Detection of Amphetamine and Methamphetamine Following

Journal of Analyt ical Toxicology, Vol. 22, July/August 1998

Detection of Amphetamine and Methamphetamine Following Administration of Benzphetamine*

John T. Cody* and Sandra Valtier Clinical Research Squadron, 59th Medical Wing, Lack/and AFB, Texas 78236-5319

Abstract I

Interpretation of urine drug-testing results is a challenging endeavor for several reasons. Effects of pH, dilution, legitimate and illicit sources of the drugs, and, perhaps the most challenging, the possibility of the methampbetamine and/or amphetamine being the result of the use of some other drug. Although it is known that 14 different compounds are metabolized to methamphetamine or amphetamine or both, there is little information on the metabolic profile of many of these compounds, making interpretation of results difficult. Benzphetamine, administered as a single Didrex tablet, was given to 10 subjects (7 male and 3 female) and urine samples collected for the next 7 days. Gas chromatography-mass spectrometry results showed 3 of the 10 subjects did not have a single urine sample that exceeded a 500-ng/mL cutoff for amphetamine or methamphetamine. The other subjects had between one and six samples that tested positive at or above that level. Two subjects excreted more methamphetamine than amphetamine, whereas the other eight excreted greater amounts of amphetamine than methamphetamine. The observed ratio between amphetamine and methamphetamine was significantly different than what would be expected from the use of methamphetamine. Results of this study indicate the metabolism of benzphetamine to desmethylbenzphetamine is a major pathway in the metabolism of the drug. Enantiomer analysis of the methamphetamine and amphetamine revealed only the d-enantiomer. Results of this study add significant information useful to interpret the possibility of benzphetamine as the origin of methamphetamine and amphetamine in urine samples.

Introduction

Metabolism of so-called "precursor drugs" to methamphetamine and/or amphetamine has been described for 14 different compounds used in various parts of the world (1). Published

"The views expressed in this article are those of the authors and do not reflect the official policy of the Depa~ment of Defense or other Deportments of the U.S. Government. The voluntary, fully informed consent of the subjects used in this research was obtained as required by AF140403.

* Address for correspondence: John T. Cccly, Ph.D., Commander, Clinical Research Squadron, 1255 Wilford Hall Loop, Lackland AFB, TX 78236-5319. E-mail: cody@whmc-la?o.af.mil.

reports clearly establish the metabolism to amphetamine and methamphetamine but often do not provide complete descriptions of pharmacokinetic parameters. Although these studies have significant forensic application in establishing the metabolic products of the drugs and provide some information regarding concentrations found in urine and/or tissues, limitations do exist that min- imize their utility in interpreting other cases. Many reports describe findings based on a small number of or single observa- tions, and specific details regarding the use (dose, time since administration, etc.) are not available.

Benzphetamine is used as a diet pill and is available by pre- scription in the U.S. and other countries. The recommel~ded starting dose is 25-50 mg/day. Benzphetamine has been shown to be metabolized to both methamphetamine and amphetamine (2-5). The metabolic pathway has not been well described in humans, and the number of subjects reported is small. A report by Budd and Jain (2) described the urine excretion profile of the drug and metabolites over time in a single subject given a single 20-mg dose. The maximum concentrations of amphetamine and methamphetamine reported in that case (n = 1) were 690 and 1060 ng/mL, respectively. The amount of methamphetamine exceeded amphetamine in the urine in that study, but amphetamine was reported to exceed methamphetamine concentration in two subjects in another study (5). With the small number of subjects reported in these studies, the proportion of amphetamine to methamphetamine and its variability is difficult to assess. Benzphetamine was either not detected (2) or detected at very low levels for a short period of time (3,4). In a multidose (30 rag/day for 5 days) study by Kikura and Nakahara (5), one (of two) subjects had detectable benzphetamine in a single sample collected 1 h after the fifth dose of the drug.

Materials and Methods

Materials Amphetamine, methamphetamine, amphetamine-d5 (1-phenyl-

2-aminopropane-l,2,3,3,3-ds), methamphetamine-d5 (1-phenyl-2- methyl-d3-aminopropane-l,2-d~), methamphetamine-ds

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Journal of Analytical Toxicology, Vol. 22, Jury/August 1998

Table I. Sample pH, Creatinine, Specific Gravity, Amphetamine and Metharnphetamine Concentrations, and Amphetamine- to-Methamphetamine Ratio*

Specific Crealinine Hours Concentration (ng/mL) Subject pH gravity (mg/dL) post-dose Ratio Amphetamine Methamphetamine

1 5.81 1.010 38.6 00:00 0 0 1 6.28 1.010 30.8 02:00 114 116 1 6.97 1.010 61.5 05:00 305 255 1 5.84 1.010 51,1 08:00 361 284 I 5.34 1.020 226,0 13:00 1135 753 I 5.30 1.010 74.6 17:00 419 251 I 5.73 1.010 80.3 21:00 263 141 "L 5.97 ~ .0"i 0 55,4 25:00 140 68 1 5.36 1.025 160.0 27:00 331 148 I 5.49 1.029 188.0 34:00 367 155 1 5.59 1.027 358,5 41:00 249 85 1 5.51 1.022 129.0 45:00 116 34 1 6.46 1.006 17.7 47:30 13 0 1 5.68 1.011 56.3 52:00 18 6 1 5.33 1.010 68.0 59:00 15 0 1 7.13 1.025 151,0 66:00 10 0 1 5.46 1.010 48,5 70:00 9 0 1 5.43 1.010 47.3 73:00 5 0 I 5.36 1.012 45.2 76:00 0 0 I 5.24 1,015 81.3 83".00 5 0 1 7.13 1.015 71.0 91:00 0 0 1 5.17 1.010 54.1 94:00 0 0 1 6.33 1.012 66.6 98:00 0 0 1 6.49 1.010 50.6 101:30 0 0 1 5.83 1.020 98.0 107:30 0 0 1 5.79 1.010 45.9 113:30 0 0 1 6.08 1.010 59.2 119:00 0 0 1 6.70 1.015 76.5 122:30 0 0 1 6.10 1.020 102,0 132:00 0 0 1 5.70 1.015 72,7 139:00 0 0 1 5.70 1.010 38.6 143:00 0 0

