR) method to enable non-contact mapping of the thickness and distribution of coatings such as lubricants across the surface of items such as hard disks. Factors to be discussed include: sensitivity and precision of the method, reproducibility, automation, measures to minimize the spectroscopic influence of interfering factors such as atmospheric water vapor, and the challenges of interpreting and calibrating spectroscopic results from dielectric surfaces such as glass. This work builds on extensive experience in the use of grazing-angle FTIR for in-situ cleaning validation and verification.

Sustainable Magnetically Retrievable Nanoadsorbents for Selective Removal of Pb2+ and Pd2+ Ions from Different Charged Wastewaters Sriparna Dutta, University of Delhi, 105, Block B, Department of Chemistry, New Delhi 110007, India A reliable access to clean and adequate water represents one of the biggest global challenges of the present century. In a quest to explore sustainable solution for combating problems related to water contamination caused by the discharge of toxic heavy metals, we have effectively integrated nanotechnology with superior adsorption technology and developed magnetically retrievable nanoadsorbents for the cyclic recovery of heavy metals such as Pb2+ and Pd2+ from different charged wastewater. This is a‘double green’ approach, as it aims at reduction of hazardous waste with simultaneous reduction in the usage of virgin resources. These nanoadsorbents have been synthesized using a facile covalent immobilization approach wherein metal specific chelating ligands like acetoacetanilide and 2,6-diacetylpyridine have been grafted onto the surface of amine functionalized silica encapsulated magnetite nanoparticles. A wide range of sophisticated analytical techniques such as X-ray diffraction, transmission electron microscopy, field emission scanning electron microscopy, Fourier transform infrared spectroscopy, energy dispersive spectrometer, energy dispersive X-ray fluorescence and vibrating sample magnetometer have been utilized for gaining an insight into the morphology and structure of the synthesized nanoadsorbents. Crucial experimental parameters such as the amount of nanoadsorbent, pH, adsorption time, type of eluting agent and its volume on adsorption and desorption have been investigated in detail. Notably, these nano-adsorbents exhibited higher adsorption capacity, took lesser time for adsorption-desorption cycle, required mild elution conditions, showed ease of separation and displayed enhanced reusability.

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Probing Nanoscale Hydrophobicity and Chemical Distribution of Surface Modified Polyethersulfone (PES) Membranes Wanyi Fu, New Jersey Institute of Technology, John A. Reif, Jr. Dept. of Civil and Environmental Engineering, Newark, NJ 07102, Wen Zhang Chemical modifications bring unique properties into polymeric membranes that may have enhanced filtration or separation efficiencies, antifouling, antimicrobial activity and selectivity. However, there is a lack of nanoscale characterization of chemical additive distribution and impacts of chemical modifiers or additives on membrane surface properties, especially those at nanoscale. In this study, a series of industrially relevant polyethersulfone (PES) membranes modified with poly (ethylene glycol) (PEG) and polyvinylpyrrolidone (PVP) were analysed systematically. Particularly, hydrophobicity and chemical distribution were scrutinized by atomic force microscopy (AFM) and AFM coupled with infrared analysis capability (AFM-IR) for the first time that successfully resolved nanoscale structural and chemical properties of the chemically modified PES membranes. Our results indicated the heterogeneous spatial distribution of PVP and PEG based on their characteristics IR bands and the resulting hydrophobicity distribution on modified membrane surfaces at nanoscale. Particularly, we established a linear correlation (R2=0.994) between the measured adhesion force and water contact angles, which enabled the examination of local surface hydrophobicity. The PES membranes became more hydrophilic with the increasing blend of PVP and PEG. With AFM-IR, trace amounts (1-4%) of PVP could be identified sensitively on PES membranes based on their unique characteristic IR bands, which were not achieved by Fourier transform infrared or IR mapping. Overall, these novel characterization approaches hold paramount importance for the design and quality control of polymer membrane modification and manufacturing.

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Fundamentals and Applications of Matrix-Assisted Ionization: Zero Energy Input Ionization Charles N. McEwen, University of the Sciences, Chemistry Dept., 600 South 43rd St., Philadelphia, PA 19104, Khoa Hoang, Milan Pophristic Common to all methods of converting nonvolatile/ thermally unstable compounds to gas-phase intact ions efficiently is a matrix, either liquid as in electrospray ionization (ESI) or solid as typically used in matrix-assisted laser desorption/ionization (MALDI). Initially, attempts to extend mass spectrometry (MS) to large nonvolatile compounds mostly applied high energy input such as particle bombardment, high heat, or radiation with lasers. ESI and MALDI are the most successful ionization methods for mass spectrometry to use a matrix, where ESI applies a high voltage to a solution and MALDI uses laser ablation of a solid matrix. A newly discovered matrix-assisted ionization (MAI) method transports nonvolatile compounds into the gas-phase from a solid matrix with high sensitivity and without external energy input. Our experiments and literature searches asks; 1) what is the charge separation process, 2) what is the mechanism for transport of analyte from the solid to the gasphase, and 3) are the ions transported directly or through matrix clusters/particles. While sublimation seems to be a firm requirement for MAI, how analyte molecules, regardless of volatility, move from the solid to gas phase ions is still a mystery. Both a charge separation process and a process to transfer even proteins into the gas phase are required. Experiments, as well as theory, suggest that the process occurs through spontaneous particle expulsion. Intriguing results related to analyte incorporation and mixed matrices will be discussed. Additionally, to fundamental aspects of MAI, applications of this simple and highly sensitive ionization process will be discussed, including direct ionization of biological fluids and tissues.

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The Interactions between Amine-Rich Poly [oxo-norbornenes]Coated Gold Nanoparticles and Phospholipid Membranes Zeev Rosenzweig, University of Maryland Baltimore County, Dept. of Chemistry and Biochemistry, Baltimore, MD 22250, Zheng Zheng, David Boschert, Karen Lienkamp, Bo Zhi, Christy L. Haynes The paper describes a quantitative study of the interactions between gold nanoparticles, which are coated with poly [oxo-norbornenes], a recently introduced class of cationic polyelectrolytes, with phospholipid membranes. Our ability to manipulate and control the chemical structure of poly [oxo-norbornenes], specifically, the density of amine-functionalized side chains and the surface coverage of poly [oxo-norbornenes] on gold nanoparticles, enables the determination of the role of amine density and surface coverage of the conjugated polymers on the anti-membranal activity of the polymer-coated gold nanoparticles. The study makes use of a toolkit of analytical methods including ultraviolet-visible (UV-Vis) spectroscopy, dynamic light scattering and zeta potential measurements, transmission electron microscopy, and fluorescence assays to study the interactions between the nanoparticles and phospholipid vesicles (liposomes) quantitatively. The study reveals that the conjugation of poly [oxo-norbornenes] to gold nanoparticles results in increasing anti-membranal activity compared to the anti-membranal activity of free poly [oxo-norbornenes] in solution. This increase in activity is attributed to increased amine charge density on the gold nanoparticles’ surface, which increases their impact when the nanoparticles are in contact with the liposome membrane. Our study also shows that the anti-membranal activity of poly [oxo-norbornenes]-coated gold nanoparticles depends on the molecular structure of the oxo-norbornene monomers which affect the surface charge and polymer coverage on the nanoparticles. Importantly, the anti-membranal activity of poly [oxo-norbornenes]-coated gold nanoparticles increases with increasing amine density but overly packing the nanoparticles with amine groups leads to a significant reduction in their anti-membranal activity. This is attributed to

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Imaging Mass Spectrometry in Drug Development: Visualizing Tissue with a Molecular Lens Reid Groseclose, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA 19406 Discovery and development of safe and efficacious medications is a challenging and complex process. A critical aspect of this endeavor is continuous evaluation and adaptation of new analytical technologies that can serve as vehicles to fill our knowledge gaps in biology and chemistry. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) provides the unique capability to determine the spatial distribution of a drug and its metabolites as well as endogenous compounds in tissue samples without a label. This methodology allows for the correlation of analyte tissue distributions with histology images, thereby integrating chemical structures with tissue morphology. MALDI IMS can generate high spatial resolution images, is highly sensitive and selective, and can be quantitative. This imaging modality offers the potential to further our mechanistic understanding of drug disposition, disease progression and pharmacology (including toxicology) by providing snap shots of temporal and causal changes. In addition to the tissue distribution of drugs and their metabolites, it is clear that a more detailed understanding

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Novel Instrument Solution for Content Uniformity Sample Preparation Ishai Nir, Distek, Inc., 121 North Center Dr., North Brunswick, NJ 08902, Andrew Kielt, Jeff Seely The Uniformity of Dosage Units by Content Uniformity test covered in United States Pharmacopeia chapter is required for most dosage forms. Unlike dissolution, disintegration or physical parameters testing, the actual details of how to perform content uniformity testing are largely unspecified, and thus, unlike the other tests, have not lead a preponderance of dedicated instruments. The solutions that have been offered have attempted to automate the entire test, including sample extraction/prep and analysis. As a result, these solutions have been very complicated and expensive. This talk presents an innovative instrument that addresses only the sample preparatiom portion of this test. Limiting the scope of the functionality of this instrument greatly reduces its complexity and cost. This unit reduces the multiple steps and tools conventionally used for content uniformity sample prep to a single ultra-efficient process, reducing the sample prep time by as much as 90%. The disposable sample tubes used in this design further eliminate errors due to sources such as cross-contamination and incomplete sample incorporation, preventing mistakes which necessitate tedious Stage 2 testing. Examples of suitability and sample preparation times reduction in actual pharmaceutical products are demonstrated. Data showing comparable content uniformity results from this instrument versus existing sample prep methods are also presented.

of biological systems can be gained by also evaluating the changes in endogenous compound (e.g., lipids) distributions as a function of disease and pharmacology. This presentation focuses on our efforts using MALDI IMS and histology in drug development to better understand tissue disposition and gain mechanistic insights into drug correlated toxicities and efficacy. Case studies from early and late stage drug development, where MALDI IMS was employed to investigate the mechanisms of adverse events, provide insights into disposition, and PK/PD relationships are presented.

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Improving Quantitation through a Fundamental Understanding of the MALDI Sample Preparation Process Kevin G. Owens, Drexel University, Dept. of Chemistry, 3141 Chestnut St., Philadelphia, PA 19104 Matrix-assisted laser desorption/ionization (MALDI) is a soft-ionization technique for the analysis of a broad range of analytes. Work on basic mechanisms of the MALDI technique suggests that the matrix plays a number of important roles in the analysis, including desorption of analyte from the surface, ionization, as well as protection of the analyte from interaction with the sample surface. While recognized for its qualitative strengths, quantitative analysis by MALDI is often problematic. Much current quantitative work relies on the addition of an internal standard (often isotopically labeled) to the sample. Even with an internal standard, use of the dry-drop method often results in inhomogeneous samples exhibiting poor shot-to-shot and sample-to-sample reproducibility. We have used electrospray deposition to produce more homogeneous samples; this allows us to investigate subtle properties of the MALDI process. One key contributor to analyte signal strength is the matrix-to-analyte ratio (M/A). As the amount of analyte increases, analyte signal is found to increase up to a point, plateau and then decrease. The shape of the M/A plot is similar for different materials, although the location of the saturation point varies with the mass of the analyte and matrix selected. Analyte signal is further affected by the chemical properties of the matrix and analyte; e.g., for peptides/proteins, the basicity of the analyte and acid/base properties of the matrix. For synthetic polymers, the identity and quantity of cations present affect the analyte signal. This talk shows that understanding the fundamentals of the MALDI sample preparation process can yield better quantitative results.

