THE DETERMINATION AND THE URINARY EXCRETION OF [PDF]

the use of the ultraviolet absorption spectrum of caffeine for its determina- ... absorption spectrum of caffeine and fo

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THE

DETERMINATION

AND CAFFEINE

THE URINARY IN ANIMALS*

EXCRETION

OF

BY R. S. FISHER, E. J. ALGERI, AND J. T. WALKER (From the Department of Legal Medicine, Harvard Medical School, Boston) (Received for publication,

January

14, 1949)

Analytical

Method

Apparatus---A Beckman DU quartz photoelectric spectrophotometer, equipped with 10 mm. quartz cells, is used. * This research was supported by a grant from the Eastern Racing Association, Inc., Revere Racing Association, Inc., Massasoit Greyhound Association, Inc., and the Taunton Greyhound Association, Inc. 71

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Caffeine, its physiological actions, and its metabolic fate have been of great interest to the medical profession for many years becauseof the therapeutic value of the drug as a respiratory and circulatory stimulant, and its wide-spread consumption in beverages. Despite this, a suitable method for the detection and quantitative determination of small amounts of caffeine in body fluids has not been available. It is the purpose of this paper to present such a technique, along with certain observations concerning the excretion of caffeine by the dog and horse. Chronologically, caffeine determination has passedfrom the stage of ultimate analysis of extractive residues early in this century (1, 2), through calorimetric procedures in the 1930’s (3), to the use of ultraviolet spectrophotometry (4). The former are not applicable to urine and blood because of the impracticability of obtaining caffeine-containing extracts of sufficient purity for routine carbon and nitrogen analyses to yield significant results. The calorimetric procedure, with use of the murexide reaction, as described by Tanaka and Ohkubo (3), has, in our experience, failed to yield sufficient precision and specificity to allow its use in analysis of caffeine-containing urines. Ishler, Finucane, and Borker (4) have reported the use of the ultraviolet absorption spectrum of caffeine for its determination in coffees and crude caffeine preparations. This laboratory has been studying the ultraviolet absorption spectra of various methylated xanthines during the past year and has developed a technique based on these phenomena which allows detection of caffeine in amounts as small as 2.5 y per cc. of urine. Ishler et al. have described the ultraviolet absorption spectrum of caffeine and found that it conforms to the Beer-Lambert law. We also confirm these findings and have obtained a molecular extinction of 10,800 (log lo/l = 0.635 at 273 rnp for 12.5 y per cc.). Fig. 1 shows the spectrum of pure caffeine.

72

URINARY

EXCRETION

OF

CAFFEINE

Reagents1. Saturated aqueous solution of lead acetate (Pb(C2H30&.3Hz0). 2. Anhydrous powdered Na2C03, reagent grade. 3. Powdered NaHC03, reagent grade. 4. Saturated aqueous NaHC03 solution. 5. 0.05 N HCl; prepared from redistilled HCl. 6. Specially purified CHCla. Reagent grade CHCls, washed serially with 10 per cent NaOH, concentrated HzS04, two portions of distilled water, and then redistilled from glass.

3110 WAVE

FIG. 1. Ultraviolet in HCl at pH 3.

absorption

spectrum

LENGTH

mp

of caffeine, concentration

12.5 ‘y per cc.,

Procedure Urine-A 40 cc. specimen of the urine is treated by the dropwise addition from a burette of a saturated solution of lead acetate until precipitation is complete. The solution is filtered and 1.0 gm. of NaHC03 is added to the filtrate. Na2C!03is then added to pH 9.0 f 0.5 (universal indicator paper). This results in the precipitation of excess lead, and the solution is again filtered. A 20 cc. aliquot of the filtrate is shaken serially with two 20 cc. portions of CHCI, in a separatory funnel. The combined CHC4 extracts are washed once with 5 cc. of 0.05 N HCl and dried by filtering through anhydrous sodium sulfate suspendedon a cotton pledget in a small funnel. The solvent is then removed by vacuum distillation at room tem-

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-l

R.

S.

FISHER,

E.

J.

ALGERI,

AND

J.

T.