0.98 1.20 1,27 1.51 1.67 1,87 2.06 2,24 2.37 2.93 3.41

3.00

2 7.~ 3 ~ ,0~ 0 85.0 00"00 0 0 2 7.31 1.005 ] 1.3 00:30 0 0 2 7.17 1.010 15.7 01:11 60 48 1.25 2 6.96 1.010 41.4 03:30 198 114 1.74 2 6.82 1.005 29,9 05:00 151 92 1.64 2 5.72 1.017 178.0 07:30 586 248 2.36 2 5.70 1.021 206.0 09:30 646 383 1.69 2 6.10 1.011 81.4 11:30 271 159 1.70 2 5.70 1.020 149,0 17:45 537 301 1.78 2 6.00 1.019 185,0 20:00 300 170 1,76 2 6.80 1.017 46,0 21:30 76 44 1,73 2 6.20 1.020 182.0 25:30 235 133 1,77 2 5.90 1,015 260,0 27:30 154 g7 1.77 2 5.70 1.018 309,0 30:00 140 76 1,84 2 5.80 1.023 190.0 33:30 235 123 1.91 2 5.70 1.025 176.0 41:30 224 112 2,00 2 7.30 1.021 148,0 46:30 36 16 2.25 2 7.30 1.010 68.7 49:00 19 8 2.38 2 6.99 1.003 17.8 49:30 11 4 2.75 2 7.30 1.012 95.0 51:30 19 8 2.38

* Maximum measurable reading for specific gravity was 1.035. Samples that gave that reading were reported as such and not diluted and reanalyzed. LOD for amphetamine and methamphetamine was 5 nglmL. LOQ for amphetamine and methamphetamine was 5 ng/mL. Bolded numbers indicate samples were positive by 500-nglmL cutoff criterion. NT = not tested, Ratio was calculated as amphetamine divided by methamphetamine.

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Journal of Analytical Toxicology, Vol. 22, July/August 1998

Table I. continued. Sample pH, Creatinine, Specific Gravity, Amphetamine and Melhamphetamine Concentrations, and Amphetamine-to-Methamphetamine Ratio*

Specific Creatinine Hours Concentration (ng/mL) gravity (mg/dL) post-dose Subject pH Amphetamine Methamphetamine Ratio

2 6.40 1.023 227.0 56:00 63 26 2 5.50 1.027 218.0 66:00 94 35 2 6.90 1.022 136.0 70:30 17 6 2 6.50 1.029 240,0 74:30 28 10 2 6.10 1.025 242.0 78:30 39 12 2 6.20 1.010 69.1 80:00 9 0 2 5.90 1.025 228.0 83:00 29 9 2 5.60 1.025 200.0 92:30 22 8 2 5.60 1,025 169.0 94:30 15 5 2 5.60 1.015 99.5 97:00 5 0 2 5.70 1.028 268.0 102:00 10 0 2 5.60 1.035 294.0 108:30 13 5 2 5.60 1.030 175.0 111:30 7 0 2 5.70 1.030 180.0 116:30 5 0 2 6.40 1.020 125.0 118:30 0 0 2 5.80 1.020 164.0 121:30 0 0 2 5.50 1.030 219.0 126:30 0 0 2 5.50 1.025 222.0 137:30 0 0 2 6.60 1.020 128,0 142:30 0 0

2,42 2.69 2.83 2.80 3.25

3.22 2.75 3.00

2.60

3 5.80 1.010 51.2 00:00 0 0 3 5.95 1.010 26.5 02:00 116 170 0.68 3 5.65 1.010 42.0 05:00 222 312 0.71 3 6.27 1.010 47.2 07:00 159 228 0.70 3 5.76 1.010 87.4 11:30 233 335 0.70 3 6.12 1.015 270.0 14:30 259 378 0.69 3 6,43 1.010 140.2 21:30 94 132 0.71 3 6.04 1.010 34.5 24:00 72 100 0.72 3 6.44 1.010 41.4 26:00 39 70 0.56 3 6.72 1,010 38.2 27:30 31 49 0.63 3 6.64 1,010 72.1 30:00 39 64 0.61 3 6.11 1.005 45.7 33:00 35 55 0.64 3 7.22 1.008 47.8 35:00 17 28 0.61 3 7.34 1,017 246.0 38:00 9 15 0.60 3 6,32 1.009 57.0 42:30 23 36 0.64 3 5.80 1.006 55.2 45:30 32 47 0.68 3 5.83 1,007 60.4 48:00 14 20 0.70 3 6.94 1.005 27.4 49:30 6 9 0.67 3 6.98 1.005 23.9 50:30 7 9 0.78 3 7.00 1.005 20.8 52:00 6 8 0.75 3 6.08 1,010 78.6 56:00 19 27 0.70 3 6.62 1.005 23.6 57:30 8 10 0.80 3 6.00 1.010 81.0 60:30 13 19 0,68 3 6.17 1.009 71.3 63:00 9 13 0.69 3 6.46 1.007 58.1 67:00 9 10 0.90 3 5.97 1.005 37.0 69:30 8 9 0.89 3 5.53 1.005 21.9 72:30 0 0 3 5.30 1.006 37.4 74:30 0 7 0.00 3 5.50 1.010 71.5 78:00 5 7 0.71 3 5,82 1,005 64,9 82:30 0 5 0.00 3 6.72 1.005 30.4 83:30 0 0 3 7.37 1.005 31.4 86:30 0 0 3 5.82 1.015 114.0 89:00 0 0

