Journal of Analytical Toxicology,Vol. 28, September2004
Enantiomeric Separationand Quantitation of
(_+)-Amphetamine,(_+)-Methamphetamine,(_+)-MDA,
(_+)-MDMA, and (_+)-MDEAin Urine Specimensby
GC-EI-MS after Derivatization with (R)-(-)- or
(S)-(+)- Methoxy-(x-(trifluoromethy)phenylacetyl
Chloride (MTPA)*
Buddha D. Pault, John Jemionek, David Lesser, and Aaron Jacobs
Division of Forensic Toxicology, Office of the Armed ForcesMedical Examiner, Armed Forces Institute of Pathology,
Rockville, Maryland 20850
Douglas A. Searles
Navy Drug ScreeningLaboratory, San Diego, California 92134
Abstract[
In drug testing, the presence of methamphetamine in urine is
generally confirmed by a gas chromatography-mass spectrometry
(GC-MS) method. Derivatization of the compound to a
perfluoroalkylamide, prior to confirmation, typically yields better
chromatographic separation. Once methamphetamine is detected,
a second GC-MS test is necessary to distinguish positive results
from the use of over-the-counter medication, Vicks inhaler, or
from use of a prescription drug, selegiline (Deprenyl).
R-(-)-Methamphelamine is the urinary product from legitimate
use of these medications. The second GC-MS test is to confirm
illicit use of (S)-(+)-methamphetamine. In the procedure, the
two methamphetamine isomers are changed to the
chromatographically separable diastereomers by a chiral
derivatizing agent, (S)-(-)-trifluoroacetylprolyl chloride (TPC).
But the method has inherent limitations. Racemization of the
reagent produces mixed diastereomers even from pure
(S)-(+)-methamphetamine. Instead of using TPC, we utilized
(R)-(-)-~methoxy-o,-(trifluoromethyl)phenylacetyl chloride
(MTPA) to prepare the amides of diastereomers of
methamphetamine. No racemization was observed with this
reagent. The method was extended to resolve GC peaks of
(R)-(-)- and (S)-(+)-isomers of amphetamine, 3,4methylenedioxyamphetamine (MDA), N-methyI-MDA (MDMA),
and N-ethyI-MDA (MDEA). Three ions from the drug and two ions
from the deuterated internal standard were monitored to
characterize and quantitate the drugs. For MDEA, only one ion
was used. The quantitation was linear over 25 to 5000 ng/mL for
9 The opinions expressed herein are those of the authors and are not to be construed as the
official or as reflecting the views of the Department of the Army or the Department of
Defense.
t Author to whom correspondence should be addressed. E-mail: [email protected].
MDEA and 25 to 10,000 ng/mt for all other drugs. Correlation
coefficients were > 0.996. Precision calculated as the coefficient of
variation at the calibrator concentration of 500 ng/mL was within
+ 11% for all drugs. The method was applied to test 43 urine
specimens. In 91% of the methamphetamine-positive specimens,
only the (S)-(+)-isomer was detected. In all MDMA-positive
specimens, the concentrations of (R)-(-)-isomer were greater than
the (S)-(+)-isomer indicating longer retention of (R)-(-)-isomer in
the human body. The specimen concentrations (R + S)
compared well with that of a non-chiral method that used
4-carboethoxyhexafluorobutyryl chloride as derivatizing agent. But
the MTPA method has some advantage. It alone can replace the
two GC-MS methods needed to confirm the presence of
(S)-(+)-isomers of amphetamine and methamphetamine.
Introduction
Amphetamines are psychostimulants and widely abused. In
many countries their uses are countered by urine drug testing.
Initially an immunoassay method is used to screen the specimens, and if positive, the presence of the drugs is confirmed by
a gas chromatography-mass spectrometric method (GC-MS).
For better chromatographic resolution the amphetamines are
generally derivatized to amides prior the confirmation (1-12).
Sometime, an additional test is necessary to distinguish positive results from the over-the-counter medication (Vicks inhaler) or from the use of a prescription drug, selegiline (Deprenyl). (R)-(-)-Methamphetamine (R-meth) is the urinary
compound from use of these medications. The second test is to
Reproduction(photocopying)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.
449
Journal of Analytical Toxicology, Vol. 28, September 2004
confirm the illicit use of (S)-(+)-methamphetamine (S-meth).
