amphetamine enantiomer excretion profile following administration of adderall

Journal of Analytical Toxicology, Vol. 27, October 2003

Amphetamine Enantiomer Excretion Profile Following Administration of Adderall*

John T. Codyl, *, Sandra Valtier 2, and Stephen L. Nelson 3 1Academy of Health Sciences, MCCS-HMP PA Branch, Fort Sam Houston, Texas 78234-6138; 2Clinical Research Squadron, Wilford Hall Medical Center, Lackland AFB, Texas; and 3Department of Pediatrics, Wilford Hall Medical Center, Lackland AFB, Texas

I Abstract I

Amphetamine remains a widely abused drug throughout the world. It is also used therapeutically for weight loss, narcolepsy, and attention deficit disorder with hyperactivity (ADHD). ADHD has grown dramatically recently both in terms of diagnosis and treatment. Increasingly, older individuals are diagnosed and treated for ADHD, and treatment often continues into adulthood. Of the available treatments for ADHD, Adderall is widely prescribed. Despite its widespread use, there are no published data regarding the expected amphetamine excretion profile following its use. This is problematic because, in this case, medical review officers (MRO) and forensic toxicologists are asked to assess results in terms of use pursuant to valid medical prescription without specific data on which to base a sound decision. To address this situation, a study to determine the concentration and enantiomer composition of amphetamine excretion following administration of Adderall was undertaken. Adderall (20 rag) was administered to five healthy subjects with all subsequent ad lib urine samples (total urine void) collected for seven days. Adderall is a 3:1 mixture of d- and /-enantiomers of amphetamine salts. Peak amphetamine concentrations ranged from 2645 to 5948 ng/mL. Samples containing > 500 ng/rnL of amphetamine (the administrative cutoff for a positive result by gas chromatography-mass spectrometry) were seen up to 47:30 h post dose. The number of samples that contained amphetamine concentrations of_> 500 ng/mL ranged among individuals from 7 to 13. As anticipated, analysis showed the d.enantiomer to be in excess of the/-enantiomer, with the proportion of/-enantiomer increasing over time. Because of the mixture of enantiomers, not all samples that contained > 500 ng/mL of amphetamine were positive when tested by immunoassay. The drug concentration profiles were quite variable within and between subjects because of dilution and fluctuations in pH of the samples. These results are the first to describe the excretion of amphetamine following administration of Adderall. The presence of the/-enantiomer separates this drug from other preparations of the drug that are composed of only the d-enantiomer (i.e., dexedrine

�9 This work was supported by the USAF Surgeon General's Office (FWH20020056H). The voluntary fully informed consent of the subjects used in this research was obtained as required by AFI 40403. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Defense or other Departments of the US Government.

t Author to whom correspondence should be addressed: John T. Cody, Ph.O., Academy of Health Sciences, MCCS-HMP PA Branch, 3151 Scott Road, Ft. Sam Houston, TX 78234-6138.

and much illicit amphetamine), thus readily differentiating them from Adderall use. Some illicit and medicinal amphetamine is, however, a mixture of amphetamine enantiomers. Because the enantiomers are metabolized at different rates, their proportion offers the opportunity to describe excretion versus time. Coupling this data with drug concentration makes it possible for forensic toxicologists and MROs to come to an informed decision about the involvement of this drug in a positive drug test result. Using the combination of enantiomer composition and quantitative data will allow MROs and forensic toxicologists to better assess the use of this drug from abuse of amphetamine.

Introduction

Amphetamine has been used for many years for a variety of clinical indications. ~picai clinical uses of amphetamine in- clude narcolepsy, attention deficient disorder with hyperactivity (ADHD), and as a short-term adjunct to a weight-reduction program (1). Although effective as part of a weight-reduction program, amphetamines can be problematic because tolerance typically develops with repeated use. This can lead to depen- dence and the use of higher doses, which leads to the appear- ance of undesirable side effects. As a result, numerous other drugs have been developed to mimic the appetite suppression of amphetamine while decreasing the undesirable effects. Their use for narcolepsy and ADHD are long standing uses for am- phetarnines and are often prescribed on a chronic basis. The use of amphetamines for ADHD is common medical practice and can involve dextroamphetamine, dextromethamphetamine, or mixed isomer salts of amphetamine.

Shortly after the introduction of amphetamines, instances of abuse were noted. The source of the abused amphetamine in- cludes both diversion of pharmaceutical amphetamine and illicit synthesis. Amphetamine contains a single asymmetric center that gives rise to two possible enantiomers. These are commonly referred to using the d- and/-designations also des- ignated as S(+) and R(-), respectively. The d-enantiomer has greater central stimulant activity than the l- form of the drug, therefore the pure d-enantiomer is more desirable to the abuser.

Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 485

Despite this preference, the form of the drug available at any given time is more dependent on the availability of the diverted drug or the synthetic starting materials than it is on the preference of the user.

Much of the work involving the metabolism and excretion of amphetamines was completed in the 1960s and 1970s (2-16). Studies involved examination of the metabolic pathway and factors that influence metabolism and excretion. The effect of pH was examined in several studies that monitored amphetamine excretion under controlled acid or alkaline conditions, as well as uncontrolled conditions (6,8,12,14,15). These studies demonstrated that under acidic conditions, little of the drug is reabsorbed from the filtrate and is therefore excreted more rapidly. Under alkaline conditions, more of the drug is reabsorbed, thus decreasing the amount excreted and increasing the metabolism of the drug. This is illustrated by a study that showed an average of 54.5% of a 10-15-mg dose of amphetamine was excreted intact in 16 h under acid urine conditions, whereas only an average of 2.9% was excreted intact under alkaline conditions (15).