* Maximum measurable reading for specific gravity was 1.035. Samples that gave that reading were reported as such and not diluted and reanalyzed. LOD for amphetamine and methamphetamine was 5 ng/mL, LOQ for amphetamine and methamphetamine was 5 ng/mL, golded numbers indicate samples were positive by 500-ng/mt cutoff criterion. hit = not tested. Ratio was calculated as amphetamine divided by methamphetamine,

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Table I. continued. Sample pH, Creatinine, Specific Gravity, Amphetamine and Methamphetamine Concentrations, and Amphetamine-to-Methamphetamine Ratio*

Specific Creatinine Hours Concentration (ng/mL) Subject pH gravity (mg/dL) post-dose Amphetamine Methamphetamine Ratio 3 6.05 1.010 65.6 92:00 0 0 3 5.78 1,010 75.5 97:00 0 0 3 7.28 1.010 77.3 102:30 0 0 3 5.60 1.025 280.0 111:00 O 0 3 6.45 1.010 84.5 119:00 0 0 3 5.79 1.010 NT 121:30 0 0

4 5.13 1.010 91.5 00:00 0 0 4 5.25 1.010 57.5 01:30 106 201 0.53 4 5.17 1.010 70.1 03:00 207 370 0.56 4 5.28 1.000 50.4 06:30 169 291 0,58 4 5.85 1.010 43,0 09:30 116 192 0.60 4 5.64 1.010 93.0 14:30 169 266 0.64 4 6.51 1.015 137.0 22:00 69 108 0.64 4 5.88 1.010 48.3 28:00 44 69 0.64 4 5.73 1.005 51.4 32:00 38 53 0.72 4 5.59 1.010 73.8 35:00 41 55 0.75 4 5.98 1.018 194.0 37:00 51 67 0.76 4 5.93 1.015 163.0 44:00 38 48 0.79 4 5.65 1.020 185.0 46:00 45 56 0.80 4 5.68 1.011 94.0 52:30 22 26 0.85 4 5.71 1.010 75.8 57:30 14 16 0.88 4 6.27 1.011 73.4 60:30 9 10 0,90 4 6,90 1.015 76,7 70:00 5 5 1 .O0 4 5.73 1.012 79.9 74:00 7 8 0.88 4 5.18 1.020 133.0 80:30 11 11 1.00 4 5.41 1.010 79.3 84:00 0 0 4 5.28 1.020 NT 86:00 12 11 1.09 4 5.48 1.010 87.5 90:30 0 0 4 5.17 1.015 89.0 96:00 0 0 4 5.29 1.020 195.0 103:00 0 0 4 5.48 1.010 80.1 107:00 0 0 4 5,27 1.025 222.O 11 O:00 0 0 4 5.29 1,025 224.0 119:30 0 0

5 6.05 1.017 110.0 00:00 0 0 5 5.65 1.019 140.0 04:36 1779 623 2.86 5 5.78 1.021 212.0 07:30 1914 637 3.00 5 5.99 1.016 108.0 12:30 845 268 3.15 5 6.68 1.016 124.0 17:00 438 142 3.08 5 6.80 1.011 77.2 19:25 330 107 3.08 5 7.06 1.010 74.9 22:00 80 28 2.86 5 7.03 1.015 11 O.O 25:00 65 23 2.83 5 6.75 1.015 91.5 28:10 169 55 3.07 5 5.73 1.020 149.0 37:00 692 218 3.17 5 6.69 1.015 91,5 43:10 100 33 3.03 5 6.17 1.005 59.5 45:25 106 33 3.21 5 6.60 1.010 80.9 48:25 76 24 3.17 5 5,49 1.019 151.0 60:45 192 60 3,20 5 6.40 1.017 134.0 67:00 74 23 3,22 5 6.52 1,017 157.0 73:45 40 13 3,08 5 5.63 1.020 161,0 78:30 68 22 3.09 5 5.60 1.025 13.0 84:30 80 27 2.96

* Maximum measurable reading for specific gravity was 1.035, Samples that gave that reading were reported as such and not diluted and reanalyzed. LOD for amphetamine and methamphetamine was 5 ng/mL. LOQ for amphetamine and methamphetamine was 5 ng/mL. Bolded numbers indicate samples were positive by 500-ng/mL cutoff criterion. NT = not tested. Ratio was calculated as amphetamine divided by methamphetamine.