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Use of Biphasic Dissolution to Improve Bioprediction for an Amorphous Low Solubility Crystallizer Gregory Johnson, Merck & Co., MS: WP78-302, 770 Sumneytown Pike, West Point, PA 19486, Wei Xu Crystallization of low solubility compounds (BCS II & IV) from the amorphous state can present a significant problem for predictive dissolution. Rapid crystallization in-vitro can lead to underestimation of bioavailability in-vivo. Further complicating matters, the pre-existence of crystalline seeds can increase precipitation rates, worsening in-vitro prediction. To alleviate these in-vitro artifacts, an absorptive sink can be introduced to mimic drug uptake from the primary media. The resulting decrease in drug concentration lowers the thermodynamic drive for crystallization in-vitro, and may thereby provide better correlation to in-vivo pharmacokinetic studies. In this study, we examine the precipitation of “Compound A,” a BCS II small molecule, using the Sirius inForm, a small-scale automated dissolution platform. Precipitation of “Compound A” is studied in both the active pharmaceutical ingredient (API) and surface dissolution imaging forms, in the presence and absence of an absorptive nonanol sink. The inclusion of an absorptive sink in the form of a nonanol biphasic system has shown significant reduction in the observed crystallization of “Compound A” within aqueous media. These results suggest a significantly decreased role of precipitation in bioavailability, when compared to aqueous dissolution alone. For dosage forms with significant precipitation, biphasic dissolution could potentially be a more biopredictive approach than traditional dissolution.

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Industrial Applications of the Bruker™ MALDI-TOF Biotyper Suzanne K. Singles, DuPont Corporate Center for Analytical Sciences, DuPont Experimental Station, E228/118; 200 Powder Mill Rd., Wilmington, DE 19803, Barbara S. Larsen The identification of micro-organisms is an important task in industrial applications, from identifying contaminant organisms in a sterile process to the identification of commercially viable organisms to be used in industrial fermentation. Using matrix-assisted laser desorption/ionization-time-of- flight (MALDI-TOF) mass spectrometry, a characteristic “mass fingerprint” or the mass and intensity distribution of the organism’s proteins is produced in a matter of minutes. This “mass fingerprint” can be compared to a reference database for rapid identification of micro-organisms in conjunction with the appropriate software, such as Bruker™ Biotyper. The MALDI Biotyper is complementary to 16S rDNA sequencing which is time-consuming, frequently, taking days to complete a sequence. The United States Food & Drug Adminstration has accepted the instrumentation with the algorithms as a medical device for diagnostic use for certain organisms. While sequencing is still a “gold standard” for absolute identification of organisms, we have applied the MALDI Biotyper to quickly screen large culture collections and rapidly identify microbial contamination in fermentations.

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Development of Solution Based Certified Reference Materials for the Analysis of Pharmaceutical Impurities in Monograph Testing Methods Uma Sreenivasan, Cerilliant Corporation - MilliporeSigma, 811 Paloma Dr., Ste. A, Round Rock, TX 78665, Sarah Aijaz, Zoe Ruan, Maysa Bakir, Nicholas Hauser Impurities can significantly impact the safety of drug products. Accurate detection and control of impurities in drug substances and drug products is an important element of quality-by-design, International Conference on Harmonization and good manufacturing practice requirements. During manufacture of a drug material impurities can arise from many different sources including drug substance residual impurities, degradation, extractables and leachables, as well as impurities present in or derived from excipients. Accurate impurity identification and quantitation requires highly characterized reference materials. Typically, Pharmacopeial Monographs identify multiple impurities for a given drug substance that require each impurity to be analytically verified by a chromatographic method. Multiple sample preparations are usually required, the process can be a time consuming burden and offer significant opportunity for increased uncertainty and error in the analysis. Solution based impurity mixtures as certified reference materials (CRM’s) have been developed to reduce dilution errors and improve accuracy and efficiency in testing for impurities. These CRM’s are compatible with the specified monograph test methods and can include several of the impurities referenced in the monographs. All requirements of CRM manufacturing according to International Organization for Standardization (ISO) Guide 34, including homogeneity and stability testing, are thoroughly investigated to ensure the highest level of confidence and characterization for their use as reference materials. In most cases, direct traceability to an available primary standard from an issuing pharmacopoeia is also achieved. Presented here is the design and supporting stability data demonstrating the viability of solution based impurity mixtures for use in monograph testing.

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Analyzing Multi Component Dissolution Samples Using Chemometrics and In-Situ Fiber Optic UV Spectrophotometry Andrew Kielt, Distek, Inc., 121 North Center Dr., North Brunswick, NJ 08902, Ishai Nir, Jeff Seely Ultraviolet (UV) spectroscopy is widely recognized as a more cost effective and simpler way of performing dissolution analysis than chromatography. However, the limits of UV’s applicability have led to the increasing reliance on high-performance liquid chromatography (HPLC) for dissolution analysis. One such category is multi-active products, where overlapping absorption in the same spectral band previously precluded the use of UV. This talk demonstrates the application of the classical least squares (CLS) chemometric technique to UV spectroscopy to overcome this obstacle. CLS is based on matrix mathematical analysis of large data sets of known and unknown spectra to calculate component concentration in unknown mixtures. As described in this talk, combining data-rich dissolution testing using a fiber optic UV dissolution system with multicomponent analysis accurately quantifies two spectrally-overlapping compounds. Examples from actual pharmaceutical products are illustrated, including comparison to HPLC data simultaneously collected from the same tests. Broader applicability and limits of the technique are then summarized.

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NMR and HPLC Investigation of the E/Z Interconversion of Hydrolysis Impurities Present in Raltegravir Drug Substance David J. Schenk, Merck & Co., MS: RY818, FL 2, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ 07065, Ryan D. Cohen, Robert Hartman, Kristine Cappuccio, Gary E. Martin, Robert A. Reamer Raltegravir is an integrase inhibitor used for the treatment of HIV. Known potential impurities of the synthetic route to the drug substance includes a pair of hydrolysis impurities that can be generated during potassium salt formation. These impurities are currently monitored by a validated reversed-phase high-performance liquid chromatography (HPLC) method. Since the level of impurities may rise above the International Conference on Harmonization (ICH) identification threshold (0.15%) structure elucidation was undertaken. The impurities could only be isolated as a mixture due to facile interconversion, and this behavior exhibited a strong solvent dependency but was not photo-mediated. High resolution mass spectrometry (MS) and tandem MS experiments indicated that the impurities had nearly identical fragmentation patterns and the same exact mass. One- and two-dimensional nuclear magnetic resonance (NMR) spectra (viz., 1H, 13C, correlation spectroscopy, heteronuclear single quantum coherence, and heteronuclear multiple bond coherence) suggested that they are E/Z isomers, and the stereochemistry was determined using nuclear Overhauser effects. Comprehensive density functional theory (DFT) calculations of chemical shifts were then used to confirm the structures. The isomer interconversion was unusual, which prompted us to perform additional kinetic and computational experiments to gain mechanistic insight, as well as to ensure that identification was consistent between NMR and HPLC. In particular, the HPLC diluent (aqueous-acetonitrile) favored the Z-isomer, whereas the E-isomer was more thermodynamically stable in the NMR solvent (dimethylsulphoxide (DMSO)-d6). Therefore, careful coordination of NMR and HPLC analyses was required to correctly assign the HPLC peak identities. Kinetic measurements in DMSO showed that interconversion was first order with a 17.6 kcal/mol activation energy. Once the isomer interconversion was understood, batch analysis was completed demonstrating consistent hydrolysis impurity content.

chemicals through thick packaging material in seconds. For normal measurements, a range of accessories can be used in the confocal configuration for different applications including microscopy and stand-off Raman. This flexibility makes the See_ Through Raman a suitable technique for a variety of applications, from material ID to demanding research.

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Using a New Horizontal Transmission Cell for FT-IR Characterization of Edible Oils Gene S. Hall, Rutgers University, Dept. of Chemistry, 610 Taylor Rd., Piscataway, NJ 08854 Edible oils such as corn, olive, fish oil, algae, vegetable, and soybean oil are valuable consumer products. The quality of these oils is affected by oxidation and rancidity, In addition, these oils contain unsaturated fatty acids that can undergo cis/trans isomerism and affect the health of individuals consuming them. Also of concern is the adulteration of geographical edible oils such as extra virgin olive oil. Fourier transform infrared (FT-IR) spectroscopy is a well-established analytical method used to characterize edible oils. However, sensitivity issues for degradation oil products is a challenge since these compounds are difficult to detect using standard IR sample preparations. Typical sample analysis includes attenuated total reflectance (ATR) and liquid (vertical) transmission cells. The latter is cumbersome and time consuming to analyze the samples. The ATR mode using a nine reflection diamond cell, still does not have the sensitivity to analyze degradation products. We report in this presentation the application of a new horizontal transmission cell Pearl by Specac to analyze the above oils. The advantages of this cell include rapid sample analyses, low detection limits of degradation products, and ease of cleaning. We have used this cell to determine the free-fatty acid content and the amount of trans isomers in edible oils. The seminar focuses on performance comparisons between the Pearl and a nine reflection diamond ATR cell using real samples. Additional information presented will be the determination of hidden spectral characteristics of degradation products that could only be observed using the Pearl horizontal transmission cell.

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Reagent Free Near-Infrared (NIR) Spectroscopic Analysis of Moisture in Lyophilized Products Kyle Hollister, Metrohm USA, 6555 Pelican Creek Circle, Riverview, FL 33578 The quality, efficacy, and shelf-life of lyophilized pharmaceutical products and ingredients depends on water content. Routine Karl Fischer (KF) titration is the standard method for measuring residual moisture content to monitor manufacturing, ensure samples are within specification, and to optimize the freeze-drying process. However, routine application of this technique is not without drawbacks, most notably the use of hazardous chemicals and the destruction of samples. Near-infrared (NIR) spectroscopy is well-suited to measuring moisture, and can do so without sample preparation or chemical consumption. In this talk, we explore how to link NIR spectroscopy to KF results for lyophilized materials and how this method can improve efficiency and tighten product specifications.

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Process Patent Protection via Analysis of Stable Isotope Ratios John P. Jasper, Molecular Isotope Technologies LLC, 8 Old Oak Ln., Niantic, CT 06357, Ann Pearson, Anthony D. Sabatelli Process patent protection via the analysis of natural-abundance stable isotopes is a demonstrated approach to extend the effective life of bio/pharmaceutical patents. The high specificity of isotope ratio analysis compared to other approaches (e.g., concentrations of organic impurities or trace metals) allows isotopic analysis to differentiate processes that were not previously resolvable by less precise analytical methods. Here we summarize the rationale behind and selected case studies of this emerging field. We review: 1) the systematics of stable isotope chemistry, 2) approaches to instrumental analysis of stable isotopes, 3) the biogeochemical origin of stable isotopic fingerprints, 3) equilibrium versus kinetic isotope effects on those fingerprints, (v) categories of application of process patent protection, and 4) case histories of application. The three reviewed cases include one of nutraceutical false advertising, one of mammalian drug product infringement, and one of wrongful accusation of human drug product infringement, which in total protected more than $2 billion United States of bio/pharmaceutical products against patent infringement. Finally, we briefly preview some new applications of stable isotopic analysis in the bio/pharmaceutical field including analysis of biologic drugs, continuous monitoring of drug reaction processes, and isotopically-directed synthesis.