In a typical test, the R- and S-meth are derivatized to the
diastereomers by (S)-(-)-N-trifluoroacetyprolylchloride (TPC)
and then separated and confirmed by a GC-MS method. But
the TPC-procedure has limitations. It is difficult to get pure
(S)-TPC because the compound tends to equilibrate with the
(R)-(+)-isomer. Apparent purity varied from 85 to 95% (13).
This is evident from the reaction products of pure (S)-meth
with (S)-TPC. Instead of (S)-meth-(S)-TPC as the only compound, some (S)-meth-(R)-TPC is formed. The GC peak of
(S)-meth-(R)-TPC appeared at the same retention time of
(R)-meth-(S)-TPC making it difficult to identify the optical
isomers of methamphetamine. Therefore, instead of reporting
the concentration of (S)-meth-(S)-TPC, the results are reported as percent of total isomers. In the U.S. military protocol,
the minimum amount of (S)-meth-(S)-TPChas to be 20% for
a specimen to be called positive for methamphetamine. We
experiencedsimilar resolution problem with optical isomers of
other amphetamines.
The reagent TPC contains an (x-proton on the chiral-carbon
atom. The isomerization (racemization) of TPC is largely due
to keto-enol tautomerization of the u-proton with the neighboring carbonyl group. Storage and reaction conditions may
also influence the racemization. To avoid racemization, it
would be prudent to search for a derivatizing agent that would
be free of the (x-protonon the chiral-carbon and then study its
effect on separation of the optical isomers. In this report we
describe the uses of (R)-(-)- or (S)-(+)-(x-methoxy-(x-(trifluoromethy)phenylacetyl chloride (MTPA)inseparation and detection of (R)- and (S)-amphetamines, including 3,4-methylenedioxyamphetamine (MDA), N-methyl-MDA (MDMA), and
N-ethyl-MDA(MDEA).
Materials and Methods
Chemicals, reagents, and supplies
All drugs and internal standards were purchased from Cerilliant (Round Rock, TX). (R)-(-)- or (S)-(+)-(x-Methoxy-(x-(trifluoromethy)phenylacetylchloride (MTPA)of 99+% purity was
purchased from Fluka Chemical Corporation (Milwaukee,WI).
All solvents and reagents were of analytical or HPLC grade.
Acetonitrile was dried over molecular sieves type 3A beads for
24 h.
Equipment
A GC-MS system consisting of an HP 5890 GC series II
plus and HP 5972 mass selective detector (MSD) from Agilent
Technology (Palo Alto, CA) was used. Helium passed through
a tube with an oxygen scrubber was used as the carrier gas.
The head pressure on the capillary column (15 m x 0.25-mm
i.d., 0.20-1Jm df, 5% phenyl polysiloxane, J&W Scientific,
Rancho Cordova, CA)was 10 psi. The instrument was operated
in splitless and temperature program modes. To avoid peak
tailing, the purge valvewas turned on at 0.40 min after sample
injection. The MSD was operated in electron impact mode at
70 eV.
450
Extraction
The (R)-(-)- and (S)-(+)- drug standards were prepared at
concentrations 250, 500, and 1000 ng/mL by adding 50, 100,
and 200 IJL of a mixture of (_+)-amphetamine, (_+)-methamphetamine, (_+)-MDA,(_+)-MDMA,and (+)-MDEA (20 IJg/mL
in 0.1M HCl) in 2 mL of drug-free urine. Asolution of (-+)-amphetamine-ds, (_+)-methamphetamine-d14,(_+)-MDA-ds,(+)MDMA-ds, and (_+)-MDEA-d5in 0.1M HCI (250 IlL, 8 IJg/mL)
was added to 2 mL of urine specimens and standards. A drugfree urine control was also used in the batch analysis. The
solutions were made basic by adding 0.5 mL of 1M NaOH.
Methylene chloride (2 mL) was added to the tubes, and the
solutions were shaken horizontally for 15 rain and centrifuged.
The upper urine layers were discarded. The free-base compounds were then extracted into 2 mL of 0.15M H2SO4,and the
organic solvent was discarded. The solutions were made basic
with 1 mL of 1M NaOH, and the drugs were extracted with
2 mL of 1-chlorobutane. The solutions were centrifuged for
5 min, and the organic solutions were transferred to another
set of tubes and used for derivatization.