Stereoselective differences in the metabolism of amphetamine have been shown by several investigators (8,12, 14,16). Some of the initial studies involved administration of racemic amphetamine and each enantiomer individually and then comparing overall pharmacokinetic parameters. Using these experimental designs, results did not indicate significant differences in the metabolic profile of the enantiomers. A lim- itation of these studies is the fact that they did not have the analytical capability available today. This is particularly true for the earliest studies which pre-date an analytical procedure that allowed direct measurement of both enantiomers in the same sample. Development of a reliable method for separation and analysis of the enantiomers made it possible to directly measure the enantiomers in the same sample. This allowed simultaneous administration of both enantiomers to assess their metabolism in the presence of one another. Even after the development of enantioselective methods, however, sensitivity was limited because of the chromatographic procedures available at the time. In addition, studies typically pooled samples over defined periods of time (i.e., 6, 12, 16, 24-h pools), which allows reasonably accurate measurement of the total amount of drug, but does not allow for determination of peak levels of individual samples or an accurate picture of the variability between individual samples. Even in the face of these limitations, some of the earliest studies demonstrated the d-enantiomer is metabolized more rapidly than its/-antipode, causing the proportion of l-amphetamine to increase in the urine over time following the administration of racemic amphetamine in several studies that monitored amphetamine.

The purpose of the present study is to examine the excretion profile of amphetamine following administration of the drug Adderall. Adderall is a single-entity (amphetamine) product composed of d-amphetamine saccharate, d,l-amphetamine as- partate, d-amphetamine sulfate, and d,l-amphetamine sulfate. The drug is available in 5, 7.5, 10, 12.5, 15, 20, and 30-mg tablets, as well as extended-release capsules (1). Adderall is designed to provide slow onset and offset, thus minimizing potential undesirable effects and the potential for abuse. It also

486


often provides effective control of ADHD with a single dose, eliminating the necessity of dosing during the school or work day making it a popular choice. The choice of stimulant used is based on a combination of perceived effectiveness, side effects, and dosing schedule, and different drugs are more or less effective in different patients. The recommended dosage of Adderall, as with most stimulant drugs, is to prescribe the lowest effective dose. Initial dosing for adults is typically 10 rag, followed by monitoring for effect for at least one week. If the desired effect is not reached, the dose is escalated until the desired effect is achieved. The typical effective dose for an adult diagnosed with ADHD is 40 mg per day in two divided doses (17).

Materials and Methods

Materials Amphetamine, methamphetamine , d-amphetamine,

d-methamphetamine, amphetamine-d6 (1-phenyi-2-aminopropane-l,l,2,3,3,3-d6), methamphetamine-dn (1-phenyl-ds- 2-methyl-d3-aminopropane-3,3,3-d3), d,l-amphetamine-d5 (1-phenyl-2-aminopropane- l,2,3,3-d5), and d,l-methamphetamine-d5 (1-phenyl-2-methyl-d3-aminopropane- 1,2-d2) were obtained from Cerilliant (Austin, TX)./-Amphetamine and l-methamphetamine were obtained from Alltech (Deer- field, IL). Heptafluorobutyric anhydride (HFBA) was obtained from Sigma Chemical Company (St. Louis, MO). The N-triflu- oroacetyl-l-prolyl-chloride (I-TPC) used for enantiomer analysis was obtained from Aldrich Chemical Company (Milwaukee, WI) and Regis Technologies, Inc. (Morton Grove, IL). Im- munoassay reagents were obtained from Roche Diagnostics Corporation (Indianapolis, IN). Adderal] (Shire Richwood, Flo- rence, KY) administered to experimental subjects, was obtained through the Wilford Hall Medical Center pharmacy (Lackland AFB, TX).

Drug administration and sample collection A single 20-rag dose of Adderall (equivalent to approximately

9.6 and 3.1 mg of d- and/-amphetamine base, respectively) in the form of a single tablet was administered to five healthy male volunteers after obtaining informed consent to participate in this Institutional Review Board approved protocol. A predose urine sample was collected to ensure no interfering substances were present. Following administration, urine was collected for the next seven days. Samples were collected ad lib in order to best simulate what would be seen in random drug testing. Likewise, no attempt was made to physiologically control urine pH to best emulate the situation normally encountered in sample collection for random drug testing. Once collected, the samples were stored at 4~ until analysis.

Sample preparation and analysis Sample pH was measured using a Fisher (Houston, TX) Ac-

cumet 50-pH meter and specific gravity determined using an AO Scientific Instruments (Keene, NY) refractometer. Creati- nine levels were determined at the Wilford Hall Medical Center clinical laboratory using standard clinical laboratory proce-


Table I. Creatinine, Specific Gravity, pH, Amphetamine Concentration, Enantiomer Composition, and Immunoassay Results for Samples Collected Following Administration of 20 mg of Adderall"

Hours Amphetamine % % Immunoassay Post Dose pH Sp. Gr. Crealinine (n~/mL) /-Amp d-Amp Result