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Molecular Separations and Vibrations of Aged Book Paper Andrew Davis, Library of Congress, 101 Independence Ave. SE, Washington, DC 20540, Amanda Jones, Fenella France We demonstrate how polymeric structures of variously aged and degraded paper materials can be characterized by the complementary use of size exclusion chromatography (SEC) and infrared (IR) spectroscopy. The collection of test books from the William J. Barrow Laboratory, now residing in the Library of Congress, offer a unique and compelling case study. Paper materials in this collection span over 500 years of natural aging, comprising 1000 individual books previously characterized by Barrow and coworkers for a range of material properties including chemical content, pH, and mechanical strength. Developments in instrumentation and experimental procedures have markedly improved the current state of cellulose characterization well beyond methods available to the original Barrow studies. Molecular weight distributions of cellulose were found to be a strong predictor of paper’s macro-scale physical integrity, enabling micro-sampling SEC methods to substitute for large scale destructive mechanical testing (a strict impossibility for general and special collections items). With the goal of obtaining a truly rapid and non-destructive method for assessing the physical condition of book papers, we paired IR spectroscopy with cellulose size and degradation insights obtained from SEC results. We show how results combined this way might be used to identify the rapidity of paper degradation, the hallmarks of artificial aging, and differences in paper sizing and composition.

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See Through Barrier Using Raman Spectroscopy with Large Sampling Volume Jun Zhao, bwtek, 19 Shea Way, Newark, DE 19713, Jack Zhou, Katherine Bakeev, A new Raman measurement method is presented that expands the applicability of Raman to see through diffusely scattering media such as opaque packaging materials, as well as to thermolabile, photolabile, or heterogeneous samples. Traditional Raman fiber optics probes employ a focused design similar to confocal microscopes. This confocal design has the advantage of maximum throughput. However, when the container diffuses the light strongly, light can no longer be focused inside the container, the confocal approach loses its effectiveness. Spatially offset Raman spectroscopy (SORS) intentionally offsets the excitation beam and collection beam, and can be used effectively to collect Raman signal generated underneath diffusive top layers while largely avoiding their signature from overwhelming that of the sample. However, probes of such design cannot be easily used in a confocal arrangement and are generally inefficient for direct sample measurements. The See_Through Raman configuration illuminates and collects the Raman scattered light from a large sample area with enhanced throughput. This greatly increases the effective sampling depth, allowing the measurement of material inside visually opaque containers via a similar mechanism as SORS. The large area also has the additional advantage of preventing sample damage by reducing the power density, as well as improving accuracy by eliminating heterogeneous effect. With its high throughput design, the See_Through analyzer provides identity of common

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Absolute Temperature Measurement by Single Material Based Twocolor Infrared Detectors Yasin Kaya, Princeton University, 351 Lemonick Court, Apt. 102, Princeton, NJ 08540, Arvind Ravikumar, Guopeng Chen, Maria C. Tamargo, Aidong Shen, Claire F. Gmachl Sensing absolute temperature of an unknown object improves classification and recognition accuracy significantly in medicine and defense industries. Measuring the infrared signal in multiple different spectrum windows by using a two-color de-

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tector is one of the most effective methods to extract a precise temperature. The most common way to implement a two-color infrared detector is to use two single band detectors centered at different wavelengths. However, one needs additional optical components; i.e., expensive, bigger and more complex optical systems, to combine and use two single band detectors as a two-color detector. An alternative method is to grow two detectors, focused at different wavelengths, on top of each other by using same material system. Such a material system should be able to cover two different spectral windows in the mid-infrared. In this work, we report two-color quantum well infrared detectors (QWIPs) centered at 4.9 um and 7.6 um based on a wide conduction band offset (~1.2eV) material system. The wide conduction offset enables to cover mid-wavelength infrared (MWIR) and long-wavelength infrared (LWIR) spectra with a single material system. Detectivities of 3x10^8 cm(Hz)^0.5/W and 2x10^7 cm(Hz)^0.5/W are obtained at for the MWIR and LWIR QWIPs, respectively. After the characterization process, the two-color detector is used to extract the absolute temperature of a standardized black body source. The extracted results are in good agreement with the actual temperatures. In summary, a two-color detector based on a single material system has been designed, characterized and successfully used in the absolute temperature detection application.

Earthquake Damage Mitigation for Collections: A Review of Principles and Recent Developments Jerry Podany, 515 South Norton Ave., Los Angeles, CA 90020 Most discussions regarding the effects of earthquakes on cultural heritage focus on the built environment, on response and recovery efforts, and on the unfortunate damage or loss suffered. In recent years however there is an increasing interest in how to control the response of “contents” to reduce damage due to ground shaking. These developments should be of particular interest to museums and other heritage collections where “contents” are defined as works of art and culturally significant objects, which are often more valuable than the structures that house them. This paper explores the most recent advances in seismic damage mitigation. Such mitigation, relatively new to the museum field, is an effort that aims to bring engineers, seismologists, conservators and mount makers together to address the challenges of the unpredictable nature of earthquakes and to develop methods to control the response of comparatively small and highly fragile objects to ground shaking. Using international case studies from museums, a broad range of preemptive measures, from simple actions based on informed common sense to technically complex approaches developed by multidisciplinary teams, are illustrated. Additionally, the next steps in developing guidelines for museums and constructing a seismic mitigation resource for conservation professionals are discussed.

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Continuous Gradient Temperature Raman Spectroscopy of Highly Polyunsaturated Lipids Critical to Brain Function Catherine L. Broadhurst, USDA Agricultural Research Service, MS: EMFSL Bldg. 303, 10300 Baltimore Blvd., Beltsville, MD 20705, Walter F. Schmidt, Julie K. Nguyen, Jianwei Qin, Kuanglin Chao, Moon S. Kim Excluding water, the brains of all mammals are 60% lipid, the majority of which are the highly polyunsaturated long-chain fatty acids docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6). One of the greatest unanswered questions in all biological science is why DHA is absolutely required for constructing fast signal processing tissues. Over 600 million years of evolution DHA has reigned supreme: everything from the plankton eye spot to the human brain uses DHA, yet it is abundant only in the cold water marine food chain. DHA and AA have vastly different biological activities but the molecular moieties (H-C=C-H)-CH2-(H-C=C-H) composing them are virtually identical. Conventional infrared (IR) and Raman techniques often fail to even distinguish among polyunsaturated lipids, and do not shed light on these questions. We developed the technique of continuous gradient temperature Raman spectroscopy (GTRS), which applies the precise temperature gradients utilized in differential scanning calorimetry to Raman spectroscopy. 20 Mb three-dimensional data arrays with 0.2 °C increments and first/second derivatives allow complete assignment of solid, liquid and transition state vibrational modes, including low intensity/frequency vibrations that cannot be readily analyzed with conventional Raman. We have analyzed DHA and AA as free fatty acids and phospholipids from -100 to 10 °C. We present novel, specific and unique three-dimensional models of these molecules, including torsion components which have not been previously considered. GTRS shows that DHA and AA do have very different vibrational modes in the identical temperature range, likely due to selective changes in elasticity at critical molecular sites.

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Vibration Research and Testing: What was the Question? W. (Bill) Wei, Cultural Heritage Agency of the Netherlands, Hobbemastraat 22, Amsterdam, NL-1071 ZC, The Netherlands Museum professionals are becoming increasingly concerned about the effect of vibrations on valuable objects of art and cultural heritage. This is due to the rapid increase in, among others, loan traffic, public activities, and construction near museums. This has led to a concomitant increase in research and testing to support the development of solutions to mitigate vibration effects. While most of this research has produced interesting results, much of it fails to properly answer the conservator’s ultimate question, “What is the allowable vibration limit for my object or collection?” In order to answer this, one must know what objects can take in terms of vibration levels and duration, coupled with a definition of acceptable amount of damage. Without this, the cost-effective development of solutions to reduce vibration risks is not possible. In the cultural heritage world, there is limited data on the effects of vibrations and/or shock on the condition of objects. The Rijksdienst voor het Cultureel Erfgoed (RCE) has been conducting research into the relationship between vibrations and damage to collections of objects for more than a decade. Based on practical experience and limited experimental testing, a conservative limit for vibration levels and duration has been introduced for museum collections. In this talk, the engineering concepts of vibrations (as opposed to shock) and fatigue damage are reviewed, and the development of the limit based on principles of fatigue is discussed. Suggestions are made for further experimental research which is required on the susceptibility of objects to damage by vibrations, but also shock.

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Vibration Related Risk in Loan Traffic Cindy Zalm, Nationaal Museum voor Wereldculturen, Steenstraat 1, PO Box 212, Leiden, 2300 AE, The Netherlands Since the end of the 1960’s, there has been a major increase in museum loan traffic. This was initially between the USA and Europe, when the Metropolitan Museum of Art (MET) in New York started using the “blockbuster model” in their schedule of temporary exhibitions. The model was soon adopted by other museums and institutions, and today more and more countries are involved in the exchange of works of art. Conservators started to worry about the deterioration of works of art involved due to transport. In the eighties it was clear that the blockbuster model was not a temporary phenomenon, but a popular way of attracting more and new public to museums. Conservators carried out research investigating the impact of changes in temperature and relative humidity, and the impact of shock and vibration during the journey. Specialized agents built their crates according to the museum conservators’ specifications. What was good enough for the National Gallery, was supposed to be good enough for other museums. The main challenge was, and still is, to build a crate that provides optimum climate insulation, and protection against shock and vibration. Most recently, the use of other materials than wood has been explored. During my lecture, I explore the main points of risks regarding shock and vibration and explain how art shippers and museums usually mitigate those. I also point out in what area’s new research could provide a better understanding of the risks involved and the possible solutions.

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Protecting Stained Glass Windows from Vibrations Caused by Construction Operations Dean Koga, Building Conservation Associates, 44 East 32nd St., 12th FL, New York, NY 10016, Erica Morasset, David Woodham The Department of Buildings of the City of New York mandates preparation and filing of construction protection plans to protect historic buildings from damage caused by construction on nearby buildings. The directive sets a threshold for vibrations, which does not take into consideration fragile objects and assemblies such as stained glass windows. Accordingly, a separate construction protection plan was prepared for the original Tiffany windows at Congregation Shearith Israel in New York City. Building Conservation Associates (BCA) documented the windows’ conditions and called for minor repair and precautionary work. Three heavily damaged windows were removed from the site for restoration during construction operations. In lieu of physical interventions to the remaining windows, BCA devised a separate vibration monitoring system mounted directly on the windows to monitor the effects of construction. ANA developed a monitoring installation that incorporates a series of triaxial geophones mounted on the vertical stabilization bars at mid height or on saddle bars where no vertical bars exist. The geophones were configured to trigger when measured peak particle velocity exceeded a preset threshold (well below the City mandated level), and send alarm emails if the displacement recorded during a trigger event exceeded the displacement thresholds. The monitoring program has thus far been highly successful – a reasonable alternative to the standard approach of invasive bracing, or even removal, while leaving the windows fully functional. Periodic checking of the window conditions against preconstruction documentation determined that no damage had occurred.