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Specific Creatinine Hours Concentration (ng/mL) Subject pH gravity (rag/elL) post-close Ratio Amphetamine Methamphetamine 5 6.87 1.020 110.0 88:45 21 7 3.00 5 6.84 1.020 122.0 92:55 17 6 2.83

6 6.38 1.025 264.0 00:00 0 0 6 8.14 1.020 160.0 01:40 140 50 6 7,18 1.015 117.0 04:30 263 100 6 6.65 1.010 66.9 06:20 279 99 6 6.66 1.011 101.0 12:00 216 67 6 6.46 1.013 125.0 18:40 258 65 6 6.79 1.010 76.0 22:30 140 30 6 5.60 1.030 318.0 28:00 803 139 6 5.50 1.026 254.0 37:00 446 63 6 5.53 1.015 122.0 43:45 85 10 6 6.06 1.010 64.8 48:00 25 0 6 5.53 1.023 201.0 52:45 76 7 6 5.55 1.026 256.0 62:00 67 6 6 6.01 1.006 50.7 65:40 11 0 6 6.84 1.016 131.0 69:45 0 0 6 5.80 1.017 190.0 79:20 15 0 6 6.10 1.002 21.9 81:00 0 0 6 6.29 1.019 189.0 89:45 0 0 6 5.69 1.024 272.0 I00:00 9 0 6 5.50 1.014 134.0 104:15 0 0 6 6.04 1.001 15.1 105:00 0 0 6 5.49 1.007 85.0 110:00 0 0 6 5.49 1.022 238.0 118:00 0 0 6 5.54 1,014 133.0 121:30 0 0 6 5.58 1.026 316,0 133:00 0 0 6 5.79 1.029 176.0 141:30 0 0

2.80 2.63 2.82 3.22 3.97 4.67 5.78 7.08 8.50

10.86 11.17

7 5.95 1.018 75.1 00:00 0 0 7 6.01 1.020 111.5 06:30 1887 510 3.70 7 7.10 1,010 31.4 09:00 253 71 3.56 7 7.05 1.010 49.7 12:15 327 93 3.52 7 654 1,015 118.5 18:30 527 155 3.40 7 5.48 1.025 115.5 23:00 960 286 3.36 7 5.66 1.025 16.9 30:15 1468 418 3.51 7 7,09 1.020 144.0 33:30 90 26 3.46 7 7.10 1.019 81.6 35:40 75 22 3.41 7 6.09 1.025 247.0 45:45 1336 386 3.46 7 5,70 1.026 200.0 54:20 295 83 3.55 7 7.20 1.020 94.0 58:20 22 7 3.14 7 6.33 1.021 120.0 66:00 36 12 3.00 7 6.30 1.026 204.0 70:45 309 84 3.68 7 6.05 1.030 290.0 80:00 117 32 3.66 7 6.46 1.026 158.0 86:10 26 7 3.71 7 7.91 1.021 94.0 92:06 0 0 7 7.96 1.025 214.0 94:30 79 22 3.59 7 6.17 1.030 175.0 105:00 41 13 3.15 7 6.03 1.023 110,0 114:30 14 0 7 6.15 1.026 172,0 120:00 18 6 3.00 7 5.44 1.028 210.0 126:25 16 5 3.20 7 6.12 1.022 98.0 131:00 0 0 7 5.53 1.028 124.0 142:30 15 0

* Maximum measurable reading for specific gravity was 1.035. Samples that gave that reading were reported as such and not diluted and reanalyzed. LOD for amphetamine and methamphetamine was 5 ng/mL. LOQ for amphetamine and methamphetamine was 5 ng/mL Bolded numbers indicate samples were positive by 500-ng/mL cutoff criterion. NT = not tested. Ratio was calculated as amphetamine divided by methampbetamine.

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Specific Creatinine Hours Concentration (ng/mL) Subject pH gravity (mg/dL) post-dose Ratio Amphetamine Methamphetamine 8 5.43 1.023 262.0 00:00 0 0 8 6.68 1.011 115.0 23:00 361 271 1.33 8 7.02 1.023 234.0 34:30 413 349 1.18 8 5.93 1.021 177.0 47:30 294 251 1.17 8 6.29 1.015 119.0 59:00 114 93 1.23 8 7.85 1.007 35.0 63:45 13 11 1.18 8 5.98 1.019 181.0 73:10 77 66 1.17 8 7.72 1.008 50.6 87:30 0 0 8 7.84 1.015 102.0 93:30 0 0 8 5.75 1.009 62.0 106:20 9 8 1.13 8 5.72 1.027 320.0 128:00 10 10 1.00 8 5.72 1.027 320.0 139:00 7 7 1.00 8 6.71 1.011 82.0 157:00 0 0 8 6.77 1.016 93.5 167:00 0 0 8 7.01 1.005 32.6 179:00 0 ' 0 8 6.09 1.006 57.3 191:30 0 0

9 5.27 1.010 58.9 00:00 0 0 9 5.48 1.004 26.7 17:46 418 232 9 5.20 1.004 47.5 20:55 696 364 9 5.34 1.004 71.9 23:40 740 374 9 5.47 1.004 43.4 31:15 273 128 9 5.45 1.011 98.0 39:20 213 85 9 5.56 1.009 79.7 43:00 132 48 9 5.69 1.004 47.5 45:00 81 29 9 5.90 1.004 64.6 53:40 54 21 9 6.15 1.004 33.7 57:30 19 6 9 6.75 1.008 53.6 62:00 21 6 9 7.48 1.011 78.7 64:30 9 0 9 6.67 1.006 49.3 70:00 9 0 9 6.47 1.014 86.0 77:45 15 0 9 5.92 1.009 54.0 81:10 12 0 9 5.39 1.010 67.3 87:15 14 0 9 5.25 1.006 39.7 90:00 7 0 9 5.16 1.005 37.5 94:00 0 0 9 5.46 1.012 93.5 99:40 0 0 9 5.43 1.010 54.0 104:20 0 0 9 5.26 1.007 37.0 108:30 0 0 9 5.29 1.021 132.0 130:00 0 0 9 5.88 1.004 28,9 135:15 0 0 9 5.40 1.011 70.0 139:33 0 0 9 5.44 1.024 181.0 144:40 0 0 9 6.19 1.015 105.0 151:30 0 0 9 6.60 1.004 33.1 153:00 0 0 9 6.16 1.012 99.5 157:00 0 0 9 7.35 1.006 38.5 160:30 0 0

