Journal of Analytical Toxicology, Vol.

22, July/August 1998

Detection of Amphetamineand Methamphetamine

FollowingAdministrationof Benzphetamine*
John T. Cody* and Sandra Valtier

Clinical ResearchSquadron, 59th Medical Wing, Lack/andAFB, Texas78236-5319

Abstract

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. Resultsof 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 viewsexpressedin this article are thoseof the authorsand do not reflect the official policy of
the Depa~mentof Defenseor other Deportmentsof the U.S. Government. The voluntary, fully
informed consent of the subjects used in this researchwas obtained as required by AF140403.
* Address for correspondence:John T. Cccly, Ph.D., Commander, Clinical ResearchSquadron,
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

methamphetaminebut often do not providecompletedescriptions
of pharmacokineticparameters. Although these studies have significantforensicapplicationin establishingthe metabolicproducts
of the drugs and providesome information regarding concentrations found in urine and/or tissues, limitations do exist that minimize their utility in interpreting other cases. Many reports
describe findings based on a small number of or single observations, 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 prescription in the U.S. and other countries. The recommel~ded
starting dose is 25-50 mg/day.Benzphetamine has been shown
to be metabolizedto 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 variabilityis difficult
to assess. Benzphetaminewas either not detected (2) or detected
at very low levels for a short period of time (3,4). In a multidose
(30 rag/dayfor 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-phenyl2-aminopropane-l,2,3,3,3-ds),methamphetamine-d5(1-phenyl-2methyl-d3-aminopropane-l,2-d~), methamphetamine-ds

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

299

Journal of Analytical Toxicology,Vol. 22, Jury/August1998

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

pH

Specific
gravity

Crealinine
(mg/dL)

Hours
post-dose

1
1
1
1
I
I
I
"L
1
I
1

5.81
6.28
6.97
5.84
5.34
5.30
5.73
5.97
5.36
5.49
5.59

1.010
1.010
1.010
1.010
1.020
1.010
1.010
~.0"i0
1.025
1.029
1.027

38.6
30.8
61.5
51,1
226,0
74.6
80.3
55,4
160.0
188.0
358,5

5.51

1.022

129.0

1
1
1
1
1
1
I
I
1
1
1
1
1
1
1
1
1
1
1

6.46
5.68
5.33
7.13
5.46
5.43
5.36
5.24
7.13
5.17
6.33
6.49
5.83
5.79
6.08
6.70
6.10
5.70
5.70

1.006
1.011
1.010
1.025
1.010
1.010
1.012
1,015
1.015
1.010
1.012
1.010
1.020
1.010
1.010
1.015
1.020
1.015
1.010

17.7
56.3
68.0
151,0
48,5
47.3
45.2
81.3
71.0
54.1
66.6
50.6
98.0
45.9
59.2
76.5
102,0
72,7
38.6

00:00
02:00
05:00
08:00
13:00
17:00
21:00
25:00
27:00
34:00
41:00
45:00
47:30
52:00
59:00
66:00
70:00
73:00
76:00
83".00
91:00
94:00
98:00
101:30
107:30
113:30
119:00
122:30
132:00
139:00
143:00

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2

7.~ 3
7.31
7.17
6.96
6.82
5.72
5.70
6.10
5.70
6.00
6.80
6.20
5.90
5.70
5.80
5.70
7.30
7.30
6.99
7.30

~,0~ 0
1.005
1.010
1.010
1.005
1.017
1.021
1.011
1.020
1.019
1.017
1.020
1,015
1.018
1.023
1.025
1.021
1.010
1.003
1.012

85.0
] 1.3
15.7
41.4
29,9
178.0
206.0
81.4
149,0
185,0
46,0
182.0
260,0
309,0
190.0
176.0
148,0
68.7
17.8
95.0

00"00
00:30
01:11
03:30
05:00
07:30
09:30
11:30
17:45
20:00
21:30
25:30
27:30
30:00
33:30
41:30
46:30
49:00
49:30
51:30