(R)-(-)-MTPAderivatization
A solution of (R)-(-)-MTPAwas prepared in dry acetonitrile
(50 IlL reagent/mL). It was stable for at least six months at
-20~ For derivatization, approximately 50 IlL was added to
the compounds in 1-chlorobutane. The tubes were capped
and heated at 70~ for 1 h. The solutions were cooled to
room temperature, and anhydrous ethanol (100 IJL) was
added to it. The tubes were capped again and heated at
70~ for 15 min. The ethanol changed the excess derivatizing agent to the ethyl ester. The solutions were evaporated
to dryness at room temperature under nitrogen. The extract
were dissolved in 100-150 IJL of ethyl acetate and tested by
GC-MS. The amide derivatives were stable for at least 60 h at
room temperature.
GC-MS parameters
Oven temperature was increased from 140~ to 215~ at
15~
and then to 285~ at 35~
The temperature was
held at 140, 215, and 285~ for 0.5, 1.5, and 1 min, respectively.
Injector and detector (transfer line) temperatures were 160
and 260~ respectively.The selected ions, relative abundances
(parenthesis), and quantitation ion (Q) were 126 (38) and 263
(100, Q) for amphetamine-ds; 118 (33), 162 (37, Q), and 260
(100) for amphetamine; 98 (100) and 281 (96, Q) for methamphetamine-du; 176 (9), 274 (100, Q), and 275 (16) for methamphetamine; 167 (100) and 211 (8, Q) for MDA-ds;162 (100), 206
(5, Q), and 260 (3.2) for MDA; 164 (100) and 278 (32, Q) for
MDMA-d5;162 (100), 200 (9), and 274 (18, Q) for MDMA;393
(Q) for MDEA-d5and 288 (Q) for MDEA.The respective(R)- and
(S)-isomers showed similar fragmentation patterns. Amphetamine, methamphetamine, and MDAions were monitored
in three separate groups, whereas both MDMAand MDEAwere
monitored in the fourth group. The injection volume was 1-3
IlL, and the electron multiplier was set at 200 V above autotune
for amphetamine, methamphetamine, 300 V above for MDA,
and 400 V above for MDMAand MDEA.The dwell time for
each ion was 50 ms.
Journal of Analytical Toxicology, Vol. 28, September 2004
agent only and could not be used (Figure 1). Abundances of two
ions of MDA were relatively low (m/z 206 at 5% and 260 at
3.2%); however, these ions showed excellent chromatographic
background and ion ratios over a range of concentration of 25
to 10,000 ng/mL. Other major ions have limited use because
they showed either the product-ion effect from the internal
standard to the drug or isotopic effect from the drug to the internal standard. These effects limit the ability to detect the
Results and Discussion
(R)-(-)-MTPA was initially introduced to prepare diastereomers of different chiral amines and alcohols and to study the
chemical shift in NMR spectra (14,15). In the preparation, the
amines and alcohols were reacted with the reagent in dry pyridine. The excess reagent was changed to the amide of N,Ndimethyl-l,3-propanediamine. The products were then purified
by acid-base separation. In our method,
the reagent was directly added to the ex189
tracted drugs in 1-chlorobutane. Derivaioooo
Amphet~mine-MTPA
tization was almost complete within 15
ilOOO
91
min at 70~ for amphetamine and MDA.
However,for methamphetamine, MDMA,
and MDEAthe reaction time was 45--60
119
260
rain. Steric hindrance from the N-substi2or162
-/'?l
I
tuted groups was the reason for longer
........
3~I.
2~
300 3~
~O
reaction time. After 60 rain reaction the
r~
excess reagent was decomposed by anhy~oooo~
drous ethanol. The solvent was then reMethamphctamine-MTPA
119
moved and the product was ready for analysis by GC-MS. For identification,
retention time and three ion ratios of the
:mooo~
unknown were compared with that of a
i
standard. Initially, a standard at a conl~xx~
centration of 1000 ng/mL was injected
c
2110
40
60
80
100
120
'140
1(10
180
200
220
240
21}0
under the scan mode (50-500 ainu)
m/z
(Figure 1).