WALKER

73

Calculations Urine-The optical density of the final washed urine extract is due to two components, viz. caffeine and other chromogens. To correct for the non-caffeine chromogens it is necessary to establish a “blank.” This may be done experimentally by treating non-caffeine-containing urines by the analytical procedure. The magnitude of the optical density of such extracts of negative urine will vary considerably (Table I), depending principally on the relative amounts of chromogenic substances in the initial specimen. The calculation is C=

0.0246

(dx

-

&)

(40 +

L)

(1)

where C = the concentration of caffeine in mg. per 100 cc., dx = theoptical density of the unknown (at 273 mp) ; cls = the optical density of the extract of the non-caffeine-containing specimen (at 273 mp); L = the volume of lead acetate solution added in the first step of the extraction procedure. With urine specimens containing more than about 5 y per cc. of caffeine, it will be necessary to dilute the final extract before spectrophotometry. In this case dx will be obtained by multiplying the observed density of the diluted extract by the dilution factor. It has been found possible to apply an alternate procedure for estimating the blank in routine analyses of urine specimens in which an initial negative urine cannot be obtained. For this purpose it may be assumed that the absorption due to chromogens other than caffeine decreases as a of straight line function in the range 245 to 300 mp. The construction such a blank absorption curve on an observed final extract spectrum is The selection of the 300 mp point (B) is based shown in Fig. 2 (line AB).

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perature. The final residue, usually crystalline if caffeine is present, is taken up in 10 cc. of warm water, filtered through cotton into a quartz cell, and the optical density recorded throughout the range 220 to 310 rnp. There will frequently be a small amount of water-insoluble amorphous material in the final residue (after chloroform evaporation), but it may safely be discarded, as we have found that this fraction, when dissolved in alcohol or chloroform, does not exhibit characteristic absorption bands in the ultraviolet. Blood-A Folin-Wu filtrate is prepared with 15 cc. of blood, 75 cc. of water, 30 cc. of 10 per cent Na2W04.2Hs0, weight by volume, and 30 cc. of $ N H&SO*. 75 cc. of this filtrate are extracted directly with two 25 CC.’ portions of CHCh and the extracts washed, dried, and further treated as in the procedure for urine, except that solution of the final residue for spectrophotometry is effected in 4 cc. of water.

74

URINARY

EXCRETION

OF

CAFFEINE

on the fact that caffeine has essentially zero absorption at 300 rnp (Fig. 1). The other point (A) is taken as the minimum of the observed curve in the region of 240 to 250 rnp. For pure caffeine (Fig. 1) the construction of such a line and observations of its ordinate at 273 mp yield a value (a) which equals 15.7 per cent of the maximum absorption (a + b). In the unknown specimensthe blank curve is constructed and its ordinate at 273 mp is deducted from the observed maximum. The resulting value is then adjusted .800

0, 220

230

240

250 WAVE

260

270

LENGTH

280

290

300

:

0

77&U

FIQ. 2. Absorption spectrum of an extract of caffeine-containing the construction of the blank absorption curve, AB.

urine, showing

for the ratio of b to (a + b) (multiply by 1.19) and this value (d,) substituted in Equation 1 for the (dx - ds) term; i.e. C = 0.0246dU (40 + L)

(2)

Blood-The same general considerations apply to blood as to urine with regard to alternate methods of estimating the blank (absorption due to chromogens other than caffeine). The equations are c = l.O5(dx - dB)

(3)

C = 1.05d,,

(4)

Comments A series of ultraviolet absorption spectra typical of those obtained in analysis of urine is shown in Fig. 3, where the spectra of the final extracts of the 1, 2, and 8 hour specimens from Experiment II (Table III) are

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.600-

R.

8.

FISHER,

E.

J.

ALGERI,

AND

J.

T.

75

WALKER

plotted. The volume of urine sample and the final volume of extract submitted to spectrophotometry were 25.0 cc. each, dilution of the extract to this final volume being necessary because of the concentration of caffeine present. The blank curve (0 hour) was obtained in this case from urine collected immediately before the caffeine was administered. Table I illustrates the significance, in the final analytical results, of the two alternate methods of estimating the non-caffeine chromogens in the exThese determinations are selected tract subjected to spectrophotometry. from the experimental results reported subsequently in this paper. They

I HR.

500 cc3

500

’ c iii z 0

400 * .300 .roo .I00 ---*--o-o-4 rio

2i.o

240

260 260 2+0 WAVE LENGTH

FIG. 3. Absorption spectra of extracts of urine 25 cc. samples; final extracts diluted to 25 cc.

-_. 280 7n,U

290

360

IO

from caffeine-stimulated

dog.

exemplify urine and blood specimens in which the caffeine concentrations varied within wide limits. Examination of the results shows that there is close agreement between the two methods when dog urine is analyzed. (The limits were from 96 to 104 per cent and the range of caffeine concenThis is to be extration was from 0.24 to 4.7 mg. per 100 cc. of urine.) pected when the magnitude of the blank is low, as is generally characteristic of dog urine. In the case of horse urine, the disparity is greater, with one pair of results differing by 38 per cent. This, however, was in a specimen with very low caffeine concentration and a relatively high non-caffeinechromogen content (0.25 mg. per 100 cc. of caffeine and ds = 0.232). Both theobromine and theophylline have absorption spectra which are

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.700

76

URINARY

EXCRETION

OF

CAFFEINE

quite similar to, though not identical with, that of caffeine and theoretically would interfere with the determination of caffeine. In actual extractions, approximately 35 per cent of theobromine present in original solutions (conI Results with Estimated Blank

TABLE

Effect of Calcutating

dB

dX

___

Concentration calculated with dB

tion calculated myestimate5 blank

Divergence’

Remarks

~ mg. 100

per cc.