Concentration(ng/mL)
Amphetamine
Methamphetamine
0
114
305
361

0
116
255
284

1135

753

419
263
140
331
367
249

251
141
68
148
155
85

116

34

13
18
15
10
9
5
0
5
0
0
0
0
0
0
0
0
0
0
0

0
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
0
60
198
151
586
646
271
537
300
76
235
154
140
235
224
36
19
11
19

0
0
48
114
92
248
383
159
301
170
44
133
g7
76
123
112
16
8
4
8

Ratio

0.98
1.20
1,27
1.51
1.67
1,87
2.06
2,24
2.37
2.93
3.41
3.00

1.25
1.74
1.64
2.36
1.69
1.70
1.78
1,76
1,73
1,77
1.77
1,84
1.91
2,00
2.25
2.38
2.75
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.

300

Journalof AnalyticalToxicology,Vol. 22, July/August1998

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

pH

Specific
gravity

Creatinine
(mg/dL)

Hours
post-dose

Concentration(ng/mL)
Amphetamine
Methamphetamine Ratio

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2

6.40
5.50
6.90
6.50
6.10
6.20
5.90
5.60
5.60
5.60
5.70
5.60
5.60
5.70
6.40
5.80
5.50
5.50
6.60

1.023
1.027
1.022
1.029
1.025
1.010
1.025
1.025
1,025
1.015
1.028
1.035
1.030
1.030
1.020
1.020
1.030
1.025
1.020

227.0
218.0
136.0
240,0
242.0
69.1
228.0
200.0
169.0
99.5
268.0
294.0
175.0
180.0
125.0
164.0
219.0
222.0
128,0

56:00
66:00
70:30
74:30
78:30
80:00
83:00
92:30
94:30
97:00
102:00
108:30
111:30
116:30
118:30
121:30
126:30
137:30
142:30

63
94
17
28
39
9
29
22
15
5
10
13
7
5
0
0
0
0
0

26
35
6
10
12
0
9
8
5
0
0
5
0
0
0
0
0
0
0

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3

5.80
5.95
5.65
6.27
5.76
6.12
6,43
6.04
6.44
6.72
6.64
6.11
7.22
7.34
6,32
5.80
5.83
6.94
6.98
7.00
6.08
6.62
6.00
6.17
6.46
5.97
5.53
5.30
5.50
5,82
6.72
7.37
5.82

1.010
1.010
1.010
1.010
1.010
1.015
1.010
1.010
1.010
1,010
1,010
1.005
1.008
1,017
1.009
1.006
1,007
1.005
1.005
1.005
1,010
1.005
1.010
1.009
1.007
1.005
1.005
1.006
1.010
1,005
1.005
1.005
1.015

51.2
26.5
42.0
47.2
87.4
270.0
140.2
34.5
41.4
38.2
72.1
45.7
47.8
246.0
57.0
55.2
60.4
27.4
23.9
20.8
78.6
23.6
81.0
71.3
58.1
37.0
21.9
37.4
71.5
64,9
30.4
31.4
114.0

00:00
02:00
05:00
07:00
11:30
14:30
21:30
24:00
26:00
27:30
30:00
33:00
35:00
38:00
42:30
45:30
48:00
49:30
50:30
52:00
56:00
57:30
60:30
63:00
67:00
69:30
72:30
74:30
78:00
82:30
83:30
86:30
89:00

0
116
222
159
233
259
94
72
39
31
39
35
17
9
23
32
14
6
7
6
19
8
13
9
9
8
0
0
5
0
0
0
0

0
170
312
228
335
378
132
100
70
49
64
55
28
15
36
47
20
9
9
8
27
10
19
13
10
9
0
7
7
5
0
0
0

2,42
2.69
2.83
2.80
3.25
3.22
2.75
3.00

2.60

0.68
0.71
0.70
0.70
0.69
0.71
0.72
0.56
0.63
0.61
0.64
0.61
0.60
0.64
0.68
0.70
0.67
0.78
0.75
0.70
0.80
0,68
0.69
0.90
0.89
0.00
0.71
0.00