Optically pure amphetamine, methamZO0(X}@ M D A - M T P A
phetamine, and MDMAwere used to ideni(0000,
tify the peaks of diastereomers. In the
chromatogram, the (-)-drug-(-)-MTPA
eluted before the (+)-drug-(-)-MTPA. Op77
105
1~5
[
tically pure MDA and MDEA were not
51
1 0
3~5
available for the peak identification. How300
9
~
~
~oo ~
~ioi~
m/2
ever, we assumed that these compounds
would follow an elution pattern similar
189
:~=~ MDMA-MTPA
to the amphetamine, methamphetamine,
or MDMA because in p-methoxyamphetamine and in other amphetamine
type of compounds, the (-)-(-)-diastereomers always eluted before the (+)-(-)-diastereomers when derivatized with (R)200214 248 274t
409
(-)-MTPA or (S)-(-)-TPC (16,17).
z= 2~ 2,0 ~o -2gO 300 3zo 340 3eo 3~0 ,~0
m/z
The fragmentation patterns of the
189
R-(-) and S-(+) isomers of the drugs were
MDEA-MTPA
almost the same. The ions that contain
major portion of the molecule were seIII00~
lected for drug identification. The structures of the fragment ions are shown in
77
105
182
Scheme 1.
4DO0
288
Fragmentation patterns of the deuter253
I
i, 63L.I, ..L. J, I , ,I.
,i ,
,
L,
.
J .
240
2/1o
28o
ated internal standard support the prom/z
posed structures. Like many other derivatives of amphetamine, none of the
compounds showed the molecular ion.
Figure I. Mass fragmentation of amphetamine, methamphetamine, MDA, MDMA, and MDEA as
The ion at m/z 189, with abundances >
(R)-MTPAderivatives.
95% is characteristic of the derivatizing
i
:!
'Z
451
Journal of Analytical Toxicology, Vol. 28, September 2004
this case (S)-(+)-drug-(S)-(+)-MTPA eluted before the (R)-(-)drug-(S)-(+)-MTPA.
A calibrator at a concentration of 500 ng/mL for (R)- or (S)was used to quantitate the isomers. It was prepared from 1000
ng/mL of the racemic mixture. Isomers (R or S) were avoided
because the purity of the compounds from commercial sources
varied. As an example, (S)-Am was found
to contain 2.7% (R)-Am, whereas (S)[(R1)-Ar-CH=CH(CH3)]
+
Meth was > 99% pure. Moreover, some of
the isomers were not available. The linAmp
118
earity was studied over the concentration
MDA and MDMA
162
range of 25 to 10,000 ng/mL in urine. All
isomers, excepting (R)- and (S)-MDEA,
were linear in this concentration range.
MDEA was linear up to 5000 ng/mL. In all
R1 = R2 = H for Amp
cases the drug concentrations were
R1 = H, R2 = CH3 for Meth
within • 20% of the expected values and
the ion ratios were within • 20% of the
R1 = 3,4-OCH20-, R2 = H for MDA
calibrator. The correlation coefficients in
R1 = 3,4-OCH20-, R2 = CH3 for MDMA
all cases were > 0.996. The drug and internal standard ratios of the calibrators
R1 = 3,4-OCH20-, R2 = C2H5 for MDEA
from five different batches were calcuAr = Aromatic phenyllated. The coefficient of variation was less
than 11% (iV= 5, 4.2 to 10.8%) indicating
excellent precision at the calibrator concentration.
compounds within a narrow range of concentration. For
MDEA, only one ion was monitored for the drug (m/z 288) and
one ion for the internal standard (m/z 293). Considerable
overlap was observed with other ions. Like (R)-(-)-MTPA,
derivatizing agent (S)-(+)-MTPA also showed similar chromatographic separation and mass fragmentation pattern. In
(R1)-Ar-CH2-CH(CHg)-N(R2)-CO§
Amp
162
Meth
176
MDA
206
+.CH(CH3)N(R2)-CO-C(OCH3)(CF3)(C6H5)
Amp and MDA
260
Meth and MDMA 274
MDEA
288
Scheme1. Structuresof fragmentions.