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Gas-Phase Ion/Ion Reactions: Oxidation and the Dehydroalanine Effect Alice Pilo, Merck & Co., MS: RY818-B208, 126 E. Scott Ave., Rahway, NJ 07065, Zhou Peng, Scott A. McLuckey Recently, several solution-phase derivatizations have been carried out in the gasphase via interactions between oppositely charged ions, viz., ion/ion reactions. One class of ion/ion reactions focuses on the oxidation of cationic biomolecules via re-

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action with periodate or persulfate anions or derivatives. The selective oxidation of methionine residues, and to a much lesser extent, tryptophan residues to the [M+H+O]+ species with periodate anion has been demonstrated. The oxidation of methionine to methionine sulfoxide results in selective loss of methane sulfenic acid that can be used to indicate and localize oxidized methionine residues. Persulfate anion is a more universal oxidizing reagent anion and oxidizes peptides both containing and lacking methionine and tryptophan residues. Furthermore, persulfate anion is capable of both oxygen transfer to generate [M+H+O]+ species and hydrogen abstraction to generate the unique [M-H]+ species. Sulfate radical anion is generated upon negative electrospray ionization (nESI) of persulfate and dominantly generates molecular radical cations upon ion/ion reactions with biomolecules. Odd-electron species undergo vastly different fragmentation pathways than their even-electron counterparts upon activation, for example, collisional activation of peptide molecular radical cations dominantly results in neutral losses from amino acid side-chains. Some of these losses result in the generation of dehydroalanine (Dha) residues, which, upon further activation undergo selective cleavage of the Dha N-Cα bond to generate c- and z-ions N-terminal to the Dha. This phenomenon can be used to incorporate residue-selective cleavages for the structural characterization of polypeptide ions. Here, we present different types of oxidation ion/ion reactions and discuss various applications of this chemistry.

matography (2D-LC) in particular has been implemented mainly as an academic research tool since the 1980’s. Recently, advances in our understanding of the gap between 2D-LC theory and practice, as well as improvements in technologies for LC in general and specifically 2D-LC, have motivated a broader audience to seriously consider implementing 2D-LC in a wider variety of workflows. In this presentation I reflect on the major milestones in the development of our current theoretical understanding of 2D-LC, and the major developments in technology that have accelerated movement of 2D-LC methodologies into the mainstream of analytical science. I then go on to describe results of current studies in my laboratory that we believe will further accelerate broader use of 2D-LC. This includes our ongoing efforts to improve the compatibility of highly complementary separation modes when used in a 2D separation format. Finally, I close with comments about exciting developments on the horizon, and areas where further research is particularly needed to improve the state of the art of 2D-LC.

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Orthogonality Measurements for Multidimensional Chromatography in Three and Higher Dimensional Separations Mark R. Schure, Kroungold Analytical, 1299 Butler Pike, Blue Bell, PA 19422, Joe M. Davis Orthogonality metrics (OMs) for three and higher dimensional separations are proposed as extensions of previously developed OMs, which were used to evaluate the zone utilization of two-dimensional (2-D) separations. These OMs include correlation coefficients, dimensionality, information theory metrics and convex-hull metrics. In a number of these cases, lower dimensional subspace metrics exist and can be readily calculated. The metrics are used to interpret previously generated experimental data. The experimental datasets are derived from Gilar’s peptide data, now modified to be three-dimensional (3-D), and a comprehensive 3-D chromatogram from Moore and Jorgenson. The Moore and Jorgenson chromatogram, which has 25 identifiable 3-D volume elements or peaks, displayed good orthogonality values over all dimensions. However, OMs based on discretization of the 3-D space changed substantially with changes in binning parameters. This example highlights the importance in higher dimensions of having an abundant number of retention times as data points, especially for methods that use discretization. The Gilar data, which in a previous study produced 21 2-D datasets by the pairing of 7 one-dimensional separations, was reinterpreted to produce 35 3-D datasets. These datasets show a number of interesting properties, one of which is that geometric and harmonic means of lower dimensional subspace (i.e., 2-D) OMs correlate well with the higher dimensional (i.e., 3-D) OMs. In the information theory work, an inconsistency is found in previous studies of orthogonality using the 2-D metric often identified as %O. A new choice of metric is proposed, extended to higher dimensions, characterized by mixes of ordered and random retention times, and applied to the experimental datasets. In 2-D, the new metric always equals or exceeds the original one.

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Top-Down Proteomics for Clinical Assay Development James Stephenson, Thermo Fisher Scientific, 168 Third Ave., Waltham, MA 02451 No abstract submitted by the author.

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Characterization and Optimization of Ion Trapping Fields in Toroidal Coordinates Stephen A. Lammert, PerkinElmer, 732 E. Utah Valley Dr., Ste. 120, American Fork, UT 84003, Robert H. Jackson III Toroidal RF ion traps have distinct advantages over other forms of ion traps (3D/ Paul, linear), however the mathematical foundation for toroidal devices is lacking. As compared to traditional spherical/cylindrical coordinate solutions of the three-dimensional (3D)/linear case, the toroidal coordinates were an intuitive choice since there is a trapping center that is offset from the axis. Currently, the trapping fields have not been fully optimized as evidenced by simulations using SIMION® that show incomplete ejection during the mass scan. Furthermore, known paradigms for conventional traps do not hold in toroidal traps. For example, while a “pure” Cartesian quadrupole field performs well as a mass analyzer, this is not the case for a “pure” toroidal quadrupole field. This difference provides a fertile field for study. We have been developing tools for the study of toroidal trapping fields in general, and for optimization of these fields for use in mass spectrometry more specifically. Enhancements to the SIMION program include the ability to construct complex fields directly from the mathematics describing toroidal multipoles. This allows study of the effects of changing specific higher order fields on ion trajectory and behavior. In addition, we have built in measurement tools to characterize the multipole component characterization of arbitrary trapping fields, as well as tools to calculate in near-real-time the ion motion frequency content characteristics. We show examples of these tools in use for optimizing our current, commercial toroidal trap geometry.

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Recent Developments in Active Temperature Control for Improved Chromatographic Performance Stephen R. Groskreutz, University of Pittsburgh, 219 Parkman Ave., Pittsburgh, PA 15260, Anthony R. Horner, Michael T. Rerick, Rachael E. Wilson, Stephen G. Weber We present on the design and application of instruments to improve performance for liquid chromatography through the use of active temperature control (ATC). ATC means to program column temperature in a time and space dependent way. Using arrays of high power Peltier elements (TECs) we have created two systems capable of complex spatial and temporal temperature programs for use with capillary and analytical scale columns. We have applied these instruments to induce temperature-assisted solute focusing (TASF), an instrumental method relying on the temperature dependence of solute retention, to enhance on-column focusing during sample loading. TASF works by cooling the column inlet to sub-ambient temperatures, ca. 5 °C, to facilitate focusing and decrease the influence of precolumn band spreading. Reduced precolumn bandspreading increases the allowable injection volume increasing method sensitivity. Following focusing the cooled portion of the column is rapidly heated, ca. 70 °C, to match the temperature of the downstream separation portion of the column. In the capillary format we have leveraged the TASF concept to induce multiple TASF focusing stages for test solutes, n-alkylparaben homologs, and the monoamine neurotransmitter serotonin. TASF effectively improved high sensitivity analyses using capillary scale (Tonset (pH5)>Tonset (pH3). IVIG started to aggregate at ~56oC according to DLS size measurement. Tertiary structure alterations were reflected in changes in the Raman aromatic side chains Trp1550, Tyr850 and 830. And with difference spectra the change in secondary structure could also be observed during heating. Bevacizumab had aggregation onset of 63.8oC and grossly precipitated at 68oC, where there were still no apparent structural alternation. Rituximab had two size transitions: 54.2 and 67.4oC, and grossly precipitated at >68oC. Raman aromatic side chains had noticeable changes before grossly precipitation. Conclusions: The new approach

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Lifecycle Management of Methods in Animal Health John Hayes, Merck Animal Health, MS: RY80-141, 126 E. Lincoln Ave., Rahway, NJ 07065 Lifecycle management of analytical methods for drug substances and drug products in animal health (AH), while similar in some ways to human health (HH), presents significant challenges. The processes for development, validation, tech transfer and revision/refinement of AH methods are technically similar to HH, but the variety of dosage forms, the speed to market, and the nature of the commercial market require a unique approach. This talk addresses the similarities and differences between AH and HH, and provide several case studies of how we as analytical chemists maintain the quality and integrity of this class of drug products and support the Science of Healthier Animals.

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combining Raman spectroscopy with DLS is capable of capturing structural change and protein aggregation simultaneously under a variety of experimental conditions. This new instrument will provide valuable insights into protein structure and stability for formulation development and comparability assessments. And it will be particularly useful for studying samples with high protein concentrations. Applications of Drop Coat Deposition Confocal Raman (DCDCR) Spectroscopy in Biopharmaceuticals Ravi Kalyanaraman, Bristol-Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903, Jeremy Peters, Anna Luczak, Eugene Park, Varsha Ganesh Drop coat deposition (DCD) is a pre-concentration sampling technique that is known to increase the signal-to-noise (S/N) ratio and has been utilized with confocal Raman spectrometer for protein secondary structure determination. This pre-concentration technique produces protein deposits in the “coffee ring” and they are known to be hydrated, thus maintaining the protein in its native structural form. The confocal Raman microscope provides highly resolved visual image of the “coffee ring” and offers the spatial resolution to collect the Raman spectrum of the protein in the ring in its native state. High quality Raman spectra from model proteins and biologics drug products were collected and the Amide I vibrational band, which is typically in the region of 1620 to 1720 cm-1 is used to determine the secondary structure of proteins. This technique has also been evaluated to monitor protein degradation, such as acid, heat and disulfide reduction. This talk features all the above applications of DCD technique coupled with confocal Raman spectroscopy.

Capillary Nanogel Electrophoresis for Analyses of Proteins and Biological Therapeutics Lisa A. Holland, WVU Chemistry, PO Box 6045, 217 Clark Hall, Morgantown, WV 26506, Cassandra L. Crihfield, Srikanth Gattu, Lloyd Bwanali Multifunctional self-assembled nanomaterials are employed an innovative alternative to support flexible and reprogrammable sample processing and tunable microscale bioseparations. The nanogels are thermally responsive, enabling the viscosity to be switched in order to steer fluids and pattern enzymes in microscale channels. This enables catalytic processing using nanoliter volumes of enzymes. These material features are leveraged to integrate sample processing and complex separations in a single platform. By harnessing nanogels, the on-board fluid is programmed to concentrate, process, steer, and then separate targeted analyte. This is significant because generic microscale channels generate a sophisticated multifunctional device for separation and selection. Nanogels provide efficient separations of biopolymers based on hydrodynamic volume as well as chemical sieving. Glycans separated based on hydrodynamic size have theoretical plate counts of 600,000. Glycan separations relevant to next generation pharmaceuticals and disease detection are modified to probe the glycan for structural information or to simplify a complex electropherogram with a plug of nanogel containing lectin or enzyme. DNA separated based on chemical sieving has theoretical plate counts from 0.9 to 2 million and are applied to human identification, pathogen recognition, and clinical biomarkers.

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Ligand-Receptor Binding Investigated by Tip-Enhanced Raman Spectroscopy Lifu Xiao, University of Notre Dame, 140 D McCourtney Hall, Notre Dame, IN 46556, Zachary Schultz Membrane receptors play important roles in regulating cellular activities. Targeting membrane receptors in cancer cells and understanding their interactions with specific ligands are key for cancer prognosis and therapeutics. However, there is a need to develop new technologies to provide molecular insight into ligand-receptor binding chemistry in cell membrane. We have developed a tip-enhanced Raman spectroscopy (TERS) approach to study the ligand-receptor interactions. Using small molecule ligand-conjugated gold nanoparticles, we can target individual receptors both at substrate surfaces and cell membranes. By using a gold nanoparticle-attached TERS tip to scan the surface, we are able to obtain specific enhanced Raman signal associated with the ligand-receptor binding with a spatial resolution of tens of nanometers. We have shown that receptors with similar structures can be differentiated in intact cell membrane, according to the different Raman signal obtained with the TERS method. This methodology suggests a route to investigate the chemical interactions associated with ligand binding to membrane receptors in cells. In addition, we have demonstrated the capability of TERS not only to study the binding chemistry but also to provide information about binding specificity in intact cell membranes, which may improve drug screening and has potential to improve early stage drug screening.

