Amphetamine excretion studies in man

TOXICOLOGY

AND

APPLIED

3, 678-688

PHARMACOLOGY

Amphetamine

Excretion

(1961)

Studies

in Man

GORDONA. ALLES AND BURNETT B. WISEGARVER Departments and

of Pharmacology, San Francisco,

and

University of California Medical Schools, Los Angeles the Laboratories of Gordon A. Alles, Pasadena Received

April

3, 1961

Studies on the extent of urinary excretion of part of the administered doses of amphetamine sulfate in man were reported by Beyer and Skinner (1940). The racemic and optically isomeric compounds were given orally and their excretion was estimated by a procedure involving the formation of a diazoamino compound. This coupling reaction was studied as to its specificity by Beyer (1942), who found that it distinguished primary amines from secondary or tertiary amines. Urinary bases that give picrate ion color in organic extraction solvents had been estimated, in terms of amphetamine before and after the compound was taken, by Richter (1938) and by Jacobsen and Gad (1940). Such estimations are subject to considerable errors due to variations in the amounts of other bases being excreted. Beyer and Skinner (1940) felt that these earlier reported estimations represented too high an average of excretion for man because of the methodology involved. Several workers in our laboratories and elsewhere have attempted to use the diazoamino color procedures detailed by Beyer and Skinner (1940). When practiced as described, unpredictable variations of color development were observed. It was not possible to use the procedure reliably. We have recognized and studied the large effects of hydrogen ion activity, temperature variations, and the time duration of the coupling reaction. Important, but simple, changes of details were then introduced into the procedure to control these variables. Other primary amines may be estimated with maximum sensitivity by other modifications of these variables. With a reliable diazoamino method for amphetamine at hand, study was made of the unchanged amounts excreted in man after the administration of large doses. We had an opportunity to study excretion in a subject 678

AMPHETAMINE

EXCRETION

679

after the ingestion of very large quantities of the racemic compound. The total amounts excreted daily were large enough to make bulk isolation from the urine also possible. Determination of the optical activity of this material afforded as estimation of the relative metabolic and excretion rates of the optical isomers. METHODS

The Diazoamino

Color

Reaction

As detailed by Beyer and Skinner (1940), a solution of p-nitrobenzenediazonium chloride is added to an HCl acidic solution of amphetamine and the mixture is allowed to stand at room temperature for 1 hour. Some Na&Oa solution is then added, and this mixture allowed to stand 1.5 minutes. Some NaOH is then added to develop a red color which is extracted into butanol; the colored butanol is valued in a calorimeter. Studies of Nolting and Binder (1887) on the diazoamino coupling of p-nitrobenzenediazonium compound with aniline showed that the coupling reaction required an alkalinity at least as great as that resulting from an excess of sodium acetate. It seemed probable that the rate of diazoamino coupling would be a direct function of the hydrogen ion activity. This was found to be true for ring diazo coupling of phenols by Conant and Peterson (1930) and of aromatic amines by Wistar and Bartlett (1941). Experiments showed that no considerable diazoamino coupling occurred during the l-hour period of standing of the p-nitrobenzenediazonium chloride with the HCl acidic amphetamine solution in the Beyer and Skinner method. Their later alkalinizing procedures were variably determinative of the coupling. The shaking or stirring rates and the addition rate while adding the Na&Oa solution variably influenced the hydrogen ion activity during coupling. The pH effect on the coupling reaction was studied by us with uniformly buffered solutions. It was also found that transfer of the red diazoamino sodium salt to butanol was undesirable and unnecessary for consistent color valuations. Mixtures of 10.0 ml of 0.0002 M amphetamine sulfate and 10.0 ml of 0.001 M p-nitrobenzenediazonium chloride in 0.1 M HCl were mixed with lO.O-ml amounts of buffer solutions of 0.10 M sodium tetraborate adjusted with HCl. These mixtures were held at 25.O”C in a thermostat for the coupling rate, and its total possible amounts were found to have large temperature coefficients. At 1, 2, 4, 8, and 16 minutes, 3.0-ml samples were removed and added to 3.0 ml M NaOH solution to terminate coupling and develop the red ion color of the diazoamino compound formed.

680

GORDON

A.

ALLES

AND

BURNETT

B.

WISEGARVER

The increased color density over a reagent blank was valued at 520 rnp with a Beckman spectrophotometer, and results were graphed (Fig. I). At constant pH the coupling rate was not uniform during the 16-minute reaction period. The initial rate was greatest with pH 8.3 in the range

I .o

1

pn

7.83

on

a27

pH 8.56

I

2

FIG.