Table I. Specimens Tested for Optical Isomers of Amphetamine and Methamphetamine by the Present (R)-MTPA Method
and the Results are Compared with that of a 4-CB Method
Amphetamine(ng/mL)
Sample
Sp-08
Sp-12
Sp-13
Sp-17
Sp-20
Sp-97
Sp-98
Sp-99
Sp-100
Sp-102
Sp-103
Sp-104
Sp-105
Sp-106
Sp-107
Sp-108
Sp-109
Sp-110
Sp-111
Sp-112
Sp-113
Sp-114
Sp-115
Sp-116
(R)-
153
Methamphetamine(ng/mL)
(S)-
(R) + (S)
4-CB*
377
377
346
64
426
2460
199
1065
1746
2225
489
251
389
1432
406
230
1253
956
382
1084
3377
4262
4139
281
64
426
2460
199
1065
1746
2225
489
251
389
1432
406
230
1253
956
382
1084
3530
4262
4139
281
489
2717
193
1049
2178
2334
629
215
336
1428
411
295
1001
973
299
992
4091
3974
3774
277
* The non-chiral 4-CB test was conducted more than a year ago by a Navy laboratory at San Diego, CA.
452
(R)-
(S)-
84
42
156
443
369
7443
4768
847
9828
10,442
11,455
2637
1081
1232
5313
2837
463
1947
2957
2325
3403
126
156
443
369
7723
4768
847
9828
10,442
11,455
2637
1081
1232
5313
2837
463
1947
2957
2325
3403
99
151
420
344
9126
4473
1158
15,517
13,326
13,158
2705
890
1083
4721
2735
569
1919
3099
2551
3650
5326
855
5326
855
4412
715
280
(R) + (S)
4-CB*
Journal of Analytical Toxicology, Vol. 28, September 2004
Table II. Specimens Tested for Optical Isomers of MDA, MDMA, and MDEA by the Present (R)-MTPA Procedure and the
Results are Compared with that of a 4.CB Procedure
MDA (ng/mL)
Sample
Sp-01
Sp-02
Sp-03
Sp-04
Sp-05
Sp-06
Sp-07
Sp-08
Sp-09
Sp-10
Sp-11
Sp-12
Sp-13
Sp-14
Sp-15
Sp-16
Sp-17
Sp-18
Sp-19
Sp-20
Sp-21
Sp-22
Sp-23
Sp-101
(R)-
($)-
162
185
470
189
307
57
299
4474
90
98
316
2952
83
86
248
269
916
134
402
72
69
218
2146
1192
32
54
28
43
166
32
119
2155
34
18
166
2790
124
48
651
323
20
95
44
52
251
510
234
MDMA (ng/mt)
(R) + (S)
4-CB*
194
239
498
232
473
89
418
6629
124
116
482
5742
207
134
899
269
1239
154
497
116
121
469
2656
1426
189
230
514
196
500
470
8649
165
140
497
4856
148
128
992
375
1303
187
477
118
90
359
2883
1421
(R)-
($)-
(R) + (S)
MDEA (ng/mL)
4-CB*
810
68
1761
168
1690
604
42
58
338
51
3124
118
77,952
9908
714
55
336
4417
830
37,985
8185
1765
773
661
61
27,488 22,303
2360
230
878
764
1929
1705
1690
1637
646
546
58
49
389
504
3242
3074
87,860 83,772
769
1196
336
548
5247
5560
46,170 47,400
2538
2027
722
786
49,791 42,277
2590
2278
710
3501
1196
830
3173
23,096
6178
737
822
3644
4003
1323
1322
956
1010
4272
4420
23,569 20,400
6544
7,638
27
143
127
126
1099
473
366
(R)-
($)-
(R) + (S)
4-CB*
1420
371
1791
1882
744
744
* The non-chiral4-CB testwas conducted more than year ago by a Navy laboratoryat San Diego, CA.
The procedure was applied to test 43
Table III. Active Duty Military Personnel Tested for Amphetamines*
urine specimens received from a navy laboratory in San Diego (SD). The specimens
Specimens
were tested by the SD laboratory (7) and
Tested
Amphetamine Methamphetamine* MDA
MDMA MDEA
stored frozen (-20~ for 12 to 18 months.