* Maximummeasurablereadingfor specificgravitywas1.035.Samplesthatgavethatreadingwerereportedassuchand notdilutedand reanalyzed.LOD for amphetamineand

methamphetaminewas5 ng/mL,LOQfor amphetamineand methamphetaminewas5 ng/mL,goldednumbersindicatesampleswerepositiveby 500-ng/mtcutoffcriterion.
hit = nottested.Ratiowascalculatedasamphetaminedividedby methamphetamine,

301

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

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

pH

Specific
gravity

Creatinine
(mg/dL)

Hours
post-dose

Concentration(ng/mL)
Amphetamine
Methamphetamine Ratio

3
3
3
3
3
3

6.05
5.78
7.28
5.60
6.45
5.79

1.010
1,010
1.010
1.025
1.010
1.010

65.6
75.5
77.3
280.0
84.5
NT

92:00
97:00
102:30
111:00
119:00
121:30

0
0
0
O
0
0

0
0
0
0
0
0

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4

5.13
5.25
5.17
5.28
5.85
5.64
6.51
5.88
5.73
5.59
5.98
5.93
5.65
5.68
5.71
6.27
6,90
5.73
5.18
5.41
5.28
5.48
5.17
5.29
5.48
5,27
5.29

1.010
1.010
1.010
1.000
1.010
1.010
1.015
1.010
1.005
1.010
1.018
1.015
1.020
1.011
1.010
1.011
1.015
1.012
1.020
1.010
1.020
1.010
1.015
1.020
1.010
1.025
1,025

91.5
57.5
70.1
50.4
43,0
93.0
137.0
48.3
51.4
73.8
194.0
163.0
185.0
94.0
75.8
73.4
76,7
79.9
133.0
79.3
NT
87.5
89.0
195.0
80.1
222.O
224.0

00:00
01:30
03:00
06:30
09:30
14:30
22:00
28:00
32:00
35:00
37:00
44:00
46:00
52:30
57:30
60:30
70:00
74:00
80:30
84:00
86:00
90:30
96:00
103:00
107:00
11O:00
119:30

0
106
207
169
116
169
69
44
38
41
51
38
45
22
14
9
5
7
11
0
12
0
0
0
0
0
0

0
201
370
291
192
266
108
69
53
55
67
48
56
26
16
10
5
8
11
0
11
0
0
0
0
0
0

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5

6.05
5.65
5.78
5.99
6.68
6.80
7.06
7.03
6.75
5.73
6.69
6.17
6.60
5,49
6.40
6.52
5.63
5.60

1.017
1.019
1.021
1.016
1.016
1.011
1.010
1.015
1.015
1.020
1.015
1.005
1.010
1.019
1.017
1,017
1.020
1.025

110.0
140.0
212.0
108.0
124.0
77.2
74.9
11O.O
91.5
149.0
91,5
59.5
80.9
151.0
134.0
157.0
161,0
13.0

00:00
04:36
07:30
12:30
17:00
19:25
22:00
25:00
28:10
37:00
43:10
45:25
48:25
60:45
67:00
73:45
78:30
84:30

0
1779
1914
845
438
330
80
65
169
692
100
106
76
192
74
40
68
80

0
623
637
268
142
107
28
23
55
218
33
33
24
60
23
13
22
27

0.53
0.56
0,58
0.60
0.64
0.64
0.64
0.72
0.75
0.76
0.79
0.80
0.85
0.88
0,90
1.O0
0.88
1.00
1.09

2.86
3.00
3.15
3.08
3.08
2.86
2.83
3.07
3.17
3.03
3.21
3.17
3,20
3,22
3,08
3.09
2.96

* Maximummeasurablereadingfor specificgravitywas 1.035,Samplesthatgavethatreadingwerereportedassuchand not dilutedand reanalyzed.LOD for amphetamineand

methamphetaminewas 5 ng/mL.LOQ for amphetamineand methamphetaminewas 5 ng/mL.Boldednumbersindicatesampleswerepositiveby 500-ng/mLcutoffcriterion.
NT = not tested.Ratiowascalculatedasamphetaminedividedby methamphetamine.