Subject 1 00:00 6.57 1.005 27.3 0 NA t NA Neg 01:05 5.89 1.018 242.1 2422 26.05 73.95 Pos 02:25 6.13 1.005 31.9 1037 22.51 77.49 Pos 04:05 6.36 1.005 32.9 1023 23.85 76.15 Pos 07:00 6.59 1.007 54.2 859 25.19 74.81 Pos 09:40 5.85 1.007 83.2 1732 25.60 74.40 Pos 20:40 5.66 1.024 364.3 5948 28.32 71.68 Pos 25:25 5.90 1.008 77.0 1021 28.96 71.04 Pos 29:10 5.67 1.009 80.2 911 30.12 69.88 Pos 31:10 5.82 1.006 57.1 562 30.71 69.29 Neg 34:40 5.28 1.015 163.9 1256 31.43 68.57 Pos 37:25 5.55 1.017 183.1 862 32.25 67.75 Pos 45:10 5.41 1.020 235.5 1129 33.21 66.79 Pos 48:40 5.77 1.006 53.4 202 33.79 66.21 Neg 50:00 5.71 1.003 25.0 96 34.27 65.73 Neg 51:40 5.52 1.006 39.8 122 34.85 65.15 Neg 58:25 5.47 1.011 79.6 170 36.86 63.14 Neg 60:40 5.58 1.012 110.3 147 39.02 60.98 Neg 68:40 5.64 1.019 195.3 198 39.36 60.64 Neg 73:10 6.14 1.014 93.3 79 41.89 58.11 Neg 74:40 5.72 1.012 75.1 66 41.03 58.97 Neg 78:40 6.58 1.008 57.2 31 41.11 58.89 Neg 82:10 6.17 1.014 140.4 49 42.63 57.37 Neg 87:25 5.83 1.022 192.4 107 44.12 55.88 Ne 8 94:55 5.57 1.026 281.0 106 44.56 55.44 Neg 99:10 5.29 1.023 170.6 30 46.99 53.01 Neg 104:10 5.50 1.022 156.5 28 47.13 52.87 Ne 8 108:00 5.44 1.019 119.2 20 47.04 52.96 Neg 111:20 5.37 1.022 205.7 49 46.15 53.85 Neg 121:10 5.27 1.024 195.6 25 46.98 53.02 Neg 124:00 5.12 1.018 I17.9 9 NA NA Neg 128:10 5.10 1.020 154.0 11 NA NA Neg 132:55 5.28 1.024 168.8 10 NA NA Neg 141:25 5.22 1.025 216.2 12 NA NA Neg 145:40 5.15 1.020 135.2 6 NA NA Neg 149:20 5.45 1.017 102.3 0 NA NA Neg 154:20 5.18 1.019 146.9 0 NA NA Neg 164:40 5.36 1.024 241.9 6 NA NA Neg Subject 2 00:00 6.17 1.016 133.0 0 NA NA Neg 02:30 6.93 1.008 52.4 599 24.68 75.32 Neg 05:00 6.92 1.004 30.1 1015 24.82 75.18 Pos 09:00 6.74 1.014 129.4 2024 25.62 74.38 Pos 13:00 6.59 1.015 107.1 1701 26.11 73.89 Pos 17:00 5.44 1.017 156.3 3598 27.15 72.85 Pos 28:30 5.00 1.019 252.5 3565 28.71 71.29 Pos 33:00 5.55 1.022 180.0 1406 30.66 69.34 Pos 36:00 5.46 1.022 187.3 673 31.66 68.34 Neg 40:00 5.78 1.019 131.2 543 32.99 67.01 Neg 45:00 5.30 1.021 179.4 866 33.69 66.31 Pos

"Only a single sample is presented following the last detected amphetamine for each subject. All samples for each of the subjects collected after that time contained no detectable (> 5 ng/mL) amphetamine.

t NA indicates enantiomer analysis was not completed because of the low concentration or lack of drug in the sample.

dures. Immunoassay analysis was accomplished using Roche Diagnostics Corporation on-line reagents on an Olympus (Melville, NY) AUS00 automated analyzer using standard procedures. Gas chromatography-mass spectrometry (GC-MS) analyses were performed using a Hewlett-Packard (Palo Alto, CA) 6890 gas chromatograph (GC) coupled with an HP 5973 mass spectrometer (MS) using a 7683 autoinjector.

Quantitative analysis. Two-milliliter aliquots were extracted, derivatized with heptafluorobutyric anhydride, and analyzed by GC-MS as previously described (18), with the addition of using a ZB-50 (DB-17 equivalent) capillary column (15 m x 0.25-ram i.d., 0.25- mm film thickness, Phenomenex, Torrance, CA), as well as the HP-1 column (12 m • 0.2- mm i.d., 0.33-pro film thickness, Hewlett- Packard). Instrumental conditions were as follows: splitless injection with injection port and interface temperatures at 270~ Condi- tions for the HP-1 consisted of a temperature program with an initial time of I rain at 80~ programmed to 180~ at 20~ with a 2- rain final time. Conditions for the ZB-50 column consisted of a temperature program with an initial time of 2 rain at 80~ programmed to 125~ at 20~ then programmed to 150~ at 25~ with a final time of 0 rain. Quantitation was based on single-point calibration using a calibration standard containing 500 ng/mL of amphetamine and methamphetamine, with 500 ng/mL of deuterium-labeled isotopomer in- temal standards. Low concentration samples were quantitated based on single-point calibration using a standard at 25 ng/mL amphetamine and methamphetamine and with 50 ng/mL of each internal standard. Limits of detection (LOD) for this assay were 5 ng/mL for amphetamine and methamphetamine. The assay is linear to 10,000 ng/mL for amphetamine and methamphetamine, with a limit of quantitation of 5 ng/mL for both amphetamine and methamphetamine. Ions monitored were as follows: amphetamine, m/z 240, 118, and 91; methamphetamine, m/z 254, 210, and 118; amphetamine-d6, m/z 244 and 123; and methamphetamine-dlz, m/z 260 and 213. Acceptance criteria were as follows: quantitative values within • 20% of the target concentration with proper qualitative identifi- cation as determined by ion ratios within • 20% and retention time within • 2% of the calibration standard, while exhibiting acceptable chromatography.