Year
In the test, a non-chiral derivatizing agent,
1998 2,532,535
1126
1395
72
106
0
4-carboethoxy-hexafluorobutyryl chloride
1999 2,488,330
805
819
377
495
2
(4-CB), was used. The results were con2ooo 2,632,207
1301
1483
905
1218
3
1575
1883
703
1744
24
cealed to us until all tests were complete
2OOl 2,850,702
2143
2415
471
1368
31
and are summarized in Tables I and II. All
20o2 3,045,146
2,856,890
2030
2024
216
670
9
drugs were properly identified by the pre2003
sent MTPA-method.The total (R + S) con* Specimensmay be positivefor multiple drugs.
* Numbers representS-(+)-methamphetamineonly.
centrations were comparable with the SDresults excepting specimen-08 for MDEA
(R-MDEA 744 ng/mL) and specimen-17 for amphetamine (Samphetamine, methamphetamine, MDA, and MDMA were
amp 64 ng/mL). It appeared that specimen-08 was diluted for
0.650, 0.154, 0.527, and 0.183, respectively. These values were
strong MDMAconcentration (83,772 ng/mL) and the MDEA
less than the corresponding critical values (tcrit2.069, N = 24,
was below the limit of detection. Specimen-08 on reanalysis
95% confidence) indicating that there is no difference between
showed R-MDEA at a concentration of 843 ng/mL. The amthe procedures.
phetamine in specimen-17 was below the LOD in the SDThe total ion chromatogram (TIC) and the selected ions of
testing procedure. Most concentrations were within • 20% of
specimen-20 are shown in Figure 2. The specimen showed deSD-values (85% for amphetamine and methamphetamine, and
tectable levels of (S)-amp, and (R)- and (S)-isomers of metham71-79% for MDAand MDMA).The difference in concentrations
phetamine, MDA,and MDMA.It appeared that the donor conmay be due to instability of the drugs, heterogeneous nature of
sumed both methamphetamine and MDMA. In most
urine, and interlaboratory variations. The results of the two
methamphetamine-positive specimens, only the (S)-isomer
procedures were analyzed by the t-test. The t-values for
was detected (20/22, 91%). This indicates that the compound
453
Journal of Analytical Toxicology, Vot. 28, September 2004
6.6~ Total Ion Chromatogram
500000
400000
3OOO0O
.g
200000
8.67
~8.,
100000
0
5.50
6.50
6.00
7.00
7.50
8.00
.
.
.
.
.
.
.
.
9.~
8,50
.
.
.
.
.
9.50
]~me (min)
.
I|
'4~176176
.
.
40000c Ion 17@00
35o00r Ion 274.00
32ooor Ion 275.00
.
,~mpnetamme-
o,r~ II
....
38o001 Ion 1UZ.UU
S,R A Methamphetamine-
Rs.,R~ I
II
/A/
I@100
1
~1
'00~
5.75
500oo1Ion 263.0(
5.85
As.'5
5.95
R,R
8.05
8.25
e.15
e.45
5.55
Time (min)
8.35
Time (min)
-t
5 ,o
[I~ 126"0CIIR,I~-
Ion 98.00
32000 Ion 281.00
i r"-Amphetamine-'
MTPA -
40000
MTPA
2 ~
I/II
J kL
R,R
_~
5.85
6.81
5.93
,:, ~, ~
40000 Ion 2 6 0 . 0 0
28000[
-
e.ss
8.75
Methamphetaminrdl4S,R MTPA
R,R
2,0o01
~= 20000
"g=
10000
'=r
80o0
12000
8.25
635
6.45
Time (mill)
8.21
1
/ I R,R
~ 8000
.=
S
-~
MDMA-MTPA
40000
4OOO
30000
2OOO
20000
10000
0
6.75
~ 60000
"10 50000:
80O0
"~
R,R
90000 ion 274.00
Ion 200.00
80000
Ion 162.00
70000
MDA-MTPA
(~
Ion 206.00
10000 Ion 162.00
6 65
8.67
100000
12000 Ion 260.00
6.55
Time (rain)
8.05
8.15
8.25
835
8.45
8.45
8,55
8.55
44000 Ion 211.00
.20
40000 Ion 167.00
30000
32000
:~,R
~= 2s00o
24000
,~ 20000
8.75
8.65
Time (min)
6.85
8.95
Time (min)
835
MDA-ds-MTPA
S,R
Ion 278.00
24000 Ion 164.00
3.66
6.71 MDMAd5-MTPA
S,R
R,R
20000
16000;
i
12000
6o~
120O0
4o0o
8,05
8.15
625
8.35
Time (min)
8.45
8.55
6.45
055
8.65
5.75
5.85
6.95
Time (rain)
Figure 2. Total ion and SIM chromatogram of specimen-20 showing detectable amount of (ag-(+)-amphetamine and (R)-(-)- and (S)-(+)-isomers of methamphetamine, MDA, and MDMA.