302

Journalof Analytical Toxicology,Vol. 22, July/August1998

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

pH

Specific
gravity

Creatinine
(rag/elL)

Hours
post-close

Concentration(ng/mL)
Amphetamine
Methamphetamine Ratio

5
5

6.87
6.84

1.020
1.020

110.0
122.0

88:45
92:55

21
17

7
6

6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6

6.38
8.14
7,18
6.65
6.66
6.46
6.79
5.60
5.50
5.53
6.06
5.53
5.55
6.01
6.84
5.80
6.10
6.29
5.69
5.50
6.04
5.49
5.49
5.54
5.58
5.79

1.025
1.020
1.015
1.010
1.011
1.013
1.010
1.030
1.026
1.015
1.010
1.023
1.026
1.006
1.016
1.017
1.002
1.019
1.024
1.014
1.001
1.007
1.022
1,014
1.026
1.029

264.0
160.0
117.0
66.9
101.0
125.0
76.0
318.0
254.0
122.0
64.8
201.0
256.0
50.7
131.0
190.0
21.9
189.0
272.0
134.0
15.1
85.0
238.0
133.0
316,0
176.0

00:00
01:40
04:30
06:20
12:00
18:40
22:30
28:00
37:00
43:45
48:00
52:45
62:00
65:40
69:45
79:20
81:00
89:45
I00:00
104:15
105:00
110:00
118:00
121:30
133:00
141:30

0
140
263
279
216
258
140
803
446
85
25
76
67
11
0
15
0
0
9
0
0
0
0
0
0
0

0
50
100
99
67
65
30
139
63
10
0
7
6
0
0
0
0
0
0
0
0
0
0
0
0
0

7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7

5.95
6.01
7.10
7.05
654
5.48
5.66
7,09
7.10
6.09
5,70
7.20
6.33
6.30
6.05
6.46
7.91
7.96
6.17
6.03
6.15
5.44
6.12
5.53

1.018
1.020
1,010
1.010
1,015
1.025
1.025
1.020
1.019
1.025
1.026
1.020
1.021
1.026
1.030
1.026
1.021
1.025
1.030
1.023
1.026
1.028
1.022
1.028

75.1
111.5
31.4
49.7
118.5
115.5
16.9
144.0
81.6
247.0
200.0
94.0
120.0
204.0
290.0
158.0
94.0
214.0
175.0
110,0
172,0
210.0
98.0
124.0

00:00
06:30
09:00
12:15
18:30
23:00
30:15
33:30
35:40
45:45
54:20
58:20
66:00
70:45
80:00
86:10
92:06
94:30
105:00
114:30
120:00
126:25
131:00
142:30

0
1887
253
327
527
960
1468
90
75
1336
295
22
36
309
117
26
0
79
41
14
18
16
0
15

0
510
71
93
155
286
418
26
22
386
83
7
12
84
32
7
0
22
13
0
6
5
0
0

3.00
2.83

2.80
2.63
2.82
3.22
3.97
4.67
5.78
7.08
8.50

10.86
11.17

3.70
3.56
3.52
3.40
3.36
3.51
3.46
3.41
3.46
3.55
3.14
3.00
3.68
3.66
3.71
3.59
3.15
3.00
3.20

* Maximummeasurablereadingfor specificgravitywas1.035.Samplesthatgavethatreadingwerereportedassuchand notdilutedand reanalyzed.LOD for amphetamineand

methamphetaminewas5 ng/mL.LOQfor amphetamineand methamphetaminewas5 ng/mLBoldednumbersindicatesampleswerepositiveby 500-ng/mLcutoffcriterion.
NT = not tested.Ratiowascalculatedasamphetaminedividedby methampbetamine.