Enantiomer analysis. Two-milliliter urine

487


Table I. (continued) Creatinine, Specific Gravity, pH, Amphetamine Concentration, Enantiomer Composition, and Immunoassay Results for Samples Collected Following Administration of 20 mg of Adderall*

Hours Amphetamine % % Immunoassay Post Dose pH Sp. Gr. Creatinine (rig/mr) /-Amp d-Amp Result

47:30 5,41 52:00 6,19 57:00 5.62 61:00 6.32 69:00 5.16 71:30 6.19 78:30 5.75 83:00 6.43 87:00 6.93 91:00 5.38 96:30 7.32 99:00 7.38 102:00 7.30 104:00 7.30 108:00 7.35 111:00 6.38 116:00 4.89 120:30 7.29 123:30 5.71 127:45 5.31 131:00 5.98 Subjed3 00:00 7.30 02:00 6.56 04:30 6.89 06:30 7.31 09:00 6.23 11:00 6.07 12:30 6.79 14:00 7.05 15:00 7.06 18:30 6.69 22:00 6.75 22:30 7.23 25:00 6.75 26:00 5.92 27:30 6.11 28:45 6.23 30:00 6.81 31:00 6.76 32:00 6.79 33:00 6.84 34:00 6.87 35:30 6.97 41:30 6.31 44:00 5.30 47:00 5.55 49:15 5.86 52:00 5.76 54:00 5.64 55:00 6.12

1.020 170.1 712 34.46 65.54 Neg 1.021 168.4 167 36.88 63.12 Neg 1.021 207.3 265 37.09 62.91 Neg 1.019 152.0 96 39.59 60.41 Neg 1.018 140.7 186 39.49 60.51 Neg 1.019 179.6 93 40.39 59.61 Neg 1.017 205.4 54 42.87 57.13 Neg 1.019 146.5 31 44.03 55.97 Neg 1.018 85.5 12 43.93 56.07 Neg 1.015 113.1 45 44.55 55.45 Neg 1.006 112.0 14 NA t NA Neg 1.010 60.4 4 NA NA Neg 1.013 98.0 0 NA NA Neg 1.008 70.4 0 NA NA Neg 1.012 92.0 0 NA NA Neg 1.013 83.7 7 NA NA Neg 1.016 123.4 19 49.98 50.02 Neg 1.012 79.6 0 NA NA Neg 1.018 129.7 8 NA NA Neg 1.021 216.7 9 NA NA Neg 1.017 144.5 0 NA NA Neg

1.002 19.0 0 NA NA Neg 1.006 45.4 883 23.62 76.38 Pos .007 62.6 1090 24.31 75.69 Pos .005 43.3 390 24.68 75.32 Neg .005 40.8 1352 25.20 74.80 Pos .006 62.9 860 25.88 74.12 Pos .004 37.7 734 26.17 73.83 Neg .003 23.9 361 26.50 73.50 Neg .002 20.3 328 27.37 72.63 Neg .006 59.0 653 27.58 72.42 Neg .021 207.9 2645 29.66 70.34 Pos .006 49.8 516 29.47 70.53 Neg .010 95.3 805 30.57 69.43 Neg .013 112.6 2069 31.60 68.40 Pos .005 37.5 621 31.42 68.58 Neg .007 57,3 659 32.10 67.90 Neg .003 30.4 288 32.29 67.71 Neg .005 35.5 294 32.81 67.19 Neg .004 29.9 224 33.11 66.89 Neg .004 28.9 178 33.96 66.04 Neg .005 34.9 186 33.91 66.09 Neg .003 24.5 135 34.33 65.67 Neg .007 59.5 321 35.36 64.64 Neg .016 157.2 1088 36.31 63.69 Pos .008 87.3 443 37.26 62.74 Neg .007 77.8 269 38.21 61.79 Neg

1.008 59,9 215 39.55 60.45 Neg 1.009 66.7 213 39.32 60.68 Neg 1.003 22.0 77 39.83 60.17 Neg

* Only a single sample is presented following the last detected amphetamine for each subject. All samples for each of the subjects collected after that time contained no detectable (_> 5 ng/mL) amphetamine.

r NA indicates enantiomer analysis was not completed because of the low concentration or lack of drug in the sample.

488

samples containing 500 ng/mL each of amphetamine-d5 and methamphetamine-ds were extracted and derivatized with I-TPC as previously described (19). Instrumental conditions were as follows: splitless injection, with injection port and interface temperatures set at 270~ Conditions for the HP-I column (12 m x 0.2-mm i.d., 0.33-pm film thickness, Hewlett-Packard) consisted of a temperature program with initial temperature at 130~ programmed to 170~ at 4~ then to 240~ at 35~ with a final time of 0 min. Given that Adderall is amphetamine only, the run time for sample analysis was modified Her elution of amphetamine to decrease overall run time. Ions monitored were m/z 237, 241,251, and 255 for d- and/-amphetamine, d,l-amphetamine-ds, d- and l-methamphetamine, and d,l-methamphetamine-ds, respectively. This assay was developed to provide qualitative determination of the enantiomeric composition of amphetamine, methamphetamine, and re- lated analogues. Each batch of samples was calibrated using a sample containing 50% of both enantiomers of amphetamine and methamphetamine and analyzed with control samples containing 0% l-enantiomer plus 100% d-enantiomer of amphetamine and methamphetamine and 100% l-enantiomer plus 0% d-enantiomer of amphetamine and methamphetamine along with a control containing no amphetamine or methamphetamine. Acceptance criteria were as follows: enantiomer ratios of the deuterated internal standards for all control and unknown samples within • 20% of calibrator; drug enantiomer ratios of controls within • 20% of target percentages; and negative control (0 ng/mL) showed no detectable amphetamine or methamphetamine, with acceptable chromatography and retention times within • 2% of calibrator.