Fast, High Resolution Size Exclusion Chromatography of Monoclonal Antibodies (mAbs) and Antibody Drug Conjugates (ADCs) Stacy L. Shollenberger, Tosoh Bioscience, Mail-stop: #100, 3604 Horizon Dr., King of Prussia, PA 19406, Phu T. Duong, Atis Chakrabarti, Keegan Gike Numerous types of monoclonal antibody (mAb) species, such as intact mAbs, mAb fragments, and antibody-drug conjugates (ADCs), are currently in development for the treatment of cancer and other diseases. Extensive characterization of these molecules is required to obtain the necessary approvals for clinical trials and eventual commercialization. Size exclusion chromatography (SEC) is the method of choice for qualitative and quantitative evaluation of mAb fragments and aggregates in process development and commercial production. Current SEC methods utilize columns with larger particles and geometries requiring long analysis times, typically greater the 30 minutes. As biopharmaceutical companies implement design-of-experiments (DOE) based approaches to monitor aggregate and fragment variants, the number of samples to be analyzed has increased exponentially. Thus, there is a need for high-throughput SEC methods to streamline the drug development process. This study demonstrates the use of shorter and narrower columns packed with smaller diameter particles to improve speed and resolution of mAb and ADC separations. Additionally, it investigates the use of mobile phase modifiers to improve resolution of mAbs and ADC variants possessing greater hydrophobic character. Compared to traditional SEC columns, the use of a 4.6 mm x 15 cm SEC column containing 2 µm particles demonstrated a decrease in analysis times to less than 5 minutes without compromising method accuracy. Resolution of hydrophobic samples was improved through the addition of organic solvents and arginine without compromising sample integrity. These results offer robust methods that can be quickly and effectively applied to the analysis of various mAbs and ADCs.

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Confocal Raman Microscopy Tracks Flufenamic Acid Delivery Using Lipophilic versus Hydrophilic Penetration Enhancers Qihong Zhang, Rutgers University-Newark, 73 Warren St., Newark, NJ 07102, Yelena Pyatski, Richard Mendelsohn, Carol R. Flach Recent technological advances have led to an increase in the use of confocal Raman microscopy to tackle biophysical and pharmacological issues in topical delivery. Advantages of the technique include the capability to optically section full thickness skin noninvasively. In addition, both exogenous agents and perturbation to the endogenous agents can be monitored without the introduction of probe molecules. For effective topical delivery, a drug must cross the stratum corneum (SC) barrier into viable tissue. The use of penetration enhancers is a widespread approach for barrier modification. In the current study, flufenamic acid (FluA), a non-steroidal anti-inflammatory drug, is employed as a model agent for investigating the influence of lipophilic versus hydrophilic enhancers. In separate experiments, FluA in octanol or propylene glycol/ethanol (75/25) is applied for varying time followed by confocal Raman microscopic mapping of drug and enhancer spatial distribution. The exogenous enhancers are spectroscopically differentiated from the endogenous lipids by utilizing deuterated species. The FluA is tracked by the C=C stretching mode at 1618 cm-1. Discrete, small inclusions of both enhancers are observed throughout the SC. High concentrations of FluA are co-localized with octanol domains which appear to provide a pathway to the viable epidermis for the drug. In contrast, FluA concentrates in the upper SC when using the hydrophilic agent and endogenous lipids appear unperturbed in regions outside the enhancer pockets. The ability to monitor perturbations to endogenous ultrastructure and molecular structure in skin while tracking penetration pathways provides insight into different delivery mechanisms for the penetration enhancers used herein.

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Determination of pH-Induced Oligomerization of a Lipidated Peptide by IMS-MS Elizabeth E. Pierson, Merck & Co., MS: RY80T-B164, 126 E. Lincoln Ave., Rahway, NJ 07065, Nicholas A. Pierson, Justin P. Pennington Peptide therapeutics are a rapidly growing class of drugs, however, they often exhibit poor pharmacokinetic profiles due to their short half-lives and metabolic instability. One strategy to combat these issues is to conjugate peptide therapeutics, typically with lipid chains, to induce self-association and increase half-life. However, formation of oligomers must be monitored with caution, since uncontrolled self-association can lead to the formation of large, irreversible aggregates or fibrils. Formulation efforts to monitor and control the oligomerization of peptides can be precluded by the lack of suitable detection methods. Dynamic light scattering (DLS), multiangle light scattering (MALS), and small-angle X-ray scattering (SAXS) are often employed to estimate size, molecular weight, and structure. Here, we provide definitive oligomer identification with IMS-MS without relying on size standards. Liraglutide, a lipidated peptide active pharmaceutical ingredient (API), was dissolved in ammonium acetate buffer. Three separate liraglutide solutions were adjusted to pH values associated with known oligomerization transformations [Y. Wang et al, Mol. Pharmaceutics 2015, 12, 411-419]. Solutions were analyzed by electrospray ion mobility spectroscopy mass spectrometry (ESI-IMS-MS) using flow injection with an Agilent 6560 ion mobility QToF MS. Instrument conditions such as temperature and voltages were tuned to facilitate preservation of non-covalent complexes. Oligomer

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states were measured shortly after pH adjustment and after various storage times at 5 °C, 25 °C and 40 °C. The intact MS data was processed and analyzed using Intact Mass software (Protein Metrics, Inc.).

Protecting a Cultural Icon: Moving the Liberty Bell with Minimal Vibration Karie Diethorn, Independence National Historical Park, National Park Service, US Department of the Interior, 143 S. 3rd St., Philadelphia, PA 19106 No abstract submitted by the author.

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An Immuno-Analytical Separation Instrument for the Determination of Bioactive Peptides in Biosamples Norberto Guzman, Princeton Biochemicals, Inc., P.O. Box 7102, Princeton, NJ 08543 There is an abundance of bioactive peptides contained in a wide range of food sources, which are generated by fermentation, enzymatic cleavage, chemical hydrolysis, or gastrointestinal digestion processes from food proteins. Many of these peptides have beneficial properties to humans while others may adversely affect human health. In the case of milk and derived dairy products, which are considered important constituents of a balanced diet, they are also the source of bioactive peptides capable of acting on particular cellular receptors. Among these peptides, beta-casomorphin 7 (BCM7), derived during hydrolysis of milk A1 variant beta-casein, contains a histidine residue at position 67 and has been of significant interest. It has been suggested that BCM7 contribute to an increase risk of certain non-communicable diseases, including schizophrenia, autism, cardiovascular diseases, and type-1 diabetes. BCM7 and related peptides are known as opioid peptides because they behave as opioid receptor ligands. The role of beta-casomorphins remains controversial and more research with improved diagnostic techniques is needed to unravel the mechanism and to study the physiological functions of beta-casomorphins. In this presentation, I discuss the determination of opioid peptides in biofluids using a miniaturized immuno-analytical portable instrument. The principle by which this instrument operates is based on a two-dimensional technology known as immunoaffinity capillary electrophoresis (IACE). IACE combines the use of antibodies, or other affinity ligands, as highly selective capture agents with the superior resolving power of capillary electrophoresis and capable of improved sensitivity, specificity and throughput.

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Strategy of Measurement and Data Analysis for Monitoring during the Transportation of Paintings Matthias Läuchli, Museum of Fine Arts Bern, Hodlerstrasse 8-12, Bern 3000, Switzerland, Nathalie Bäschlin, Cornelius Palmbach Based on the findings revealed by the research project Transportation of Fragile Paintings (www.gemaeldetransport.ch), a concept for the monitoring of the transportation of paintings has been developed for the practical application in a museum context. A number of different painting transports were monitored for the Museum of Fine Arts Bern. Two-point measurements were carried out to determine acceleration (1600Hz) in three axes as well as for collecting climate data (1/300Hz). The first measuring point was positioned in the immediate proximity of the work. The second was located on the outer shell of the crate. Different time segments and transportation modes (handling, truck, airplane) were tagged following the reports of the courier. The complete acceleration record of a full transport path was analyzed by counting all shocks. Shocks were thereby defined as the segment between two zero crossings and include vibrations in terms of the amplitude and duration of single oscillations. The large quantity of single shocks is filtered into a reasonable number of relevant values. This analysis enables the documentation and comparison of transport-related loads on the artwork. Furthermore, the two-point measurement concept enables the assessment of the damping capacity of the crates with respect to shock and vibration impact, as well as climatic insulation. The clear representation of the data helps compare and assess the different transportation modes regarding the number of their occurrence and duration. The trend of increasing loan activities clearly demands better correlation of experience-based observations with the physical data of logging-based risk assessment.

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Chemical Identification of Subvisible Particles in Protein-Based Formulations and Impact of Silicone Oil on the Protein Aggregation Olga Laskina, rapID, 11 Deer Park Dr., Ste. 201, Monmouth Junction, NJ 08852, Oliver Valet, Markus Lankers Subvisible particles in protein-based formulations can have different origins: particles can be extrinsic, intrinsic, and inherent. Silicone oils are often applied to the inner surfaces of syringes to form lubricating films. However, protein based therapeutics can have strong interactions with the silicone oils used in prefilled syringes and lead to a formation of particles. Here, we used a wet cell to enable characterization of particles in-situ and to avoid sample preparation that can introduce undesired changes to the samples. Additionally, we monitored changes in silicone layer in prefilled syringe with a layer explorer (LE) instrument during two months of storage and the subsequent formation of particles. The particle analysis in the wet cell was performed using a single particle explorer (SPE) instrument. The SPE is an automated microscope combined with Raman spectroscopy that takes pictures, counts, and determines the chemical composition of particles. The majority of subvisible particles were fatty acid particles that formed upon polysorbate degradation. The silicone oil layer thickness decreased after two months of storage. Analysis of the particles from the formulation shows the presence of protein particles and silicone oil droplets, as well as intrinsic/extrinsic particles of polyamide, cellulose, and dolomite. This work demonstrates the importance of maintaining the silicone oil layer homogeneity to insure the quality of protein based formulations in prefilled syringes. The use of the wet cell enables characterization of pristine protein particles and helps to avoid sample preparation that can introduce undesired changes to the samples.

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Transport of Pastel Paintings: Fatigue Damage due to Vibrations Leila Sauvage, Rijksmuseum, Museumstraat 1, Postbus 74888 Amsterdam, 1070DN, The Netherlands Pastel paintings are considered as one of the most fragile works of art because the medium is poorly bound to the support. Each handling and transport represents thus a risk of losing pastel agglomerates. In 2014, a collaboration between the Rijksmuseum, the Cultural Heritage Agency of the Netherlands and Delft Technical University was initiated. This research aims at predicting fatigue damage of 18th century pastel paintings subjected to vibrations during transport. This paper discusses important initial results of this study. Fatigue tests were conducted on mockups representative of 18th century pastel portraits. These mock-ups were made using handmade paper and pastel sticks were selected after a technical survey of the Rijksmuseum collection. The fatigue tests were stopped when failure, or unacceptable damage, was reached. The results were plotted as Wöhler diagrams or S-N curves, which relate vibration levels to the number of cycles (duration) to failure. The first fatigue tests indicate that damage due to vibration is cumulative. The S-N curves obtained are typical for other materials and objects, where pastel mock-ups subjected to high vibration amplitudes reach failure before the ones subjected to lower vibration amplitudes. Further, the results suggest that the properties of the pastel mock-ups, such as the roughness of the support, the morphology of the pastel particles and the presence of fixative, play an important role in the fatigue behavior for a given vibration environment.