303

Journalof Analytical Toxicology,Vol. 22, July/August1998

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

pH

Specific
gravity

Creatinine
(mg/dL)

8
8
8
8
8
8
8
8

5.43
6.68
7.02
5.93
6.29
7.85
5.98
7.72

1.023
1.011
1.023
1.021
1.015
1.007
1.019
1.008

262.0
115.0
234.0
177.0
119.0
35.0
181.0
50.6

7.84

1.015

102.0

8
8
8
8
8
8
8

5.75
5.72
5.72
6.71
6.77
7.01
6.09

1.009
1.027
1.027
1.011
1.016
1.005
1.006

9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9

5.27
5.48
5.20
5.34
5.47
5.45
5.56
5.69
5.90
6.15
6.75
7.48
6.67
6.47
5.92
5.39
5.25
5.16
5.46
5.43
5.26
5.29
5.88
5.40
5.44
6.19
6.60
6.16
7.35

10
10
10
10
10

6.29
6.37
6.35
5.85
5.98

Hours
post-dose

Concentration(ng/mL)
Amphetamine
Methamphetamine

62.0
320.0
320.0
82.0
93.5
32.6
57.3

00:00
23:00
34:30
47:30
59:00
63:45
73:10
87:30
93:30
106:20
128:00
139:00
157:00
167:00
179:00
191:30

0
361
413
294
114
13
77
0
0
9
10
7
0
0
0 '
0

0
271
349
251
93
11
66
0
0
8
10
7
0
0
0
0

1.010
1.004
1.004
1.004
1.004
1.011
1.009
1.004
1.004
1.004
1.008
1.011
1.006
1.014
1.009
1.010
1.006
1.005
1.012
1.010
1.007
1.021
1.004
1.011
1.024
1.015
1.004
1.012
1.006

58.9
26.7
47.5
71.9
43.4
98.0
79.7
47.5
64.6
33.7
53.6
78.7
49.3
86.0
54.0
67.3
39.7
37.5
93.5
54.0
37.0
132.0
28,9
70.0
181.0
105.0
33.1
99.5
38.5

00:00
17:46
20:55
23:40
31:15
39:20
43:00
45:00
53:40
57:30
62:00
64:30
70:00
77:45
81:10
87:15
90:00
94:00
99:40
104:20
108:30
130:00
135:15
139:33
144:40
151:30
153:00
157:00
160:30

0
418

0
232
364
374
128
85
48
29
21
6
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

1.020
1.019
1.020
1.024
1.030

133.0
120.0
165.0
211.5
262.5

00:00
08:30
10:00
11:30
16:45

0
25

696
740
273
213
132
81
54
19
21
9
9
15
12
14
7
0
0
0
0
0
0
0
0
0
0
0
0

1343
2892
2462

0
16
328
610
596

Ratio
1.33
1.18
1.17
1.23
1.18
1.17

1.13
1.00
1.00

1.80
1.91
1.98
2.13
2.51
2.75
2.79
2.57
3.17
3.50

1.56
4.09
4.74
4.13

* Maximummeasurablereadingfor specificgravitywas1.035.Samplesthatgavethatreadingwerereportedassuchand not dilutedand reanalyzed.LOD for amphetamineand

methamphetaminewas5 ng/mkLOQfor amphetamineandmethamphetaminewasSng/mL.Boldednumbersindicatesampleswerepositiveby 500-nglml_cutoffcriterion.
NT = nottested.Ratiowascalculatedasamphetaminedividedby methamphetamine.

304

Journal of AnalyticalToxicology,Vol. 22, July/August1998

Table I. continued. Sample pH, Creatinine, Specific Gravity, Amphetamine and Methamphetamine Concentrations, and
Amphetamine-to-Methamphetamine Ratio*
Subject
10
10
10

pH
5.73
5.80
6.12

Specific
gravity
1.030
1.035
1.030

Creatinine
(mg/dL)
243.0
390.0

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

6.37
6.52
6.55
6.23
6.59
6.00
5.93
6.64
6.04
5.98
6.51
6.90
7.19
7.17
7.50

1.026
1.028
1.022
1.020
1.024
1.030
1.020
1.022
1.026
1.026
1.020
1.025
1.016
1.009
1.007

244,5
236.0
203.0
127.0
122.0
163.0
231.0
182.0
180.0
196.0
199.5
155.0
212.0
102.0
57.3
44.2