4

I.

Diazoamino

a

coupling

I6

reaction

rates

at 25” C.

studied, but maximum color development per unit of amphetamine was maximum at pH 7.5-7.8 for the total reaction period used. This is due to p-nitrobenzenediazonium hydroxide also entering into other reactions, as noted by Cain and Nicoll (1902). These other reaction pathways almost instantaneously take over the course of the reaction after NaOH alkalinization. The diazoamino red color formed in strong alkaline solution is quite stable and due to an ionized salt form, as formulated by Hantsch and Hein ( 1919) for the diazoamino compound formed with aniline. Estivnation of Amphetamine A procedure was developed for estimating Smaller amounts can be estimated by diminishing color dilution volumes. p-Nitroaniline solution: 690 mg (0.005 mole) 12.0 N HCI, then made up to 100 ml with water

20-500 pg per sample. sample and diazoamino is dissolved with 8.3 ml and filtered.

AMPHETAMINE

EXCRETION

681

Sodium nitrite solution: 1.40 g (0.020 mole) is dissolved, then made up to 100 ml with water. Disodium phosphate solution: 10.72 g NaZHPO1. 7H20 (0.040 mole) is dissolved and made up to 100 ml with water. Diazo solution: Made fresh daily by adding 5.0 ml of the 0.20 M sodium nitrite solution to 10.0 ml of the p-nitroaniline solution at room temperature in a volumetric flask. After 10 minutes the mixture is made up to 100 ml and can be kept at room temperature until used on that day. Procedure for urine: 50 ml urine with 10 ml 5 N NaOH is placed in allglass distillation apparatus and steam distilled until 50 ml distillate is obtained. To this are added 0.4 ml 5 N NaOH and 50 ml of 6% 110” C petroleum ether; and mixture is shaken 1 minute then separated. The organic layer is then shaken with 20 ml 0.10 A’ HCl, then separated. The organic layer is washed with 5 ml water, then separated. The acidic extract and the water wash are combined, neutralized to about pH 7.0-7.5 with 0.10 N NaOH, then made up to 100 ml. Comparison standards are prepared with 20-500 pg amphetamine in a total volume of 50 ml and run concurrently through the distillation and extraction methods. A solution of 13.6 mg anhydrous amphetamine sulfate in 500 ml contains 20.0 yg amphetamine base per 1.00 ml. Reagent blanks are run throught the procedure to give the zero settings for color density in the spectrophotometer. After allowing complete room temperature equilibration of all extracts and reagent solutions, 20 ml of the 100 ml extracts are put into a flask with 2.0 ml of the 0.4 I4 disodium phosphate solution. Then 2.0 ml of diazo solution is added; the mixture is shaken and allowed to stand 60 minutes. The pH should be 7.4 or the disodium phosphate reagent solution adjusted so that this pH results. At the end of the hour, 1.0 ml of the 5.0 N NaOH solution is rapidly added, and after 10 minutes or longer the color density of 520 mp is valued. Study of the specificity of the diazoamino reaction for amines showed that while the rate and extent of coupling under the conditions given varied per mole unit of amino compounds, all aliphatic and phenylaliphatic primary amines and primary amino alcohols tested gave diazoamino red colors. Corresponding aliphatic and phenylaliphatic secondary amines, or quaternary ammonium compounds did not give the red color reaction. It was noted that under the conditions of the procedure the extent of color formation with primary-carbin primary amines (such as phenethyla-

682

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ALLES

AND

BURNETT

B.

WISEGARVER

mine) was greater than with secondary-carbin primary amines (such as phenisopropylamine or a-methylphenethylamine) . These secondary-carbin compounds in turn gave greater color formation than did the corresponding tertiary-carbin primary amines (such as phenterbutylamine or a,adimethylphenethylamine) . RESULTS