Results and Discussion

Summary results of the samples collected from subjects in this study (see Table [) showed peak levels ranging from 2645 to 5948 ng/mL. Peak concentrations were reached anywhere from 5:25 h to 22:00 h post dose. The first sample provided by each subject contained amphetamine at concentrations over 500 ng/mL, the administrative cutoff for a positive result. The last positive samples were seen between 35:30 and 47:30 h post dose. The number of samples containing _> 500 ng/mL of am-



Hours Amphetamine % % Immunoassay Post Dose pH Sp. Gr. Creatinine (ng/mt) /-Amp d-Amp Result

56:00 6.58 1.004 23.2 54 40.15 59.85 Neg 57:30 5.99 1.007 47.4 113 40.41 59.59 Neg 60:00 6.09 1.011 81.2 131 40.61 59.39 Neg 61:00 5.99 1.004 31.5 87 40.61 59.39 Neg 61:30 6.11 1.001 17.1 46 38.97 61.03 Neg 63:30 5.98 1.002 22.1 54 37.32 62.68 Neg 70:00 5.49 1.016 157.3 244 41.76 58.24 Neg 72:45 6.45 1.007 73.6 48 41.76 58.24 Neg 74:00 7.18 1.004 27.9 19 41.72 58.28 Neg 76:00 6.98 1.009 59.7 23 41.52 58.48 Neg 77:30 5.88 1.007 51.5 36 46.64 53.36 Neg 81:00 6.80 1.009 62.9 16 47.57 52.43 Neg 83:30 7.14 1.014 90.2 12 47.57 52.43 Neg 85:00 6.20 1.018 122.5 54 47.47 52.53 Neg 86:30 6.36 1.003 21.6 10 NA t NA Neg 90:00 5.60 1.015 119.8 35 48.37 51.63 Neg 93:30 6.36 1.010 120.8 49 48.16 51.84 Neg 94:30 5.91 1.010 56.7 25 48.35 51.65 Neg 96:15 7.20 1.008 56.7 11 48.88 51.12 Neg 99:30 5.75 1.015 112.5 25 49.30 50.70 Neg 102:00 5.86 1.011 79.7 17 48.83 51.17 Neg 104:00 6.20 1.004 26.2 6 NA NA Neg 105:30 6.17 1.006 40.0 8 NA NA Neg 107:15 6.24 1.015 92.0 13 49.15 50.85 Neg 110:00 6.96 1.008 47.7 0 NA NA Neg 116:30 5.85 1.016 110.4 16 47.11 52.89 Neg 118:00 5.14 1.015 102.0 15 46.19 53.81 Neg 118:45 5.48 1.006 38.1 7 NA NA Neg 120:00 6.54 1.010 59.9 0 NA NA Neg Subject 4 00:00 5.23 1.009 61.2 0 NA NA Neg 05:25 5.18 1.012 100.4 3451 25.14 74.86 Pos 08:45 5.57 1,008 62.6 2468 25.33 74.67 Pos 11:30 6.86 1.009 74.7 1062 26.65 73.35 Pos 18:00 6.33 1.011 107.7 1492 28.85 71.15 Pos 21:30 5.66 1.007 53.5 1054 29.12 70.88 Pos 27:00 5.66 1.007 192.0 1562 31.66 68.34 Pos 28:30 6.02 1.007 43.6 383 32.65 67.35 Neg 31:00 5.73 1.005 35.4 327 33.42 66.58 Neg 35:30 5.54 1.015 123.8 690 34.94 65.06 Pos 37:00 7.14 1.010 76.5 109 36.51 63.49 Neg 42:00 6.64 1.006 83.3 178 37.67 62.33 Neg 46:30 5.67 1.008 78.1 315 38.51 61.49 Neg 48:25 5.58 1.007 57.2 199 39.29 60.71 Neg 50:15 6.06 1.004 29.0 82 40.59 59.41 Neg 52:10 5.69 1.006 45.1 105 41.32 58.68 Neg 53:25 5.95 1.004 28.9 62 41.96 58.04 Neg 57:00 5.53 1.006 54.7 90 42.89 57.11 Neg 59:10 5.13 1.009 80.9 147 43.81 56.19 Neg 65:30 5.35 1.025 207.9 218 44.83 55.17 Neg 70:30 6.40 1.026 197.7 66 46.37 53.63 Neg

�9 Only a single sample is presented following the last detected amphetamine for each subject. All samples for each of the subjects collected after that time contained no detectable (> 5 ng/mL) amphetamine.

t NA indicates enantiomer analysis was not completed because of the low concentration or lack of drug in the sample.

phetamine ranged from 7 to 13 among the subjects. Not all samples that contained ~ 500 ng/mL of amphetamine were positive by immunoassay. This is expected given the reported cross reactivity of these immunoassay reagents to d,l-amphetamine (62%) and/-amphetamine (5%) when compared with d-amphetamine (100%) (20). Amphetamine could be detected (LOD = 5 ng/mL) in at least one subject, up to approximately 160 h post dose. The total amount of amphetamine excreted intact was 32.0%, 32.4%, 44.8%, 45.9%, and 46.3% of the initial dose representing an average of 40.3% • 7.4%.

With uncontrolled urine pH, Beckett and Rowland (8) found an average of 32.9% d-amphetamine and 49.2%/-amphetamine following the administration of 5-15 mg of racemic amphetamine in the first 48 h. In a similar study, 15-40% d-amphetamine and 30-66%/-amphetamine were excreted in 48 h. This study also used doses of 5-15 mg of racemic amphetamine (12). The current study found 35-53% (mean = 46% • 9.4%) of the l- amphetamine was excreted intact and 30--44% (mean = 38.4% • 6.7%) of the d-enantiomer. Examining individual subjects, the percentage of l-enantiomer excreted intact was greater than the d-enantiomer in each.