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Generation of Mouse and Rabbit Monoclonal Antibodies Q. Julia Zhao, Bowen Bioscience, LLC, 11 Southwood Dr., Southboro, MA 01772 Monoclonal antibodies are widely used in the fields of therapeutic, diagnostic, and biological applications due to their high specificity and affinity. Most monoclonal antibodies (mAbs) currently used in diagnostic and therapeutic were generated in mice, new trends are emerging for utilizing rabbit monoclonal antibodies (RMAs) in these applications. In general, MMAs have lower affinity and specificity towards antigens, narrower epitope recognition, shorter half-life time in serum, and are more immunogenic for human. In comparison, RMAs have higher affinity and specificity toward antigens, broader epitope recognition, greatly improved response to smallsize epitopes and mouse antigens, and are less immunogenic for human. Here, we describe and summarize the technologies of generation of MMAs by hybridoma, generation of RMAs by single B cell sorting and sequencing, generation of RMAs with single B cell sorting following by in vitro cell expansion and characterization. Optimization of these steps with subsequent humanization, affinity testing as diagnostics and biological testing as anti-tumor therapeutics will also be discussed. With advances of new antigen epitopes and antibody production techniques, we believe the application of rabbit monoclonal antibody will rapidly explode in near future.

Conundra in Analysis of Damage Causation John C. Scott, Conservator of Art and Architecture, 17 Battery Pl., Ste. 1226, New York, NY 10004 In the course of conservation analytical consulting, particularly in consulting for art damage insurance claim adjustors and attorneys, cases arise in which damaging events have no direct witnesses, and in which absent clear evidence, elements of causation are unconfirmed or unknown. Causes often considered include shock and vibration. Such anomalous damage can occur under circumstances and during activities which principals do not recognize as dangerous or may not notice at all. It can be difficult to identify, test and rank risk and damage factors in specific cases, but as conservators we hope to support fair allocation of responsibility and to help prevent recurrence. Some such questions are conundra: complex, perplexing, and difficult questions with no obvious solution. Since insurance adjustment negotiation and litigation move inexorably toward responsibility allocation, these conundra may become dilemmas, when difficult choices compel unsatisfying outcomes. I present a few cases where consulting conservator-analysts have offered creative solutions despite minimal information.

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Handheld LIBS for Pharmaceutical Raw Material Identification: Delivering Solutions that Maximize Value Qun Li, B&W Tek, 19 Shea Way, Ste. 301, Newark, DE 19713, Katherine A. Bakeev, Dan Liu, Jack Zhou Pharmaceutical companies are required to inspect 100% of their incoming raw materials before they can be used, a safeguard to ensure the integrity and safety of the medicine they deliver. Mobile spectroscopic solutions such as handheld laser-induced breakdown spectroscopy (LIBS) and handheld Raman are invaluable in providing rapid analysis that can be done outside of the traditional laboratory environment with small amounts of sample. We have developed a handheld LIBS system based on high-repetition rate micro-pulse laser, giving a high dynamic range. The advantage of the atomic spectroscopic tool of LIBS is that it can be used for the identification of ionic salts such as NaCl, which are not identifiable with molecular spectroscopic tools that have been adopted for sample identification. Ionic salts represent a large volume of raw materials for manufacturers of injectable pharmaceuticals, and handheld LIBS provides a rapid means of identifying these materials without the need for extensive sampling and sample preparation techniques associated with traditional wet chemical methods of identification.

study and liquid chromatography mass spectrometry (LC-MS) technology. This work established the correct RRT for each impurity in the finished product and identifies some unknown degradation products. In this presentation a detailed discussion is presented on how to set up a model to identify complicated impurity profiles in the general high-performance liquid chromatography (HPLC) method development.

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New GC Inlet Liner Deactivation Exhibits Excellent Response for Active Compounds Cathy S. Hetrick, Restek Corporation, 110 Benner Circle, Bellefonte, PA 16823, Linx Waclaski, Brian Jones, Mark Badger The choice of an inlet liner is critical to gas chromatography (GC) analysts, since it impacts the quality of the chromatographic data. Inertness is one of the most important factors to consider, as active analytes can easily be degraded or adsorbed within a hot GC inlet. A new liner deactivation was developed with a high level of inertness towards sensitive compounds. This liner deactivation was tested with a variety of analytes, including various classes of pesticides, as well as acidic and basic probes. Other liner deactivations were also analyzed to compare performance for active compounds. Liners used in this study were single taper with wool performed in split-less mode. This provides one of the most rugged assessments of deactivation quality.

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Characterization on Non-Compendial Reference Standards for Impurities: How Good is Good Enough? Christian Zeine, LGC Standards, Mercatorstr. 51, Wesel 46485, Germany, Omar Mneimne While the guidelines for the characterization of an active pharmaceutical ingredient (API) are numerous and readily available, the information and guidelines for a pharmaceutical impurity are not easily found and often less detailed. The national regulatory authorities (United States Environmental Protection Agency, International Conference on Harmonization (ICH), and United States Pharmacopeia) provide minimal guidance on this issue and the protocols vary between the regulatory bodies. The ICH guidelines simply require that an impurity reference standard be evaluated and characterized according to its intended usage. These vague guidelines result in the development of a variety of analytical methods from both the manufactures and end users that can lead to audit findings. This poster demonstrates different approaches to the analytical characterization of impurity reference standards. It will help answer the question of what level of characterization is acceptable for the corresponding analytical purpose and look at the risks of using a qualitative standard when a quantitative standard is required.

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Measurement of Polychlorinated Biphenyls in Serum by HRGC-IDHRMS Songyan Du, New Jersey Department of Health, 3 Schwarzkopf Dr., Ewing, NJ 08628, Norman Patterson, David Riker Polychlorinated biphenyls (PCBs) are persistent pollutants and detectable amounts are constantly found in blood of most populations that have been examined. As part of efforts of New Jersey Department of Health State Biomonitoring program, a highly sensitive and selective high-throughput method for trace-level PCBs detection in human serum was developed and validated according to the Centers for Disease Control and Prevention (CDC) guidelines for biological sample analysis. Forty ortho-substituted PCBs are measured in serum samples by using high-resolution gas chromatography-isotope-dilution high-resolution mass spectrometry (HRGCID-HRMS). By the employment of liquid-liquid extraction and automated removal of coextracted biogenic materials using modular solid phase extraction (SPE), this analytical method provides a rapid measurement of PCBs with high precision and accuracy. The effectiveness of the cleanup methodology was evaluated by fortifying sample with calibration standards, and the appearance of sample free of matrix effects was demonstrated. The reproducibility for the analyses of three batches of samples spiked with PCBs at low, medium, high levels (N=21) was below 10% for all of the analytes. Mean recoveries of the 13C-labeled internal standards ranged from 40% to 90% for the 40 monitored PCB. The limit of detection (LODs) ranged from 0.01 to 0.05ppb for different PCB congeners. The method development and validation results demonstrated that this method was precise, accurate and robust with high-throughput that suitable for large-scale biomonitoring studies.

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Considerations for Designing Stable Certified Reference Materials Lesley S. Owens, Inorganic Ventures, 300 Technology Dr., Christiansburg, VA 24073, Brian W. Alexander, Paul R. Gaines, Thomas J. Kozikowski, James A. King Jr. The stability of certified reference materials (CRMs) is of utmost concern for reference material producers (RMPs). Under International Organization for Standardization (ISO) Guide 34, RMPs must account for stability when assigning product uncertainties and expiration dates (long-term stability) as well as when assessing product integrity during the shipping process (transport stability). The focus of this presentation is long-term stability since transport stability is not a concern in solutions that exhibit long-term stability. Inorganic Ventures utilizes a “Stable by Design” approach, supported by years of chemical expertise and technical knowledge, when developing new stock and custom CRMs. Since there are an infinite number of analyte, concentration, and matrix combinations for CRMs, each unique solution is carefully scrutinized under the “Stable by Design” guidelines to ensure long-term stability. Critical considerations for solution stability include analyte concentration, inter-element compatibility, matrix compatibility, and container properties, such as container material and cleanliness. Issues such as troublesome elements (e.g., Hg and Ag), matrix limitations, and cleanliness of container materials are presented. Analysts are encouraged to be mindful of these considerations when preparing working solutions as well as when assessing lifetime of working solutions.

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Improved Quantitation for EPA Method 8015C Diesel Range Organics Tom J. Mancuso, Perkin Elmer Inc., 11 Albertson Ave., Blairstown, NJ 07825, Dawn May United States Environmental Protection Agency (EPA) 8015C is a gas chromatographic method used to establish concentrations of a variety of non-halogenated volatile organic compounds, semi volatile organic compounds, and petroleum hydrocarbons. For the purpose of this application, a Clarus® 690 GC was used for the analysis of petroleum hydrocarbons, specifically Diesel Range Organics (DRO). The Clarus 690 gas chromatography (GC) uses a wide range flame ionization detector (WR-FID), which is designed with a large dynamic range to be able to detect high and low concentrations of analytes during a single chromatographic run. TotalChrom® chromatography data system (CDS) software will calculate the hydrocarbon envelope between nC10 and nC28 to accurately quantitate the diesel range organics concentration in each sample. Equipped with a 0.011” jet ionization detector, the wide range FID offers more sensitive detection of analytes and can cover a broad range of reporting limit requirements.

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Identify Complicated Impurity Profiles in the Stability Indicated HPLC Method Development Yan Wang, Apotex Pharmaceutical, 150 Signet Dr., Toronto, ON M2J 4E4, Canada A stability indicating method was developed to monitor the degradation products in an antiviral drug product for releasing test for research & development (R&D) and quality control. The development work was started on the basis of a United States Pharmaceutical (USP) monograph method with the modification to accommodate the proposed formulation. When verifying the USP Organic Impurity method, it was found that the obtained relative retention times (RRTs) from the injection of relevant USP SS mixture are different than the RRTs listed in the USP monograph. This makes it difficult to correctly label individual impurity peaks in the chromatogram. There are 19 potential impurities while the USP SS mixture only identifies three of them. The standards for most impurities are not commercially available. The identification by RRT only for those impurities without standards is in question. An investigation has been conducted to identify all the impurities by the combination of stress

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Flavors, Odors, and Contaminants in Alcoholic Beverages Using Vacuum Assisted Sorbent Extraction and GC-MS Analysis Victoria L. Noad, Entech Instruments, 2207 Agate Ct., Simi Valley, CA 93065, Daniel B. Cardin Analysis of flavor compounds in alcoholic beverages presents challenges for extraction, separation, and quantitation. Matrix affinities for volatile organic compounds (VOCs) are high, and matrix interferences may saturate adsorbents. Additionally, aromas may represent multiple compounds of different volatilities and polarities, which often dictates the extraction technique. A new technique for analyzing the full range of VOC to SVOCs in the headspace of alcoholic beverages using vacuum assisted sorbent extraction (VASE) paired with gas chromatography mass spectrometry (GC-MS) is presented. Application of VASE to samples allows reliable extraction

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of VOCs to SVOCs with minimal matrix effects due to the high phase ratio and surface area of the adsorbent. A tube containing a solid adsorbent, called a sorbent pen, is placed directly into the headspace of a vial holding a liquid or solid sample. A seal is formed between the sorbent pen and the top of the vial to create a closed system for extraction. The top of the sorbent pen contains a seal which allows the vial to be evacuated through the adsorbent immediately after insertion. Once under vacuum, VOCs and SVOCs can diffuse into the headspace and onto the adsorbent faster than when extraction is performed at atmospheric pressure. Results from several high-alcohol containing beverages are presented. Variations in aroma profiles are shown for light, dark, and aged rums. Wine has been analyzed for aromas and for trihaloanisole quantitation to part per quadrillion levels using single quad GC-MS systems. Cognac and whiskey have been studied to provide a quantitative method to monitor phthalate levels for regulatory compliance.