1.80 1.91 1.98 2.13 2.51 2.75 2.79 2.57 3.17 3.50

10 6.29 1.020 133.0 00:00 0 0 10 6.37 1.019 120.0 08:30 25 16 1.56 10 6.35 1.020 165.0 10:00 1343 328 4.09 10 5.85 1.024 211.5 11:30 2892 610 4.74 10 5.98 1.030 262.5 16:45 2462 596 4.13

* Maximum measurable reading for specific gravity was 1.035. Samples that gave that reading were reported as such and not diluted and reanalyzed. LOD for amphetamine and methamphetamine was 5 ng/mk LOQ for amphetamine and methamphetamine was S ng/mL. Bolded numbers indicate samples were positive by 500-nglml_ cutoff criterion. NT = not tested. Ratio was calculated as amphetamine divided by methamphetamine.

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Specific Creatinine Hours Concenlration (ng/mL) gravity (mg/dL) post-dose Subject pH Amphetamine Methamphetamine Ratio

10 5.73 1.030 243.0 19:30 2415 607 10 5.80 1.035 390.0 22:30 3776 965 10 6.12 1.030 244,5 31:30 1549 396 10 6.37 1.026 236.0 33:00 427 112 10 6.52 1.028 203.0 36:45 379 107 10 6.55 1.022 127.0 38:45 251 71 10 6.23 1.020 122.0 40:30 234 65 10 6.59 1.024 163.0 44:30 195 54 10 6.00 1.030 231.0 47:30 318 89 10 5.93 1.020 182.0 55:00 215 60 10 6.64 1.022 180.0 59:30 64 20 10 6.04 1.026 196.0 64:30 67 22 10 5.98 1.026 199.5 69:00 61 20 10 6.51 1.020 155.0 79:30 27 8 10 6.90 1.025 212.0 83:00 0 0 10 7.19 1.016 102.0 86:00 0 0 10 7.17 1.009 57.3 89:00 0 0 10 7.50 1.007 44.2 89:30 0 0 10 6.08 1.021 212.0 104:00 12 0 10 6.01 1.021 203.0 106:15 0 0 10 6.42 1.021 161.0 109:00 0 0 10 5.79 1.021 160.0 111:00 6 0 10 5.98 1.022 124.0 115:45 0 0 10 5.87 1.015 107.0 127:00 0 0 10 5.63 1.022 138.0 130:30 0 0 10 6.04 1.018 82.5 133:45 0 0 10 6.20 1.029 180.0 138:30 0 0 10 5,98 ] .026 180.0 142:00 0 0

3.98 3.91 3.91 3.81 3.54 3.54 3.60 3.61 3.57 3.58 3.20 3.05 3.05 3.38

Maximum measurable reading for specific gravity was 1.035. Samples that gave that reading were reported as such and not diluted and reanalyzed. LOD for amphetamine and methamphetamine was 5 ng/mL. LOQ for amphetamine and methamphetamine was 5 ng/mL. Bolded numbers indicate samples were positive by 500-ng/mL cutoff criterion. NT = not tested. Ratio was calculated as amphetamine divided by methamphetamine.

(1-phenyl-2-methyl-da-aminopropane-l,2,3,3,3-ds), and methamphetamine-dl] (1-phenyl-ds-2-methyl-d3-aminopropane-3,3,3-d3) were obtained from Radian Corp. Amphetamine-d6 (1-phenyl-2- aminopropane-l,l,2,3,3,3-d6) was obtained from A1Rech. d-Amphetamine, d-methamphetamine,/-amphetamine, l-methamphetamine, and benzphetamine were obtained from Sigma Chemical. The internal standards (amphetamine-d5 and methamphetamine-ds) used for enantiomeric characterization were racemic. The defivatizing reagents, heptafluorobutyric anhydride (HFBA) and N-trifluoroacetyl-l-prolyl chloride (L-TPC), were obtained from Sigma and Regis Chemical, respectively. The benzphetamine.HCl administered to experimental subjects, in the form of Didrex (Pharmacia & Upjohn), was obtained through the medical center pharmacy.

Drug administration and sample collection Fifty milligrams of benzphetamine .HC1, in the form of a

single Didrex tablet, was administered orally to 10 healthy vol- unteers (7 male and 3 female, all more than 18 years of age) with no history of amphetamine, methamphetamine, or benzphetamine use. A pre-dose urine sample was collected from each subject shortly before administration of the drug. Following

administration, urine samples were provided ad lib for seven days, and total void volume was measured to assess percentage conversion to methamphetamine and amphetamine. Following collection, samples were refrigerated until analysis. No attempt was made during this study to physiologically control urine pH.