As is typical for amine drugs, the excretion of amphetamine was greatly affected by urine pH. Measured concentrations of amphetamine also varied because of dilution of the urine as re- flected by creatinine concentrations. Although the concentration of many drugs can be re- lated to creatinine concentration to "correct" for dilution, the influence of urine pH is too great to allow this with drugs such as the amphetamines. The dramatic effects of changes in pH and creatinine concentration on the concentration of amphetamine are revealed by review of Table I. The proportion of d- and l-enantiomers, however, is more predicative and showed fairly consistent changes, despite vast changes in specific gravity, creatinine, and pH. The ratio of d- to/-amphetamine in the Adderall tablet is 3:1. Given that ratio, one would expect nearly the same 3:1 proportion of enantiomers in the urine shortly following administration, which is in fact what was seen in this study. Results of this changing proportion are shown in Figure 1. Although the concentration of amphetamine varied dramatically over time, the proportion of d- to l-amphetamine followed a much more predictable pattern. Because the change in proportion of the enantiomers is based on the metabolism of the drug rather than its rate of excretion and

489



Hours Amphetamine % % Immunoassay Post Dose pH Sp. Gr. Creatinine (ng/mt) /-Amp d-Amp Resull

73:35 5.92 1.024 154.8 72 46.32 53.68 Neg 75:30 5.70 1.009 57.4 31 47.07 52.93 Neg 79:10 5.32 1.012 81.5 34 48.10 51.90 Neg 81:00 5.78 1.005 38.8 13 49.47 50.53 Neg 83:30 6.13 1.008 66.7 16 50.71 49.29 Neg 85:45 7.20 1.008 56.6 0 NA t NA Neg 91:00 6.67 1.008 59.6 7 NA NA Neg 92:40 6.17 1.009 67.4 11 51.17 48.83 Neg 94:30 5.26 1.006 40.1 8 NA NA Neg 96:00 5.40 1.005 38.3 0 NA NA Neg Subject 5 00:00 7.23 1.006 22.2 0 NA NA Neg 02:43 6.87 1.010 62.7 1246 22.94 77.06 Pos 04:13 6.57 1.005 33.7 1235 23.09 76.91 Pos 07:43 7.41 1.015 154.3 1616 24.03 75.97 Pos 10:43 6.73 1.011 101.8 2078 24.62 75.38 Pos 15:43 6.25 1.018 251.0 3225 25,82 74.18 Pos 17:13 7.07 1.004 41.8 725 25.42 74.58 Pos 18:43 7.18 1.003 25.8 435 25.82 74.18 Neg 20:43 6.14 1.005 43.8 995 26.47 73.53 Pos 22:43 5.80 1.005 41.9 998 27.15 72.85 Pos 24:31 6.63 1.005 32.5 449 27.21 72.79 Neg 25:58 5.83 1.011 95.8 1314 27.89 72.11 Pos 31:13 6.35 1.017 147.7 945 28.89 71.11 Pos 38:43 6.17 1.016 154.9 956 30.05 69.95 Pos 39:43 6.17 1.017 226.7 945 31.05 68.95 Pos 42:13 7,65 1.003 18.3 47 31.10 68.90 Neg 44:33 6.91 1.006 46.5 182 31.91 68.09 Neg 46:43 6.72 1.007 49.7 207 31 ~97 68.03 Neg 48:13 6~65 1 ~006 36.8 175 34~31 65.69 Neg 49:43 6.88 1.017 132.0 134 34.92 65.08 Neg 50:13 6.90 1.011 68.0 161 33.45 66.55 Neg 56:44 6.62 1.018 148.7 287 35.42 64.58 Neg 64:43 7.11 1.021 214.3 221 36.21 63.79 Neg 69:43 7.32 1.019 174.5 71 38.62 61.38 Neg 72:43 8.01 1.018 107.6 13 NA NA Neg 76:43 7.21 1.022 162.0 119 41.08 58.92 Neg 80:23 6.05 1.007 47.4 89 39.92 60.08 Neg 80:44 6.17 1.012 67.3 29 46.12 53.88 Neg 85:43 6.85 1.013 104.6 64 42.84 57.16 Neg 87:13 6.01 1.012 100.7 107 43.47 56.53 Neg 90:13 6.70 1.009 65.9 45 43.28 56.72 Neg 91:13 7.66 1.005 28.1 10 NA NA Neg 95:43 6.37 1.023 208.7 90 44.07 55.93 Neg 103:43 5.85 1.021 124.1 63 44.51 55.49 Neg 111:43 5.65 1.009 64.2 21 43.93 56.07 Neg 112:43 6.69 1.002 11.7 0 NA NA Neg 113:13 7.36 1.001 8.9 0 NA NA Neg 114:13 7.25 1.004 19.8 0 NA NA Neg 116:43 6.25 1.007 35.3 12 NA NA Neg 118:43 7.77 1.005 23.5 0 NA NA Neg

' Only a single sample is presented following the last detected amphetamine for each subject. All samples for each of the subjects collected after that time contained no detectable (> 5 ng/mL) amphetamine. NA indicates enantiomer analysis was not completed because of the low concentration or lack of drug in the sample.

given the d-enantiomer is metabolized more rapidly than the 1-, the amount of/-amphetamine excreted intact increases pro- portionately over time. These results are consistent with Gunne (6), who suggested there was no effect on enantiomer ratios whether the urine was acidified or made alkaline.