Biosensors were built using the most active ME in each type. All showed catalytic effect and linearity in concentration.

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Controlling Surface Modification Through the Use of Mixed AzideTerminated Self-Assembled Monolayers Ruth M. Mandel, University of Delaware, Department of Chemistry and Biochemistry, 114 Lammot Dupont Lab, Newark, DE 19716, Mackenzie G. Williams, Andrew V. Teplyakov The ability of mixed self-assembled monolayers (SAMs) to incorporate different surface functional groups was studied using mixed azide-terminated SAMs. The surface coverage of azide within SAMs on a gold substrate was varied and used as a method of establishing a high degree of control over subsequent surface reactions. Mixed SAMs were formed from known ratios of methyl-terminated alkylthiol to azide-terminated alkylthiol chains in solution. By varying the percentage of azide-terminated alkylthiol, calibration curves could be constructed showing a linear dependence of the resulting azide-functionalized surface on initial solution concentration. Of particular interest were low ( 90% for all analytes, except Δ8-THC (~80%). The optimized method was applied for analysis of 635 cannabis samples with 68 strain types collected from different growers over 4 years. Although all the samples contained < 10% Δ9THC, in compliance with the NJ Medicinal Marijuana Program regulation, a large variation in cannabinoids content was observed across cultivars. This suggested that establishing a cannabinoid profile for strains produced by different cultivators may be helpful to provide accurate information to the physicians and patients.

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Downstream Process Control Using Real Time Molecular Weight with Light Scattering Bhumit Patel, Merck & Co., Biologics and Vaccines, BioProcess Development, 2000 Galloping Hill Road, Kenilworth NJ 07033, Adrian Gospodarek, Michael Larkin, Sophia Kendrick, Izhar Medalsy, Mark Brower, Douglas Richardson, David Pollard For many proteins including monoclonal antibodies (mAbs), aggregate removal can be a challenge for the purification process. In development, fractions are typically collected during purification and pooling decisions are made after offline analysis using analytical size exclusion chromatography. This is laborious as well as time consuming. This work demonstrates connecting the AKTA Avant purification system directly to a Wyatt TREOS- an inline multi-angle light scattering (LS) detector that uses first principle measurements to calculate average molecular weight, Mw. The TREOS sends a start/stop fractionation trigger signal directly to the AKTA Avant system when a preset Mw criteria is met or unmet. This is in real-time with a 15s delay in trigger events that eliminates the need for analysis after purification. We applied this unique process analytical technology (PAT) tool for monitoring and control of the average Mw in various purification applications. We have performed both bind and elute hydrophobic interaction chromatography (HIC) purification and flow-through HIC purification, where only protein that meets a preset Mw criterion is collected in real time. The TREOS was also connected to a mAb continuous production process to monitor an anion exchange chromatography separation for multiple days. Any deviation from the expected Mw was controlled by signaling the AKTA Avant to discard the material. This strategy has the potential to be scaled to production allowing

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The Effect of Environmental Conditions and Substrate Material on the Weathering of Gasoline and Light Petroleum Distillates Brooke W. Kammrath, University of New Haven, 300 Boston Post Rd., West Haven, CT 06516, Matthew Ciano, Robert Powers, Erika Chen In arson investigations, it is often crucial to determine if an ignitable liquid was present at the scene, as they are often used as accelerants. A variety of ignitable liquids are used as accelerants. The most common is gasoline, in part because it is widely available and cost effective. Light petroleum distillates, such as paint thinners or lighter fuels, are also a commonly used class of ignitable liquids because they too are easily obtainable and highly flammable. Both of these classes of ignitable liquids contain many highly volatile compounds, and are particularly susceptible to weathering. Weathering is the effect of evaporation on the chemical makeup of an ignitable liquid. Samples recovered from fire scenes rarely show the characteristics of unweathered materials, so criminalists must be capable of recognizing ignitable liquids in their weathered forms. 
It has long been stated that a wide variety of conditions can have effects on the weathering process of ignitable liquids. These conditions can include extent, temperature, air composition, airflow, exposure to

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for direct control of aggregates across scales. In conclusion, real time Mw by light scattering is the only non-destructive inline PAT for monitoring the critical quality attribute of average Mw during purification.

es and achieve company objectives. The chosen approach must align well with business needs. Determining the right option is a complicated and daunting task. This session explores various strategies scientific managers are utilizing to achieve their organization’s goals.

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Raman-Based Nutrient and Metabolite Control in Bioprocessing Optimizes Product Quality and Peak Viable Cell Density Karen A. Esmonde-White, Kaiser Optical Systems Inc., 371 Parkland Plaza, Ann Arbor, MI 48103, Maryann Cuellar, Alexander Pitters, Sean Gilliam, Ian Lewis, David Strachan, Herve Lucas, Bruno Lenain In-situ Raman spectroscopy enables simultaneous measurement of multiple bioprocess parameters within a cell culture or fermentation bioprocess. Raman spectra directly report on physical and chemical information, which enable predictive models for real-time control of multiple components. In-situ Raman has opened up many avenues in upstream and downstream bioprocess analytics because it is robust, scalable, and transferable between cell lines, media feedstocks, and process conditions. In this paper, we describe developments in Raman spectroscopy for real-time understanding, monitoring, and control in upstream bioprocess operations. Using case studies, we show how Raman-based control of glucose and lactate in cell culture bioprocesses improved understanding for process robustness and realization of optimal product quality. During process development, Raman enables adoption of quality-by-design principles to define manufacturing design spaces in order to demonstrate process robustness and quality. Improvements to viable cell density profile and therapeutic product quality has been demonstrated in cell culture bioprocesses employing a Raman-based control strategy. We also describe turnkey models developed to account for matrix complexity and facilitate cross-scale model transferability.

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Lab Coat Safety 101: Common Misconceptions Debunked – Top 4 Things Every College or University Must Know Pascual Laguerra, Cintas Corporation, 990 Washington St., Dedham, MA 02026 In December 2008, a tragedy occurred at the University of California, Los Angeles (UCLA) that completely changed the way universities, safety professionals and others view laboratory safety. A young researcher was transferring air-sensitive chemicals into a sealed container when the syringe broke and the compound spilled onto her sweater, instantly igniting. After more than two weeks in the hospital, the woman died from serious burns to more than 40 percent of her body. She was not wearing a protective, flame-resistant lab coat at the time of her accident. I cover the three other topics during my presentation.

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Leveraging CRO Relationships to Accelerate New Product Development Scott Hanton, Intertek Allentown, MS: RD1, Dock 5, 7201 Hamilton Blvd., Allentown, PA 18195 Contract research organizations (CRO) provide many advantages to enable faster new product development, especially when a key hurdle to the new development requires skills or capabilities that are not core to the company developing the product. CROs can provide access to specialized expertise and capabilities, and enable the company to focus on what they do best. This presentation explores different aspects of the relationship with a CRO. We intend to emphasize these elements of the relationship: Flexibility, Benefits of ‘renting’ over ‘ownership’ for non-core skills, Speed, Collaboration, Project management, Cooperative learning, and show how finding the right CRO partner can deliver significant benefits to the project.

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Raman-Based Bioreactor Control: Building Sustainable Applicationss: High Speed Video Analysis Using Common Spectroscopy Tools John Bobiak, Bristol Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903 Robustness and sustainability are goals for any analytical method. As Raman techniques are adopted across different bioprocessing locations, a number of spectral artifacts could enter the historical library of spectra. Such sources of spectral variation may lead to prediction error, and ultimately constrain feedback control applications. In this presentation, spectral residuals and background simulation exercises are discussed as the technical foundation for developing robust methods. A bioprocess control loop is described which uses a combination of paced feeding with small, on-demand boluses to maintain a desired glucose concentration.

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Merck Strategy on Handling Hybrid Assay Coupling Immunoaffinity Purification with LC-MS/MS for Peptide Quantification in Regulated Bioanalysis to Support GLP TK and Clinical PK Studies Yang Xu, Merck & Co., MS: WP75B-300, 770 Sumneytown Pike, West Point, PA 19486 Technological advances have led to a shift in the preferred bioanalytical strategy for peptide quantification from immunoassays to liquid chromatography tandem mass spectrometry (LC-MS-MS) based analysis. The major challenges in applying this approach to regulated bioanalysis are assay sensitivity, specificity, and ruggedness. To overcome these challenges, a hybrid method coupling immunoaffinity purification (IAP) with LC-MS/MS has been developed, successfully validated, and implemented in assays supporting good laboratory practice (GLP) toxicokinetics (TK) and clinical pharmacokinetics (PK) studies. Due to the lack of regulatory guidance for handling hybrid assays at the current stage, our strategy is presented and discussed. First of all, it is critical to develop a reliable analytical method for successful validation and study support. Our systematic approach allowed us to efficiently evaluate assay performance across different species, and improve assay sensitivity, specificity, carryover and reproducibility during method development. Secondly, it is important to specify the acceptance criteria in a validation plan before initiating validation. The data from our validated assays suggested that the proposed criteria were reasonable and achievable. Thirdly, incurred sample reanalysis (ISR) is a challenging indicator that reflects overall assay reproducibility. Our ISR data from the in-study support confirmed our confidence in the hybrid assays we developed. This strategy is exemplified with successful case studies to demonstrate that the LC-MS/ MS assay significantly improved assay performance and PK characterization of the investigational drugs, and contributed significantly to the program development.

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Simultaneous Monitoring of Reactions by NMR, Raman, IR, and NIR Xiaoyun Chen, The Dow Chemical Company, 1897 Bldg. Analytical Sciences, Midland, MI 48642, Donald Eldred, Xianghuai Wang, Siyu Tu, Li Cui, Paul LaBeaume, Mark Rickard, Jing Liu, Hsu Chiang, Kwan Skinner In this talk we showcase a recent example where we carried out in-situ monitoring of a complex network of reactions by combining the power of nuclear magnetic resonance (NMR), Raman, infrared (IR) and near-infrared (NIR). A wealth of information can be obtained by such a comprehensive analytical approach. The strength and weakness of each technology are discussed.

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Comparative Review of Keeping Special Microbiology in a Hospital Lab vs. Out-Sourcing to Reference Labs Margaret E. Blaetz, East Coast Clinical Consultants, 977 Reed Ave., Franklinville, NJ 08322 Determining when to perform testing in-house or outsourcing to another laboratory is a decision laboratory managers make on a regular basis. This question is asked not only when evaluating new analysis, but should also be asked periodically of all the testing you currently perform in-house. Many factors, including a detailed analysis on cost, personnel, time, and other factors, should be reviewed. And every decision should account for intangibles such relationships and non-monetary risks and benefits. In this session, we walk through the factors to make the decision, using a real-life experience in a hospital special microbiology laboratory. At the end of this session, you will have developed a template to performing these calculations for any test in your laboratory.