Sample preparation and analysis Sample pH was measured using a Fisher accumet model 50 pH

meter and specific gravity was determined using an AO Scientific refractometer. Creatinine levels were determined at the Medical Center's clinical laboratory using standard clinical laboratory procedures. Gas chromatographic-mass spectrometric (GC-MS) analyses were performed using a Hewlett-Packard 5890 II GC coupled to an HP 5971 MS using a 7673 autoinjector.

Quantitative analysis. Quantitation was based on single-point calibration using a calibration standard at 500 ng/mL of amphetamine, methamphetamine, benzphetamine, and internal standard. Amphetamine and methamphetamine were quantitated using their deuterated isotopomer as internal standard. Benzphetamine was quantitated using deuterated methamphetamine. Low concentration samples were quantitated based on single-point calibration using a standard at 25 ng/mL of each

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of the analytes of interest and 50 ng/mL of the internal standards. Aliquots (2 mL) containing 500 ng/mL each of amphetamine-d6 and methamphetamine-d8 or methamphetamine-du were extracted by addition of 0.3 mL 1 M NaOH and 5 mL 1-chlorobutane. Tubes were shaken for 10 min at approximately 120 cycles per min (cpm) then centrifuged for 5 rain at approximately 1500 rpm using a Sorvall RC3C centrifuge using a H6000A rotor to separate the layers. The organic layer was transferred to a clean, dry, glass tube and the drugs back extracted by addition of

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Figure I. Chromatography of amphetamine, methamphetamine, and benzphetamine. A, Chromatography of amphetamine and amphetamine-d6 (peak I), methamphetamine and methamphetamine-du (peak 2), and benzphetamine (peak 3) from calibration standard containing 500 n~mL of each constituent. B, Chromatography of amphetamine and amphetamine- d~,, methamphetamine and methamphetamine-d11 and benzphetamine from control sample containing 50 nB/mL of the internal standards and 2 n~/mL of benzphetamine. Benzphetamine ions at m/'z 148 and 91 are shown in detail in insert. C, Chromatography of amphetamine and amphetamine-d6, methamphetamine-du, methamphetamine and benzphetamine ions at m/z 148 and 91 from a urine sample collected following administration of benzphetamine.

2.0 mL 0.15M sulfuric acid. The tubes were again shaken and centrifuged as described. The top organic layer was aspirated to waste followed by the addition of 1 mL of 1M NaOH and 5 mL 1-chlorobutane to the bottom aqueous layer. Samples were then shaken and centrifuged as described. The top organic layer was then transferred to a clean, dry glass tube to which 200 IJL of 1.0% HCI in methanol was added. Samples were then placed in a water bath (50-60~ and evaporated to dryness under a stream of nitrogen. Derivatization was accomplished by reconstitution

of the dried extract in 100 IJL of ethyl acetate, addition of 25 ]JL of HFBA, and incubation at

A 60-70~ for 15 min. The extract was then evaporated under a stream of nitrogen, reconstituted in ethyl acetate, and injected into the GC-MS. Instrumental conditions were as follows: splitless injection; injector and interface temperature, 270~ An HP-1 (12 m x 0.2-mm i.d., 0.33-1Jm film thickness) column was used with a tempera- tureprogram of 80~ for 1 min, programmed to 210~ at 20~ with a 2 rain final time. Ions monitored were as follows: m/z 240, 118, and 91 for amphetamine; 244 and 123 for amphetamine- d6; 254, 210, and 118 for methamphetamine; and 258 and 213 for methamphetamine-ds or 260 and 213 for methamphetamine-du and 91 and 148 for detection of benzphetamine (Figure 1). Each analytical batch of samples was calibrated at 500 ng/mL and analyzed with control samples at 0 ng/mL and concentrations above and below the calibration standard. The assay is linear up to 10,000 ng/mL for amphetamine and methamphetamine, with a limit of detection (LOD) of 5 ng/mL for both amphetamine and methamphetamine (6). The LOD for benzphetamine was 2 ng/mL. Acceptance criteria for the assay were as follows: mass ion ratios for all control and unknown samples were within _+ 20% of calibrator; quantitation of controls were within + 20% of target concentration; and negative control (0 ng/mL) quantitated less than the LOD, with acceptable chromatography and retention times within + 2% of calibrator. For samples with low concentrations (i.e., < 25 ng/mL), accurate quantitation was obtained by using a calibration standard at 25 ng/mL of each of the analytes of interest and 50 ng/mL of the internal standards. This allowed accurate quantitation to 5 ng/mL for each of the analytes.

Enantiomer analysis. Urine samples (2 mL) were analyzed using amphetamine-d5 and methamphetamine-d5 as internal standards. Extraction was accomplished by addition of 0.3 mL 1M NaOH and 5 mL 1-chlorobutane. Tubes were shaken for 10 rain at approximately 120 cpm then centrifuged for 5 min at approximately 1500 rpm to separate the layers. The organic layer was transferred to a clean, dry, glass tube; 50 IJL of N-trifluoroacetyl-l-prolyl chloride was added,

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and the mixture was then allowed to stand at room temperature for 15 rain. Three milliliters of 0.01M NaOH was then added, and the samples were shaken and centrifuged as described here. The organic layer was transferred, evaporated under nitrogen at 50--60~ reconstituted in ethyl acetate, and injected into the GC-MS. Instrumental conditions were as follows: splitless injection; injector temperature, 220~ interface temperature, 270~ oven temperature program, 120~ for 2 rain then 4~ to 200~ 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 is a qualitative determination of the enantiomeric composition of amphetamine and methamphetamine enantiomers. 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-enantiorner plus 0% d-enantiomer of amphetamine and methamphetamine along with a control containing no amphetamine or methamphetamine. Acceptance criteria for the assay were as follows: enantiomer ratios of the deuterated internal standards for all control and unknown samples were within + 20% of calibrator; enantiorner ratios of controls were 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.