10

6.08

1.021

10

6.01

1.021

10

6.42

10
10
10
10
10
10
10

5.79
5.98
5.87
5.63
6.04
6.20
5,98

Hours
post-dose
19:30
22:30
31:30

Concenlration(ng/mL)
Amphetamine
Methamphetamine
2415
607
3776
965
1549
396

33:00
36:45
38:45
40:30
44:30
47:30
55:00
59:30
64:30
69:00
79:30
83:00
86:00
89:00
89:30

427
379
251
234
195
318
215
64
67
61
27
0
0
0
0

112
107
71
65
54
89
60
20
22
20
8
0
0
0
0

212.0

104:00

12

203.0

106:15

1.021

161.0

109:00

1.021
1.022
1.015
1.022
1.018
1.029
] .026

160.0
124.0
107.0
138.0
82.5
180.0
180.0

111:00
115:45
127:00
130:30
133:45
138:30
142:00

6
0
0
0
0
0
0

0
0
0
0
0
0
0

Ratio
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

Maximummeasurablereadingforspecificgravitywas1.035.Samplesthatgavethatreadingwerereportedassuchand notdilutedandreanalyzed.LODforamphetamineand

methamphetaminewas5 ng/mL.LOQfor amphetamineandmethamphetaminewas5 ng/mL.Boldednumbersindicatesampleswerepositiveby 500-ng/mLcutoffcriterion.

NT= nottested.Ratiowascalculatedasamphetaminedividedby 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-2aminopropane-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 volunteers (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
305

Journal of Analytical Toxicology,Vol. 22, July/August1998

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. Tubeswere 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 H6000Arotor
to separate the layers. The organic layer was transferred to a
clean, dry, glass tube and the drugs back extracted by addition of

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 evaporatedto 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
75000
A
60-70~ for 15 min. The extract was then evapo3
rated under a stream of nitrogen, reconstituted in
60000
ethyl acetate, and injected into the GC-MS.
Instrumental conditions were as follows:splitless
45000
injection; injector and interface temperature,
270~ An HP-1 (12 m x 0.2-mm i.d., 0.33-1Jm
2
30000
film thickness) column was used with a temperatureprogram of 80~ for 1 min, programmed to
15000
210~ at 20~
with a 2 rain final time. Ions
monitored
were
as
follows:
m/z 240, 118, and 91
=
r~
~
.
.
.
.
0
for amphetamine; 244 and 123 for amphetamine4.00
8.0o
8.00
10100
d6; 254, 210, and 118 for methamphetamine; and
258 and 213 for methamphetamine-ds or 260 and
320000
B
213 for methamphetamine-du and 91 and 148
for detection of benzphetamine (Figure 1). Each
analytical
batch of samples was calibrated at 500
240000
ng/mL
and
analyzed with control samples at 0
4ooot~z 91.oo~ ~
ng/mL and concentrations above and below the
160000
calibration standard. The assay is linear up to
10,000 ng/mL for amphetamine and metham8.00
9.00
10.00
phetamine, with a limit of detection (LOD) of 5
80000
ng/mL for both amphetamine and methamphetamine (6). The LOD for benzphetamine was 2
-A A
4.00
8.00
ng/mL. Acceptance criteria for the assay were as
8.00
~oloo
follows: mass ion ratios for all control and
unknown samples were within _+ 20% of cali1400000
C
brator; quantitation of controls were within +
1200000
20% of target concentration; and negative control
20000
(0 ng/mL) quantitated less than the LOD, with
1000000
15000
acceptable chromatography and retention times
10000
800000
within + 2% of calibrator. For samples with low
5000
concentrations (i.e., < 25 ng/mL),accurate quan600000
0
titation was obtained by using a calibration stan8,00
9.00
10.00
400000
dard at 25 ng/mL of each of the analytes of interest
and 50 ng/mL of the internal standards. This
200000
allowed accurate quantitation to 5 ng/mL for each
i
A
A
0
of the analytes.
4.00
8.oo
8.00
~ 10100
Time (min)
Enantiomer analysis. Urine samples (2 mL)
Figure I. Chromatography of amphetamine, methamphetamine, and benzphetamine. A,
were analyzed using amphetamine-d5 and
Chromatography of amphetamine and amphetamine-d6 (peak I), methamphetamine and
methamphetamine-d5 as internal standards.
methamphetamine-du (peak 2), and benzphetamine (peak 3) from calibration standard conExtraction was accomplished by addition of 0.3
taining 500 n~mL of each constituent. B, Chromatography of amphetamine and amphetaminemL 1M NaOH and 5 mL 1-chlorobutane. Tubes
d~,, methamphetamine and methamphetamine-d11 and benzphetamine from control sample
were shaken for 10 rain at approximately 120
containing 50 nB/mL of the internal standardsand 2 n~/mL of benzphetamine. Benzphetamine
cpm then centrifuged for 5 min at approximately
ions at m/'z 148 and 91 are shown in detail in insert. C, Chromatography of amphetamine and
1500 rpm to separate the layers. The organic layer
amphetamine-d6, methamphetamine-du, methamphetamine and benzphetamine ions at m/z
was
transferred to a clean, dry, glass tube; 50 IJL
148 and 91 from a urine sample collected following administration of benzphetamine.
of N-trifluoroacetyl-l-prolyl chloride was added,