Excretion

of Full

Doses of Amphetamine

Some primary amines are continuously excreted in the urine of man, and their excretion contributes to the problem of estimation of the excretion of administered amphetamine. The amounts of endogenous primary amines are, however, small and fairly constant from day to day over short observation periods. Study of the blank excretion on one of us (G. A. A.) on two sample days before taking any drug showed the blank secretion to be about 1.6 pgjml of amphetamine equivalent on each of these days. After intake of 40 mg racemic amphetamine sulfate in 200 ml of water, the urine was collected for the following O-24- and 24-48-hour periods. Of the 29.4 mg amphetamine base thus administered, 9.60 mg was excreted in the first 24 hours and an additional 3.04 mg was excreted in the next 24 hours. These figures correspond to 33% and 43% excretion for the 24- and 48-hour periods following taking of the drug. Analyses of the same two blank excretion samples by the picrate ion color method of Richter (1938)) modified by the use of ethylene dichloride as the organic extraction solvent, showed a blank excretion of 5.0-5.6 ug/ml amphetamine equivalent on the first sample day and of 10.4-l 1.8 yg/ml on the second sample day. After the administration of amphetamine dosage on the second sample day, the extra excretion was estimated to be 16.2 mg in the first 24 hours and an additional 3.7 mg in the next 24 hours. These figures correspond to 55% and 68% excretion unchanged for the 24- and 48-hour periods. However, if the blank excretion of the first blank sample day were used as the blank value for the drug-taking experiment, well over 100% excretion would be calculated as having occurred in 48 hours. The daily variation of picrate-estimated base excretion without drug was of such magnitude that it precluded any sound conclusion being drawn from use of the picrate ion color method. Excretion

of Large

Doses

of Amphetamine

One subject (A.E.G.), stabilized at an intake around 600 mg racemic amphetamine sulfate daily, was available for study with respect to the excretion of the doses being taken.

AMPHETAMINE

EXCRETION

683

This man was an able physician, experienced in the uses of anesthetics and other drugs, who had retired following a serious coronary attack. He later developed an encephalitis and attempted to control the tremor with phenobarbital and then scopolamine with little success. Seconal as a routine medication appeared to benefit the tremor, but edema of the limbs was serious and blood pressure low. Periodic cyanotic seizures were helped by oxygen inhalation and 30-mg doses of amphetamine sulfate. Some two years later edema and cyanotic seizures increased and doses of 60-80 mg of amphetamine sulfate were required and used repeatedly to raise the systolic blood pressure to 150-160 mm, with relief of edema and tremor. During the course of three years dosage was increased as necessary to maintain a systemic amphetamine effect just short of contraction of the bladder sphincter interfering with urinary excretion. The dosage situation had progressed until an intake of as much as 150 mg in single doses and regularly around 1200 mg daily was used to maintain a satisfactory circulatory and tremor control. The higher dosage was maintained for about 6 months, but had been decreased to 600 mg total daily at the time of the excretion study. Four daily doses of three SO-mg tablets of racemic amphetamine sulfate were being taken with occasional oxygen inhalation therapy for temporary circulatory or respiratory crises. Twenty-four hour samples of urine were taken for a period of 15 days. The dosage of amphetamine was kept constant for the first 8 days, then stopped completely for the following 7 days. During the first 8 days the amphetamine content of the urine was valued by the diazoamino method with the results reported in Table 1. The results total 1.526 g amphetamine from the 8 X 0.600 X 0.733 (3.518 g) administered, representing an average 43% daily excretion. No blank correction for any endogenous primary amine excretion was available for this period. Estimation of amine excretion by the picrate ion color method of Richter, modified by the use of ethylene dichloride, gave somewhat lower total values on samples 3 through 8. Values so estimated were 55, 109, 118, 125, 158, and 107 ug/ml amphetamine equivalent. After the voluntary cessation of daily dosage, the 24-hour samples showed daily decreasing amounts as estimated by the diazoamino method (Table 2). Estimation of excretion by the picrate method on the first 4 days’ samples gave values of 55.5, 16.6, 10.2, and 8.8 ug/ml. During the fol-

684

GORDON

A.

ALLES

AND

BURNETT

B.

WISEGARVER

lowing 3 days the picric method indicated an increase to 44.5, 16.4, and 19.7 pg/ml. The cause for this was unknown, but it may have been due to some change in diet or in motor activity, as noted by von Euler (1945a,b). TABLE URINARY

EXCRETION

OF

AMPHETAMINE

I ADMINISTERED”

AT

CONSTANT

DOSAGE

FOR

S DAYS

Sampleb

Volume (ml)

1

Amphetamine

Total

(m/ml)

(md

Density

PH

2120

1.016

6.2

104

222

2 3

1040 2240

1.017 1.014

5.7 6.4

188 74

196 166

4 5

1330 1880 1110

1.022

6.0 5.8

141 148

5.6

142

188 256 158

1160

1.019

1250

1.020

5.5 5.8

173 111

6 7 8

e Four doses per b Twenty-four-hour

1.019 1.020

day

of three SO-mg samples estimated

tablets of racemic amphetamine by the diazoamino method.