Several samples were tested for the presence of benzphetamine, and the data indicated the drug was not present in appreciable amounts, a result consistent with previous pub-

lished reports. As a result, benzphetamine was not monitored in the remainder of the samples.

Results and Discussion

As reported previously (2-5), administration of benzphetamine results in measurable amounts of amphetamine and methamphetamine being excreted in urine. Amphetamine and methamphetamine levels found in urine following the administration of a single 50-rag oral dose of benzphetamine �9 HCI are shown in Table I. Examination of these data shows two distinctly different results with respect to the major metabolite excreted. Several subjects excreted higher amounts of methamphetamine than amphetamine (2 of 10 subjects), whereas others (8 of 10 subjects) excreted higher amounts of amphetamine than methamphetamine.

Following methamphetamine administration, the normal metabolic pathway includes demethylation of methamphetamine to amphetamine. Percentages of methamphetamine and amphetamine excreted following methamphetarnine use are dependent on urinary pH, and, on average, 43% of the dose is excreted intact in the first 24 h. Under acidic conditions, 76% is excreted intact, with 7% as amphetamine. Alkaline conditions drop the excretion rates to only 2% of the dose excreted intact and 0.1% as amphetamine (7-9). The methamphetamine derived from benzphetamine is similarly metabolized to amphetamine by the same pathway. From the proportions seen in this study, it is clear the metabolic production of amphetamine comes not just from methamphetamine, but also

Cl"~ -CH-N--CI"I2...( { } ~)

~ f ~ . ~ __ H O ~ C I . ~ . C ~ , , I . ~ . N _ _ H &,

Figure 2. Metabolic pathway for benzphetamine. A, benzphetamine; B, desmethylbenzphetamine; C, methamphetamine; D, amphetamine; E, HO-benzphetamine; F, HO-desmethylbenzphetamine; G, HO-methamphetamine; and H, HO-amphetamine.

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a substantial amount from desmethylbenzphetamine (see Figure 2 for proposed pathway). If the production of amphetamine were only from the sequential metabolism of benzphetamine to methamphetamine and then to amphetamine, the amount of amphetamine relative to methamphetamine would be consistent with that seen following administration of methamphetamine. In this case, the amount of amphetamine far exceeds the proportion expected from methamphetamine demonstrating the conversion of benzphetamine to desmethylbenzphetamine followed by conversion

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Figure 3. Enantiomeric composition of amphetamine derived from benzphetamine metabolism. A, Chromatography of/-TPC derivatized /- amphetamine (peak 1), d-amphetamine (peak 2), /-methamphetamine (peak 3), d-methamphetamine (peak 4) from calibration standard containing 500 ng/mL each of racemic drug and deuterated internal standard. B, Chromatography of amphetamine enantiomers from a urine sample collected following use of benzphetamine showing d-enantiomer only.

Table II. Percent of Benzphetamine Dose Excreted as Amphetamine and Methamphetamine

Subject Amphetamine Methamphetamine Ratio* TotaP

1 3.8% 2.1% 1.81 5.9%

2 4.4% 2.2% 2.00 6.6%

3 2.2% 2.8% 0.79 5.0%

4 1.8% 2.5% 0.72 4.3%

5 5.7% 1.7% 3.35 7.4%

6 3.3% 0.7% 4.71 4.0%

7 6.5% 1.6% 4.06 8.1%

8 2.3% 1.7% 1.35 4.0%

9 5.6% 2.5% 2.24 8.1%

10 6.2% 1.4% 4.43 7.6%

* Ratio is calculated as amphetamine divided by methamphetamine. t Total represents the combined total of amphetamine and methamphetamine

expresed as a percentage of the dose of benzphetamine.

308


to amphetamine is a major pathway in the metabolism of benzphetamine. Evidence for this metabolic pathway is seen with all subjects. Even those subjects that excreted greater amounts of methamphetamine than amphetamine showed amphetamine at a much higher percentage than expected from methamphetamine metabolism. These data demonstrate that pathways to both methamphetamine and desmethylbenzphetamine exist in all subjects and most reasonably reflect differences in enzy- matic acitivity for the demethylation versus debenzoylation in each subject. It has been shown in animal studies by Jeffery and Mannering (10) that the demethylation of benzphetamine is accomplished by both a constitutive and an inducible enzyme. It may be the results observed in this study represent differing amounts of these isoforms of the enzyme that determine the amount of benzphetamine demethylated before debenzylation. No evidence exists to suggest that age or gender would affect the enzyme distribution. Another possibility would be varying activ- ities of other enzymes in the pathway, which would account for the differences in ratios of the two drugs; however, delineation of the exact cause is beyond the scope of the present study.

Data from unpublished studies with radiolabeled benzphetamine involving 10 human subjects showed excretion of greater amounts of amphetamine than methamphetarnine by all 10 subjects (11). This study showed that total excretion of amphetamine and methamphetamine as a percentage of the parent drug ranged from 4.25 to 11.5%. The ratio of amphetamine to methamphetamine averaged 2.99.