;nogd 2:

per ccnl

0.414

0.232

0.21

0.13

4.070

0.232

4.3

4.3

0.313

0.093

0.24

0.23

-4

4.340

0.093

4.7

4.9

+4

0.830

0.170

0.73

0.64

-12

0.756 0.964 1.115

0.170 0.170 o.aaot

0.65 0.88 0.87

0.65 0.71 0.69

-19 -21

1.549 1.424 1.239

0.342t 0.352t 0.094

1.3 1.2 1.3

1.4 1.1 1.3

0.694 0.409

0.094 0.133

0.66 0.29

0.65 0.26

0.400 0.660

0.133 0.114

0.28 0.57

0.28 0.57

0.598

-8 0.114 0.50 0.46 of the difference between the two results and the result based on calcula&. “blank” urine specimen but analyzed on successive days. Note increase increasing age.

* Ratio tions with t Same in dB with

-38 0

0

+8 -8 0 -2 -10 0 0

(See Table II) Horse urine, 0.25 mg. caffeine added per 100 cc. Horse urine, 5.0 mg. caffeine added per 100 cc. Dog urine, 0.25 mg. caffeine added per 100 cc. Dog urine, 5.0 mg. caffeine added per 100 cc. (See Table IV, Experiment III) Horse urine. 1st hr. 2nd hr. 4th “ (See Table IV, Experiment IV) Horse urine. 1st hr. 10th hr. 19th “ (See Table III, Experiment II) Dog urine. 1st hr. 8th hr. (Experiment III) Horse blood. 10 min. after administration 70 “ “ ‘I (Experiment I) Dogblood. 1st hr. after administration 2nd hr. after administration

centrations of 0.25 and 5.0 mg. per cent) was recovered in the final residue. With theophylline in similar concentrations, less than 2.5 per cent was recovered, and it is judged that it would not interfere with caffeine determination except under very extraordinary conditions. The acid washing of the combined chloroform extracts serves to remove

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~

Concentra-

R.

S.

FISHER,

E.

J.

ALGERI,

AND

J.

T.

77

WALKER

certain organic bases from the solution and thereby prevent their interference in later spectrophotometry of the caffeine. (Among these are strychnine, benzedrine, and nicotine.) Further studies are in progresswith the intention of determining these substances,should they be present in the specimen of urine examined. If the acid extract is to be examined spectrophotometrically, a preliminary wash of the chloroform is advisable (4 cc. of saturated sodium bicarbonate solution). Results

TABLE

Recovery of Caffeine

II Added

to Urine

Horse Found concentration

0.25 5.0

R.STJe~

mg. per 100 cc.

fier cent

0.21

84 86

4.3

K,OWll concentration

Found concentration

ng. per 100 cc.

ng.

0.25 1.4 5.0

per 100 CC.

0.24 1.3 4.8

Recovery

+er cent 96 93 96

Dogs-Female greyhounds were prepared for catheterization by surgical exposure of the urethral orifice and the incisions allowed to heal. Caffeine was administered intravenously and by stomach tube and urine specimens collected at intervals and analyzed for caffeine. The results are presented in Table III. Blood specimenstaken at 1 and 2 hours after the intravenous dosagewere analyzed and showed 0.57 and 0.50 mg. per 100 cc. of caffeine respectively. It is apparent at once that only a very small fraction of an administered dose of caffeine is recoverable in the urine. Further, the magnitude of this is influenced by the relative diuresis. Thus in Experiment II, water diuresis occurred because of the administration of 625 cc. of water with the caffeine, and 7.5 mg., or 3.0 per cent, of the administered dose were recovered in 6 hours. In Experiment I, in which water was not given by stomach tube, only 0.7 per cent was recovered in 6 hours. Hors-The elimination of caffeine by a mare was studied after intravenous and oral administration of the drug. The urine specimens were collected through a large Foley catheter which was kept clamped between collections of the various samples. Table IV shows the results of these studies.

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In V&-Varying amounts of caffeine were added to samples of horse and dog urine and the specimens analyzed by the above technique. The recoveries are shown in Table II.

TABLE III and Recovery of Caffeine in Doa Urine

Concentration

-

Urine volume _

cc. 28 14 27 24

bs.