TABLE URINARY

EXCRETION

Sampleb

OF AMPHETAMINE

Volume (ml)

Density

FOR

139

sulfate.

2 7 DAYS~

AFTER

PH

CESSATION

Amphetamine h/ml)

1

1510

1.019

6.0

56.0

2 3

2290 1690

1.016

5.5 5.5

14.6 4.8

4 5 6

1430 2260 1600

1.019 1.020

5.9 5.8 j.8

2.7 1.7 1.1

7

1580

1.020

6.6

0.5

a Days following b Twenty-four-hour

201

1.020 1.019

8 days covered by Table 1. samples estimated by the

diazoamino

OF DOSAGE

Total (mg) 84.~ 33.5 8.2 3.9 3.8 1.8 0.8

method.

The condition of the person stopping the large and long-continued dosage of amphetamine was of interest. Some nine years prior to the experiment and prior to the taking of phenobarbital or amphetamine medication, the systolic blood pressure was known to be around lOO110 mm. During the stabilized period at the beginning of the excretion studies a series of blood pressures averaged 127/94 systolic/diastolic and during the 7 days of stopping of amphetamine dosage averaged 144/103.

AMPHETAMINE

EXCRETION

685

Four days after stopping medication appetite was considerably increased and 7 pounds were gained by the seventh day of stopping. Sleep was increased, particularly at night. More oxygen inhalation therapy was required than before stopping, and at times severe depression was noted. Some increase in water excretion occurred at this time with almost a 50% increase in daily sodium and chloride ion excretions. After the study period, amphetamine was not taken for 2 months, then later used only to tide over difficult times when oxygen inhalation therapy alone was inadequate. Relative

Excretion

of Optical

Isomers

Several of the large volumes of urine excreted during the period of daily intake of 600 mg racemic amphetamine sulfate were pooled and steam distilled in 2000-ml batches after being made alkaline. The steam distillates were made neutral with H2SOa, then concentrated until crystals of ammonium and other base sulfates began to separate. To a concentrate estimated to contain 1.7 g amphetamine sulfate, alkali was added, and an odor of piperidine was noticeable along with that of much ammonia (see von Euler, 1945a,b). After extraction with petroleum ether, the extract was separated, dried with anhydrous MgS04, and partially evaporated; then the residual bases were taken into dilute HCl solution. This solution made up to 100 ml was found to contain 0.83 g amphetamine with an optical rotation of -O.O15”/dm. with sodium light. A solution of this concentration of pure levo isomer would rotate -24.8” X 0.83 X 0.01 (or 0.206”) so that the mixture contained about 8cJcexcess levo over dextro isomer. The calculation used the rotation data of Leithe (1932). Preparation by microfractionation of the base liberated from this solution gave a base of refractive index at 22” C of ND 1.5176; No 1.5174 at 25” C is observed for pure Z-amphetamine. The distilled base on benzoylation gave 600 mg of its N-benzoyl derivative melting at 133-134” C, which showed a specific optical rotation of -17” in ethanol under conditions where pure I-nT-benzoylamphetamine showed a specific rotation of -67”. The excretion of amphetamine in approximately 547” levo form represents the net effect of metabolism and excretion of the orally administered racemic amphetamine. This observation is more direct as to the relative metabolism of the optically isomeric forms than any conclusion derived from the data of Beyer and Skinner (1940). They did not observe any

686

GORDON

A.

ALLES

AND

BURNETT

B.

WISEGARVER

difference between the excretion of administered levo or dextro isomers, and neither did Harris et al. (1947), who compared the excretion of racemic amphetamine with that of its dextro isomer. DISCUSSION

The study of Richter (1938) was made following a dose of 20 mg amphetamine sulfate, and this same dosage was given in the studies of Jacobsen and Gad (1940) as well as lower doses. The 24-hour excretions reported averaged 40% with a range of 15-6Oc/o, and there was a showing of excretion being dependent on urine volumes in individual studies. The percentage of excreted amine appeared to be in the same range after 5- and IO-mg doses. Beyer and Skinner (1940) most frequently gave a 20-mg dosage, but gave 30 mg in two instances. Their reported 24-hour excretions ranged from 18 to 44% after the 20-mg dose and 40-41s after the 30-mg dose. These data do not indicate what amounts of diazoamino color amines were excreted in their subjects when not receiving amphetamine or whether correction was made for such in reporting their data. The report of Harris et aE. (1947) recorded that they also had been unable to use the method as described by Beyer and Skinner (1940). Their studies were made on urine samples of persons regularly receiving daily dosage of 30 mg racemic amphetamine sulfate, or 15 mg of the dextro isomer. The method used for analysis was that of Jacobsen and Gad (1940), modified by a MgO absorption step. Using statistically determined “amine” excretion values, they found excretions of 40-SO%, with an average of 45.6% from the racemic compound, and 43119% with 47.3% average, from the dextro compound. The “amine” content of single control determinations varied as much as 6 mg between days before the start of drug administration, while the ‘(extra amine” calculated as amphetamine ranged from 8 to 18 mg with the racemic compound and from 5 to 9 mg with the dextro. The studies here reported are of interest because they are concerned with unusually large dosage administration. With these doses, some 20-30 times as high as studied previously, the proportioning between the amount metabolized and the amount excreted is still closely the same as for lower dosages. If one may accept the determinations of Jacobsen and Gad (1940) as adequate and correct for 5mg doses, the range now covered is some 120 times with similar proportioning between metabolism and excretion in man.