Amphetamine from pharmaceutical sources in the U.S. is either only d-amphetamine or a mixture of d- and/-amphetamine (1,21). Cur- rently available mixed enantiomer amphetamines are manufactured in the ratio of 3:1 d- to l-enantiomer. As a result, the proportion of enantiomers shows the d-enantiomer to exceed the l-enantiomer. This pattern is different than would be expected from the use of illicit amphetamine. Illicit amphetamine is either only the d-enantiomer or a racemic mixture. The absence of l-enantiomer clearly would demonstrate that Adderall could not be the source of the amphetamine. Initially, racemic amphetamine results in essentially equal amounts of both enantiomers shortly after administration of the drug, followed shortly thereafter by a progressively increasing proportion of the l-enantiomer. As seen in Figure 1, following administration of Adderall, the proportion of d- to l-enantiomer did not ap- proach 1:1 until at least 72 h post dose. Four of the five subjects did not actually reach a 1:1 ratio, even after as many as 132 h post dose. Prior to that time, the d-enantiomer exceeded the l-enantiomer. It is important to note that this circumstance cannot be attained by using a single-entity amphetamine. The only sce- nario that would give this proportion would be to administer d-amphetamine and racemic amphetamine in the appropriate proportions and timing to attain those proportions. Al- though unlikely, this possibility should be considered when interpreting results.

Unlike the U.S., the illicit form of amphetamine in the U.K. is racemic (22). Some treatment regimens for amphetamine addicts use d-amphetamine as a therapeutic adjunct to assist compliance with the overall treatment program. Based on these facts, Tetlow and Mer- rill (23) used amphetamine isomer ratios to monitor compliance with treatment of amphetamine abuse with d-amphetamine. Sub- ject samples were monitored for the enantiomers of amphetamine, and it was found that those that were compliant with treatment with d-amphetamine (took the prescribed d-amphetamine and did not supplement with racemic amphetamine) all showed the l-enantiomer to be less than 30% of the d-enantiomer and, therefore, selected that percentage to

490


Table I. (continued) Creatinine, Specific Gravity, pH, Amphetamine Concentration, Enanllomer Composition, and Immunoassay Results for Samples Collected Following Administration of 20 mg of Adderall*

Hours Amphetamine % % tmmunoassay Post Dose pH Sp. Gr. Creatinine (ng/mt) /-Amp d.Amp Result

120:43 5.91 1.006 39.7 9 NA t NA Neg 121:43 5.68 1.008 40.6 12 NA NA Neg 122:13 6.68 1.01 2 64.0 0 NA NA Neg 128:13 6.79 1.015 112.2 11 NA NA Neg 129:43 6.71 1.017 151.4 7 NA NA Neg 132:43 5.93 1.013 85.3 27 45.76 54.24 Neg 134:43 5.75 1.017 177.8 ] 3 NA NA Neg 136:43 6.25 1.006 45.6 0 NA NA Neg

* Only a single sample is presented following the last detected amphetamine for each subject. All samples for each of the subjects collected after that time contained no detectable (~: S ng/mL) amphetamine,

t NA indicates enantiomer analysis was not completed because of the low concentration or lack of drug in the sample,

phetamine with illicit racemic amphetamine. The third group consisted of samples that contained > 50%, which resulted from use of illicit racemic amphetamine. Using these proportions, the authors state they are able to monitor use of prescribed d-amphetamine and determine if the individual is using the drug as prescribed, supplementing the prescribed drug with illicit drug, or not complying with the treatment regimen. These evaluations are based on the common form of amphetamine in the illicit market being racemic. As a result, the illicit amphetamine available is monitored to confirm its composition (22).

Conclusions

':] a04 L

q r - - - zQ4

go !:] ~o4

l o 4

oq

Enantiomer profiles

~ I ~ ~ . _ ~ . - -

Hours post dooe

- ~ - L-1 - m - o . 1 --~--L-~ - -N--D4 - - e - L 4 --~--1>3 -,11,-1.-4 - e - 0 4 - - t - L 4 ~ 0 . 4

Figure I. Plot of enantiomer composition of samples collected over 72 h following administration of a single 20-rag dose of Adderall to five healthy male subjects. L-n represents the percent of l-enantiomer from subject n (n = subject number I-5) and D-n represents the percent of d-enantiomer from subject n.

identify patients that were using illicit amphetamine. They also showed that patients taking d-methamphetamine sometimes also used racemic amphetamine which was demonstrated by the analytical results being consistent with d-methamphetamine use and racemic amphetamine as evidenced by the presence of substantial amounts of the l-enantiomer of amphetamine in the sample. The authors also monitored exhibits of illicit amphetamine and demonstrated d-amphetamine was not available in the illicit market. As a result, their analytical data could be interpreted quite easily. In a similar study, George and Braithwaite (22) showed the average ratio of l- to d-amphetamine from abusers to be 98% • 27.5%, compared with 15% • 4.9% in subjects deemed to be compliant. The authors' evaluation of samples resulted in them falling into three different categories. Those samples with ratios of < 20% were considered "compliant", indicating they were taking their prescribed d-amphetamine and not supplementing it with illicit racernic amphetamine. The second group (> 20% and < 50%) was consistent with supplementing their prescribed d-am-

The current study presents the first published data on the excretion of amphetamine following administration of Adderall. These data allow differentiation from both of the common forms of illicit amphetamine. The presence of the l-enantiomer of amphetamine eIiminates the possibility of the individual using medicinal or illicit d-amphetamine. Use of racemic amphetamine, which is not legally marketed in the U.S., would yield enantiomer proportions different than those found following the use of Adderall or similarly compounded mixed amphetamine. Although it is theoretically possible for an abuser to use both racemic amphetamine and the pure d-enantiomer, the likelihood of this is small because it would require that both forms of the drug be available at the same time to the same individual and for them to be taken in the right proportions.