.Total.

1 2 4 6

..

Total.

Rem&S

:g.per 100cc. 1.4 1.3 2.0 1.2

nrg. 0.39 0.18 0.54 0.29

Experiment I. Weight 20.5 kilos; caffeinesodium benzoate, 400 mg. intravenously

1.40

0.7% recovery

1.2 3.2 2.2 0.9 1.0 2.2 2.0

Experiment II. Weight 25.0 kilos; caffeine citrate, 0.5 gm. in 625 cc. water per OS;water ad libitum

I

--

1 2 4 6 8 12 24

96 294 224 114 150 250 537

1.3 1.1 1.0 0.82 0.66 0.88 0.37 --

.

12.7

1665

5.1% recovery

-

TABLE IV and Recovery of Caffeine in Horse Urine

Concentration

-

-

Time after administration

Urink volume

hrs. 1 2 3 4

CC. 355 200 410 160

Total, . . .

1125

1 2 3 4 5 6 8 10 12 154 19 21 24

330 550 135 250 660 38 760 540 800 835 1335 370 840

Total....

7443

Caffeine concentration

,ng.

--

rota1 caffeine recovered

per 100 cc.

w.

0.73 0.65 0.65 0.88

2.6 1.3 2.7 1.4

-

Experiment III. 3.0 gm. caffeine sodium benzoate intravenously; mare weighing about 430 kilos

8.0

--

-_

Remarks

-

0.87 1.1 1.6 1.5 1.4 1.5 1.7 1.3 0.92 1.1 1.2 1.1 0.71

2.9 6.1 2.2 3.8 9.2 0.6 12.9 7.0 7.4 9.2 16.0 4.1 6.0 87.4 78

-

0.5% recovery Experiment IV. 3.0 gm. caffeine alkaloid orally in gelatin capsule; same mare as in Experiment III; recovery in first 4 hrs., 0.5%

__ -

2.9% recovery

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_

TOtal caffeine

93

._

10.0

Caffeine conceatration

R.

S.

FISHER,

E.

J.

ALGERI,

AND

J.

T.

WALKER

79

SUMMARY

1. A method for rapid detection and determination of caffeine in urine and blood, with an ultraviolet spectrophotometric technique, is described. It is applicable to urinary concentrations of the drug as low as 2.5 y per cc. 2. The procedure allows the isolation and detection of caffeine in the presence of other commonly used stimulants and the strongly basic alkaloids. 3. Caffeine appears promptly in the urine of dogs and horses after the oral or intravenous administration of the drug. It continues to be excreted in the urine for at least 24 hours after administration. 4. The urinary recovery of orally administered caffeine in the first 24 hour period after dosage was 2.9 per cent in a horse and 5.1 per cent in a dog. We are indebted to Dr. J. A. McComb, of the Division of Biologic Laboratories, Massachusetts Department of Public Health, for cooperation in the horse experiments reported herein. BIBLIOGRAPHY

1. Salant, W., and Rieger, J. B., U. 8. Dept. Agr., Bur. Chem., BUZZ. 167 (1912). 2. Kune, A. F., Biochem. Z., 276, 270 (1935). 3. Tanaka, U., and Ohkubo, Y., J. Coil. Agr., Tokyo Imp. Univ., 14, 153 (1937). 4. Ishler, N. H., Finucane, T. P., and Borker, E., Anal. Chem., 20, 1162 (1948).

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Examination of these results shows that after intravenous administration of caffeine the urine level of the drug is approximately the same in each of the first 4 hourly specimens. Blood samples taken 10 and 70 minutes after injection of the drug were analyzed and showed 0.29 and 0.28 mg. per 100 cc. respectively. These are to be contrasted with the 1st hour urine which contained 0.73 mg. per 100 cc., essentially 3 times that in the blood. The same general relationship was found in the dog in Experiment I (see Table III). After oral administration of caffeine to the horse, the concentration of caffeine in the urine rose progressively during the first 3 hours, remained relatively constant through the 8th hour, and then declined gradually to 0.71 mg. per 100 cc. at the end of 24 hours. At 48 hours, a urine specimen contained approximately 0.17 mg. per 100 cc. of caffeine. Thus, with dosage of the magnitude described herein, it is readily possible to detect caffeine in any single urine specimen within the first 24 hours after administration. With the horse, as with the dog, only a small fraction of a given dose of caffeine was recovered in 24 hours.

THE DETERMINATION AND THE URINARY EXCRETION OF CAFFEINE IN ANIMALS R. S. Fisher, E. J. Algeri and J. T. Walker J. Biol. Chem. 1949, 179:71-79.

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