. H

306

_ _

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

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 layerwas 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 methamphetamineenantiomers.
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% lenantiomer 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-

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

lished reports. As a result, benzphetamine was not monitored in

the remainder of the samples.

Resultsand Discussion
As reported previously(2-5), administration of benzphetamine
results in measurable amounts of amphetamine and methamphetamine being excreted in urine. Amphetamine and methamphetamine levelsfound in urine followingthe administration of a
single50-rag oral doseof benzphetamine9HCIare shown in TableI.
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

} ~)

~f~.~

__ HO~CI.~.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.

307

Journal of Analytical Toxicology,Vol. 22, July/August1998

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 followedby conversion
6ooo[

314.

4000 J

~176
20OO

14:00

16~OO

18.OO

20100

22.00

24.00

25OOO t

20ooo1
~, lsooo1
1000O"~

14.00

16.00

18.00
Time

20.00
(rain)

22.00

24,00

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, Chromatographyof amphetamine enantiomersfrom 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
2

3.8%
4.4%

2.1%
2.2%

1.81
2.00

5.9%
6.6%

3
4
5
6
7

2.2%
1.8%
5.7%
3.3%
6.5%

2.8%
2.5%
1.7%
0.7%
1.6%

0.79
0.72
3.35
4.71
4.06

5.0%
4.3%
7.4%
4.0%
8.1%

8
9
10

2.3%
5.6%
6.2%

1.7%
2.5%
1.4%

1.35
2.24
4.43

4.0%
8.1%
7.6%

* Ratio is calculatedas amphetaminedivided by methamphetamine.

t Totalrepresentsthe combinedtotal of amphetamineand methamphetamine
expresedas a percentageof the doseof 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 showedamphetamine
at a much higher percentage than expected from methamphetamine metabolism. These data demonstrate that pathways
to both methamphetamine and desmethylbenzphetamineexist
in all subjects and most reasonably reflect differences in enzymatic acitivity for the demethylation versus debenzoylation in
each subject. It has been shown in animal studies by Jefferyand
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 evidenceexists to suggest that age or gender would affectthe
enzyme distribution. Another possibilitywould be varying activities of other enzymes in the pathway,which would account for
the differencesin 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 individualsamples 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 followingadministration
of the drug. The individual results presented in Table I include
pH, specificgravity, 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

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

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.

References
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. Benzphetamine is metabolized to methamphetamine and
amphetamine;samples that contained only amphetamine (unless
it is at low levelsrepresenting terminal excretion of the metabolites) would be inconsistent with benzphetamine use. Concentrations 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 generallyseen with methamphetaminewould be consistent with the use of benzphetamine. It must also be remembered, 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-

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

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