Acknowledgments

The authors acknowledge the invaluable administrative assistance provided by Col. Mollerstrom and Dr. 5chmelz in the review, approval, and initiation of this important study. Special thanks to Cadet Rini (USAFA) for assistance with the analysis of samples in this study, particularly impressiv e given the pace at which the analysis was completed. Thanks also to Donna Hensley for her valuable assistance with processing and analysis of samples. The efforts of Major Carlisle for expeditious processing of the Adderall tablets used in this protocol are also much appreciated. Immunoassay analysis of these samples was accomplished at the Air Force Drug Testing Laboratory, and the authors wish to thank those individuals that assisted with that analysis and to Dr. Mobley and Ms. Sobecki for their administrative assistance.

491


References

1. Physician's Desk Reference, 57th ed. Medical Economics Com- pany, Montvale, NJ, 2003, pp 3138-3139.

2. E. Anggard, L.E. Jonsson, A.L. Hogmark, and L.M. Gunne. Am- phetamine metabolism in amphetamine psychosis. Clin. Phar- macoL ?'her. 14(5): 870-880 (1973).

3. M.A. Evans, G. Wimbish, L. Griffis, R. Martz, D.J. Brown, B.E. Rodda, L. Lemberger, and R.B. Fomey. Subjective responses and excretion patterns of dextroamphetamine after the administration of therapeutic doses. J. Forensic Sci. 22(1 ): 197-201 (1977).

4. M. Rowland and A.H. Beckett. The amphetamines: clinical and pharmacokinetic implications of recent studies of an assay procedure and urinary excretion in man. Arzneimittelforschung 16(11 ): 1369-1373 (1966).

5. J.M. Davis, I.J. Kopin, L. Lemberger, and J. Axelrod. Effects of urinary pH on amphetamine metabolism. Ann. N. Y Acad. Sci. 179: 493-501 (1971).

6. L.M. Gunne. The urinary output of d- and I-amphetamine in man. Biochem. PharmacoL 16:863-869 (1967).

7. M. Rowland. Amphetamine blood and urine levels in man. J. Pharm. 5ci. 58(4): 508-509 (1969).

8. A.H. Beckett and M. Rowland. Urinary excretion kinetics of amphetamine in man. J. Pharm. PharmacoL 17:628-639 (1965).

9. E. Anggard, L.M. Gunne, L.E. Jonsson, and F. Niklasson. Pharma- cokinetic and clinical studies on amphetamine dependent subjects. Eur. J. Clin. Pharmacol. 3:3-11 (1970).

10. S.H. Wan, S.B. Matin, and D.L. Azarnoff. Kinetics, salivary excretion of amphetamine isomers, and effect of urinary pH. Clin. Phar- macoL Ther. 23(5): 585-590 (1978).

11. J. Gal. Stereochemistry of metabolism of amphetamines: use of (-)- alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride for GLC resolution of chiral amines. J. Pharm. Sci. 66(2): 169-172 (t977).

12. A.H. Beckett and M. Rowland. Rhythmic urinary excretion of amphetamine in man. Nature 204(4964): 1203-1204 (1964).

13. P.S. Sever, J. Caldwell, L.G. Dring, and R.T. Williams. The metabolism of amphetamine in dependent subjects. Eur. J. Clin. Pharmacol. 6(3): 177-180 (1973).

14. A.H. Beckett, J.A. Salmon, and M. Mitchard. The relation between blood levels and urinary excretion of amphetamine under controlled acidic and under fluctuating urinary pH values using [~4C]amphetamine. J. Pharm. PharmacoL 21(4): 251-258 (1969).

15. A.H. Beckett, M. Rowland, and R Turner. Influence of urinary pH on excretion of amphetamine. Lancet 1:303 (1965).

16. L.G. Dring, R.L. Smith, and R.T. Williams. The metabolic fate of amphetamine in man and other species. Biochem. J. 116:425-435 (1970).

17. T. Spencer, J. 8iederman, T. Wilens, S. Faraone, J. Prince, K. Gerard, R. Doyle, A. Parekh, J. Kagan, and S.K. Bearman. Efficacy of a mixed amphetamine salts compound in adults with attention- deficit/hyperactivity disorder. Arch. Gen. Psychiatry 58(8): 775-782 (2001).

18. S. Valtier and J.T. Cody. Evaluation of internal standards for the analysis of amphetamine and methamphetamine. J. Anal Tox- icoL 19(6): 375-380 (1995).

19. D. Hensley and J.T. Cody. Simultaneous determination of amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), and methylenedioxyethylamphetamine (MDEA) enantiomers by GC-MS. J. Anal ToxicoL 23(6): 518-523 (1999).

20. Roche Diagnostics Corporation. Online Automated Assays for Drug Abuse: Amphetamines. Roche Diagnostics Corporation, In- dianapolis, IN, 2001, package insert.

21. Wolters Kluwer Co. Drug Facts and Comparisons. Wolters Kluwer Co, St. Louis, MO, 2002.

22. S. George and R.A. Braithwaite. Using amphetamine isomer ratios to determine the compliance of amphetamine abusers prescribed dexedrine. J. Anal ToxicoL 24(3): 223-227 (2000).

23. V.A. Tetlow and J. Merrill. Rapid determination of amphetamine stereoisomer ratios in urine by gas chromatography-mass spectrometry. Ann. Clin. Biochem. 33:50-54 (1996).

492

amphetamine enantiomer excretion profile following administration of adderall

Documents