In the present study, benzphetamine was detected, but at low concentration and only in samples collected shortly after administration of the drug (benzphetamine LOD = 2 ng/mL). Therefore, monitoring of the parent drug is of limited value in assessing the involvement of this drug. Example chromatograms are shown in Figure 1. Peak levels of methamphetamine ranged from 139 to 965 ng/mL. Amphetamine peak levels ranged from 207 to 3776 ng/mL. Concentrations from all subjects for both drugs are given in Table I. The ratio of amphetamine to methamphetamine excreted in individual samples ranged from 0.53 to 11.17 with an average of 2.4, which is dramatically different from the ratio seen when methamphetamine is used alone (Table I). The total amount of amphetamine and methamphetamine excreted as a percentage of the parent drug ranged from 4.0 to 8.1% (Table II). Using a cutoff level of 500 ng/mL for amphetamine and methamphetamine, three subjects had no positive samples. Three other subjects did not have any samples positive for methamphetamine but did have at least one sample positive for amphetamine. Positive results were not always seen shortly after administration of the drug. One subject had only one positive sample that was collected 28 h post-dose. Another subject had a sample positive more than 45 h following administration of the drug. The individual results presented in Table I include pH, specific gravity, and creatinine. The influence ofpH on the excretion of amphetamine and methamphetamine can help to interpret the fluctuations of concentration of the drugs in the urine. In addition, the effects of dilution from sample to sample can be assessed by evaluation of the specific gravity and creatinine levels reported. These data taken together can help to evaluate the drug concentrations seen, but, unfortunately, there

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is no simple formula that can be applied to account for these influences.

Enantiomer analysis of the amphetamine and methamphetamine from all subjects showed only the d-enantiomer for both drugs (see Figure 3 for example chromatograms).

merits on the metabolism of benzphetamine. Thanks also to Ms. Hensley for assistance with the processing of samples and to the Medical Center clinical laboratory staff for assistance in analysis of creatinine.

Conclusion

Interpretation of the source of amphetamine and methamphetamine in urine samples with regard to a precursor drug such as benzphetamine can be evaluated with some confidence based on the analytical results. Enantiomeric composition of the amphetamine and methamphetamine from benzphetamine shows only the d-enantiomer. Samples that contain the l-enantiomer would not be consistent with benzphetamine use. Benz- phetamine is metabolized to methamphetamine and amphetamine; samples that contained only amphetamine (unless it is at low levels representing terminal excretion of the metabolites) would be inconsistent with benzphetamine use. Concen- trations of amphetamine and methamphetamine following administration of a single dose of the drug in the urine were as high as 3776 and 965 ng/mL, respectively. The ratio of amphetamine to methamphetamine can also be very useful in interpretation. For individuals that have amphetamine levels greater than those of methamphetamine, benzphetamine is a reasonable candidate source. Likewise, samples that contain methamphetamine and amphetamine at a ratio greater than what is generally seen with methamphetamine would be consistent with the use of benzphetamine. It must also be remem- bered, however, that use of amphetamine and methamphetamine together or sequentially within a short time frame can result in the same proportions of amphetamine and methamphetarnine seen in these experimental samples.

Acknowledgments

The authors wish to thank Dr. Swanson from Pharmacia & Upjohn, Inc. for information provided on their initial experi-

References

I. J.T. Cody. Metabolic precursors to amphetamine and methamphetamine. Forensic Sci. Rev. 5:109-27 (I 993).

2. R.D. Budd and N.C. Jain. Short communication: metabolism and excretion of benzphetamine: sources of error in reporting results. J. Anal. Toxicol. 2:241 (1978).

3. A.H. Beckett, G.T. Tucker, and A.C. Moffat. Routine detection and identification in urine of stimulants and other drugs, some of which may be used to modify performance in sport. J. Pharm. Pharmacol. 19:273-294 (I 967).

4. T. Inoue and S. Suzuki. The metabolism of 1-phenyl-2-(N-methyl- N-benzylamino)propane (benzphetamine) and 1-phenyl-2-(N- methyl-N-furfurylamino)propane (furfenorex) in man. Xenobiotica 16:691-698 (1986).

5. R. Kikura and Y. Nakahara. Hair analysis for drugs of abuse. XI. Disposition of benzphetamine and its metabolites into hair and comparison of benzphetamine use and methamphetamine use by hair analysis. Biol. Pharm. Bull. 18:1694-I 699 (I 995).

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

7. R.C. Baselt and R.H. Cravey. Disposition of Toxic Drugs and Chemicals in Man, 3rd ed. Year Book Publishers, Chicago, IL, 1989.

8. A.H. Beckett and M. Rowland. Urinary excretion kinetics of methylamphetamine in man. J. Pharm. Pharmacol. 17:109S-114S (1965).

9. A.H. Beckett and M. Rowland. Urinary excretion of methylamphetamine in man. Nature 206:1260-1261 (1965).

10. E.H. Jeffery and G.J. Mannering. Interaction of constitutive and phenobarbital-induced cytochrome P-450 isozymes during the sequential oxidation of benzphetamine. Explaination for the dif- ference in benzphetamine-induced hydrogen peroxide production and 455-nm complex formation in microsomes from untreated and phenobarbital-treated rats. Mol. Pharmacol. 23" 748-757 (1983).

11. C.N.A. Swanson, Pharmacia & Upjohn, Inc., Personal communication, 1996.

Manuscript received September 15, 1997; revision received November 14, 1997.

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Detection of Amphetamine and Methamphetamine Following