AMPHETAMINE

637

EXCRETION

The methodology of quantitative maximum diazoamino color fOrmation specific for primary amines should add to the tools now available for those who wish to follow their metabolism in the body, or their appearances by dealkylation processes, such as those found for ephedrine and for methamphetamine by Axelrod (1953, 1954). SUMMARY The diazoamino coupling method for estimation of amphetamine, which is applicable to primary amines quite generally, has been much improved as a reliable method. With this method the urinary excretion of unchanged amphetamine from intakes as high as 600 mg per day has been studied in man. The percentage excretion of unchanged amphetamine appears to be relatively constant in man throughout the dosage range of 5-600mg per day. Bulk isolation of the excreted amphetamine after high dosage shows that the levo isomer was less rapidly metabolized in the body. REFERENCES J. (1935). Studies on sympathomimetic amines. I. The bio-transformation and physiological disposition of I-ephedrine and I-norephedrine. J. Pharmacot. Exptl. Therap. 109, 62-73. AXELROD, J. (1954). Studies on sympathomimetic amines. II. The bio-transformation and physiological disposition of d-amphetamine, d-p-hydroxyamphetamine and d-methamphetamine. J. Phavmacol. ExptE. Therap. 110, 31.5-326. BEYER, K. H. (1942). The color reactions of sympathomimetic amines with diazonium compounds. J. Am. Chem. Sot. 64, 1318-1322. BEYER, K. H., and SKINNER, J. T. (1940). The detoxication and excretion of betaphenylisopropylamine (benzedrine). J. Pharmacol. Exptl. Therap. 68, 419-432. CAIN, J. C., and NICOLL, F. (1902). The rate of decomposition of diazo compounds. I. Diazo-compounds of the benzene series. J. Chem. Sot. 81, 1412-1441. CONANT, J. B., and PETERSON, W. D. (1930). The rate of coupling of diazonium salts with phenols in buffer solutions. J. Am. Chem. Sot. 62, 1220.1232. HANTSCH, A., and HEIN, F. (1919). Absorption und Konstitution der farbipen Alkali-Salzen aus Nitro-triphenylmethanen und verwandten Verbindungen. Ber. deut. them. Ges. 62, 493-509. AXELROD,

HARRIS, S. C., SEARLE, L. M., and IVY, A. C. (1947). The excretion of amphetamine. J. Pharmacol. Exptl. TheTap. 89, 92-100. JACOBSEN, E., and GAD, I. (1940). Die Ausscheidung des fl-Phenylisopropylamine bei Menschen. Arch. exptl. Pathol. Pharmakol. Naunyn-Schmiedeberg’s 126, 280-289. LEITHE, W. (1932). Die Konfiguration der Ephedrine-Basen. Bev. deut. them. Ges. 65, 660-666. N~~LTING, E., and BINDER, F. (1887). Zur Kenntniss der Diazo-amido-verbindungen. Ber. deut. them. Ges. 20, 3004-3018. RICHTER,

D.

(1938).

Elimination

of amines

in man.

Biochem.

J. 32,

1763-1769.

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WISEGARVER

S. (1945a). Occurrence and determination of piperidine in human urine. Acta Pharmacol. Toxicol. 1, 29-59. VON EULER, U. S. (1945b). Piperidine output in urine during muscular work. Acta Physiol. &and. 9, 382-386. WISTAR, R., and BARTLETT, P. D. (1941). Kinetics and mechanism of the coupling of diazonium salts with aromatic amines in buffer solutions. J. Am. Chem. Sot. 63. 413.417. VON

and

EULER,

animal

U.

A. ALLES