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Antibody Drug-Target Engagement Measurement in Tissue Using Quantitative Affinity Extraction Liquid Chromatography-Mass Spectrometry Eugene F. Ciccimaro Jr., Agilent Technologies 991 Swayze Ave., Washington Crossing, PA 18977 We demonstrate a strategy using affinity extraction (AE) liquid chromatography tandem mass spectrometry (LC-MS-MS) to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as measuring the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-target antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells for measurement allowing measurement of frozen samples in a remote setting from the clinic.

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Increasing Laboratory Efficiency through In-Sourcing and OutSourcing Strategies Todd Mitchell, Pace Analytical, 1800 Elm St. SE, Minneapolis, MN 55414 In an ever changing and fast paced global marketplace, companies are challenged to respond to demand in a more cost effective and expedient way. This creates changes that trickle down into all areas of an organization. Laboratory managers and other scientific leaders are therefore challenged to get the most out of their limited resources. Out-sourcing and in-sourcing are effective ways to focus resourc-

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Considerations for Assay Platform and Reagent Selection to Quantify Endogenous Protein Biomarker; a FGF21 Case Study Yue Zhao, Bristol-Myers Squibb, Route 206 & Province Line Rd., Princeton, NJ 08543 Biomarkers play an important role in different stages of drug development to facilitate dose selection, demonstrate target engagement, monitor safety as well as stratify patients. Developing bioanalytical assays to measure endogenous protein biomarkers often poses great challenges, especially when there is a structural similar biotherapeutic analog present in the same sample. There are many analytical platforms for biomarker quantitation (eg., ligand blot analysis and liquid chromatography-mass spectrometry), and it is essential to understand the purpose of the analysis and the questions to be answered so that a suitable assay platform can be selected. For example, fibroblast growth factor (FGF21) is an endocrine hormone which acts as a regulator of glucose homoeostasis, insulin sensitivity, lipid profiling and body weight. This endogenous molecule is under wide investigation as a potential therapeutic agent for type 2 diabetes as well as other metabolic disorders. Due to the short half-life of the native molecule and its proteolytic instability, recombinant engineered FGF21 analogs have been generated to improve the pharmacokinetic properties. Endogenous FGF21 can potentially be used as a pharmacodynamic (PD) biomarker to assess drug efficacy and safety to facilitate drug development. However, the near identical sequence between the native FGF21 and the structurally similar recombinant therapeutic agent, requires a very specific bioanalytical assay to differentiate the two proteins. This presentation focuses on a high-level overview of analytical assay platform selection, antibody generation and characterization, assay development and validation, as well as data interpretation.

Incorporating Rheological and Tribological Techniques in the Food Industry James P. Eickhoff Jr., Anton Paar USA, 10215 Timber Ridge Dr., Ashland, VA 23005, Charlotte Reppich The formulation of any new food product can be a large undertaking. One most also take into account the number of steps that the formulation will see in its lifetime. These include the ease at which the formulation can be processed and most importantly the final customer perception. Through each of the steps these steps the formulation can see various environmental conditions, such as temperature and humidity, as well as different deformations to make the sample flow. Various techniques are available in order to build a framework to understand as well as rank the formulation. Two such techniques are rheology and tribology. Both are powerful tools that can be used to create this framework. Rheological techniques probe the visco-elastic balance of a material to understand the storage stability and flow properties. Tribology, on the other hand, provides an analytical technique to analyze and rank a set of formulations that can be correlated to oral perception scores used by sensory panels. In combination both techniques provide can provide a framework to understand the influence of subtle changes in a food formulation. A series of food products were measured using an array of rheological and tribological techniques on a commercially available rheo-tribometer over a range of humidity levels and temperatures.

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Quantitative Headspace Measurement of Volatiles to Semi-Volatiles in Dairy Products using Vacuum Assisted Sorbent Extraction (VASE) and GCMS Analysis Daniel B. Cardin, Entech Instruments, 2207 Agate Ct., Simi Valley, CA 93065, Victoria L. Noad The headspace of dairy products, including cheese and milk, were analyzed utilizing a new technique called vacuum assisted sorbent extraction (VASE). VASE improves the recovery of heavier and more polar volatile compounds in nearly any matrix analyzed by gas chromatography mass spectrometry (GC-MS). After introducing a sample into a 20-40mL vial, a cartridge containing 70mg of Tenax is placed into the headspace of the vial using a vacuum tight interface that allows the vial headspace to be evacuated to less than 0.01atm, or at least until the pressure needed to boil an aqueous mixture at 25 °C. Heavy volatile compounds with low vapor pressures having little to no response by classical solid-phase microextraction (SPME) are extracted 10-50x more efficiently. Unlike dynamic headspace, which uses an inert gas to sweep the volatiles of a sample through the adsorbent bed to concentrate and trap analytes, VASE is performed statically. VASE allows the sample and headspace to come to an equilibrium in a closed system, causing analytes to diffuse onto and collect at the very front of the adsorbent bed. Therefore, VASE achieves a much better recovery of heavier compounds while eliminating the common carryover issues. Once placed under vacuum, the extraction time ranges from minutes to hours until equilibrium between the sample and headspace is reached to produce complete, reproducible extractions. The results show the extensive range of compounds extracted using VASE and demonstrate its potential as a routine method for examining aroma compounds relating to flavor analysis and contaminants in difficult fatty matrices of dairy products.

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Sunit-Level and Intact Analyses of Monoclonal Antibodies from InLife Samples: LC-MS Methods for Pharmacokinetic Quantitation, Critical Quality Attributes, and Biotransformation John Kellie, GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA 19460 For decades a fundamental difference has existed in the manner by which therapeutic molecules are quantified from in-life studies based mainly on molecule size. Traditionally, small molecule therapeutics and metabolites have been quantified by liquid chromatography-mass spectrometry (LC-MS), but large molecule biotherapeutic quantitation has relied on immunoassay detection or surrogate peptide digestion-based LC-MS. One pitfall to peptide-based LC-MS approaches is loss of information from the entire protein due to measurement of only a small portion of the molecule. In an effort to analyze all molecules in the same manner regardless of compound size, we developed whole-molecule (e.g., subunit-level or intact) LC-MS methods to measure the concentration of whole biotherapeutic molecules by exact mass and allow for additional quantitation of molecular mass variants. The presented emerging techniques based on intact or subunit-level masses of biotherapeutics have multiple areas of application, for example in the critical quality attribute space or if biotransformation of the intact molecule is apparent at the intact molecular level but not at the peptide level. The data presented here sheds light on the feasibility of whole-molecule methods for pharmacokinetic utility, particularly as the data can be compared between analytical methodologies, and the reasons for equivalent or divergent data between LC-MS and immunoassay approaches can be examined.

329

Naked-Eye Electrochemical E.coli. Detection Kwok-Fan Chow, University of Massachusetts Lowell, 1 University Ave., Lowell, MA 01854, Sachintha Wijesinghe, Jung-Min Oh In this presentation, we report a naked-eye electrochemical E.coli. sensing platform that is capable of detecting E.coli. on a working electrode and providing a visual readout of the E.coli. concentration on a silver band counter electrode. The display mechanism relies on the electrode oxidation of metallic Ag as a complementary reaction to the sensing reaction. The decrement of the silver band electrode, which is clearly measurable with the naked-eye, correlates linearly with the E.coli. concentration. More importantly, this sensing platform can be operated without any electronic instrumentation.

326

UHPLC-HRMS Analysis of Theobromine in Theobroma Cacao and Chocolates Katarina Mladenovic, Kean University, 1000 Morris Ave., Union, NJ 07083, Yuriko Root, Dil Ramanathan In 2016, the global chocolate and confections industry has reached a $98.3 billion net worth. Chocolate companies have desired to present a quality, best-selling product to meet the increasing demand of chocolates. However, cacao flavonoids that contribute to exquisite taste can be lost through cacao processing. In a previous study from our laboratory, Headspace gas chromatography mass spectrometry (GC-MS) analysis was used to identify flavonoids from Costa Rican Theobroma Cacao beans through each step of cacao processing: raw, dried, roasted, and 100% cacao dark chocolate. Based on the profiling experiments completed via the National Institute of Standards and Technology library, theobromine was the major component found. Theobromine is a bitter alkaloid/flavonoid beneficial in the treatment of hypertension, arteriosclerosis, and angina pectoris. Quantitative analysis on Theobromine was completed on the Thermo Scientific ultra-high-performance liquid chromatography (UHPLC) and linear trap quadropole (LTQ) Orbitrap Discovery equipped with an electrospray ionization (ESI) source. To extract theobromine, diethyl ether was used to remove existing fats and HPLC grade water was used in isolating theobromine. Samples were vortexed and centrifuged to give a purified product with 86% recovery yield. A three-minute gradient method was developed on the UHPLC- high resolution mass spectrometry to analyze each step of processing, commercially available chocolates, and local artisan chocolates.

330

UHPLC-HRMS Analysis of Aechmea Magdalenae Rhizome’s Antibacterial Activity against E. coli Using Phospholipids as an Indicator Mirna E. Giron, Kean University, 1000 Morris Ave., Union, NJ 07083, Quintin Ferraris, Anima Ghosal, Yuriko Root, Dil Ramanathan Aechmea magdalenae rhizome is a medicinal plant used by Costa Rican natives. With a Perkin-Elmer GC Clarus 680 coupled to an MS Clarus SQ GC-MS, profiling was done on the A. magdalenae rhizome. Results showed that it contained a high probability of Acetic acid, a known antibacterial compound. In further studies, a 96well plate assay was used to determine minimal inhibitory concentration (MIC) of the plant extract against Staphylococcus aureus and Escherichia coli. Methanolic extraction of A. magdalenae rhizome was performed using a 24 hour solvent replacement method for 48 and 72 hours. The extract was centrifuged and evaporated to dryness under rotary evaporation. Then the extract was reconstituted in

49

2017 EAS Abstracts

November 2017

aqueous 20% dimethylsulphoxide (DMSO) for microbial testing. Bacterial growth of the extract treated cultures was examined using two color changing indicators at a concentration of 0.1% resazurin and iodonitrotetrazolium chloride (INT) for comparison. Due to colorific interference between the plant extract’s natural pigments and the color of the indicator, phospholipids found within the bacterial membrane have been looked at as a possible indicator to determine MIC. In E. coli, a phospholipid of high abundance is phosphatidylethanolamine (PE). For this reason, fragmentation patterns of the phospholipid and its chains were focused on during the experiment. Ongoing studies are being done to develop a rapid, sensitive ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS) method to detect a mass difference of phosphatidylethanolamine (PE) caused by using the plant extract as the chemical of inhibition.

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2018 EASTERN ANALYTICAL SYMPOSIUM & EXPOSITION

ANALYTICAL SOLUTIONS TO THE WORLD’S PROBLEMS

November 12-14, 2018 Crowne Plaza Princeton Conference Center Plainsboro, NJ

Conveniently Located in Central New Jersey for Easy Driving or Take the Train to Princeton Junction Station and Take Advantage of Our Complimentary Shuttle Service 3 Day Technical Program with 4 Days of Short Courses and Seminars to Connect You with Experts from the Field 3 Day Exposition with Access to Vendors of Analytical Equipment and Services You Can Use in Your Labs Workshops and Employment Bureau to Assist with Your Professional Development

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