454
Journal of Analytical Toxicology, Vol. 28, September 2004
was prepared from either (1R,2S)(-)-ephedrine or (1S,2S)-(+)pseudoephedrine. Presence of both isomers in MDMA-positive
specimens suggests consumption of (•
In all cases,
(R)-(-)-MDMA was the predominant compound. It appeared
that after ingestion, the (S)-isomer was metabolized or excreted faster than the (/?)-isomer. Similarly, (R)-isomer was the
major compound in MDA- and MDEA- positive specimens
(21/24 and 2/2, respectively). The urine results are similar to
that found in human plasma after (+)-MDEA administration
(18). Higher plasma concentrations of (R)- were also observed
in specimens positive for MDA and MDMA(19). Not much is
known on the differences in pharmacological activities of the
(19)-and (S)-isomers of MDA,MDMA,and MDEAin human, but
some animal studies suggest that (R)-isomers are more mescaline-type hallucinogenic than the respective (S)-isomers
(20-23).
Interferences from the isomers of ephedrine, pseudoephedrine, and phenylpropanolamine (50 ]ag/mL)were studied.
In the chromatogram, the derivatives appeared between peaks
of (S)-meth and (R)-MDA.No interferences were observed from
these compounds to the test drugs. The MTPAmethod worked
well in chiral separation and detection of amphetamine,
methamphetamine, MDA,and MDMA.When the total concentrations of (R)-(-)- and (S)-(+)-isomers of 43 specimens were
compared with the concentrations of a non-chirat method (4CB), both methods showed close correlation. Therefore, the
MTPAmethod would be a choice to replace the presently used
two confirmation methods. Although three-ion monitoring
worked well for these compounds, the method allowed only
one-ion monitoring for MDEA. In U.S. military testing, the
number of MDEA-positive specimens is relatively low (Table
III). Therefore, the MTPAmethod could be used to screen for
MDEA, and if positive, a non-chiral method could be used to
confirm its presence.
References
1. D.A. Toseland and P.H. Scott. Determination of amphetamine as
its N-acetyl derivative by gas-liquid chromatography. Clin. Chirn.
Acta 25:75-78 (1969).
2. E. Anggard and A. Hankey. Derivatives of sympathomimetic
amines for gas chromatography with electron capture detection
and mass spectrometry. Acta Chem. Scand. 23:3110-3119
(1969).
3. J.S. Noonan, P.W. Mirdick, and R.S. Ray. A method for detecting
amphetamine using gas chromatography of a halogenated derivative. J. Pharrnacol. Exp. Ther. 168:205-209 (1969).
4. G.R. Reynolds, J.D. Elsworth, K. Blau, M. Sandier, A.J. Lee, and
G.M. Stern. Deprenyl is metabolized to methamphetamine and
amphetamine in man. Br. J. Clin. Pharrn. 6:542-544 (1978).
5. M. Terada, T. Yamamoto, T. Yoshida, Y. Kuroiwa, and
S. Yoshimura. Rapid and highly sensitive method for determination of methamphetamine and amphetamine in urine by electroncapture gas chromatography. J. Chrornatogr. 237:285-292
(1982).
6. C.L. Hornbeck and R.J. Czarny. Quantitation of methamphetamine and amphetamine in urine by capillary GC/MS. Part I.
Advantages of trichloroacetyl derivatization. J. Anal. Toxicol. 13:
144-149 (1989).
7. R.J. Czarny and C.L. Hornbeck. Quantitation of methamphetamine and amphetamine in urine by capillary GC/MS. Part II.
derivatization with 4-carboethoxyhexafluorobutyryl chloride.
J. Anal. Toxicol. 13:257-262 (1989).
8. J.E. Meeker and P.C. Reynolds. Postmortem tissue methamphetamine concentrations following selegiline administration.
J. Anal. Toxicol. 14:330-331 (1990).
9. E.M. Thurman, M.J. Pederson, R.L. Stout, and T. Martin. Distinguishing sympathomimetic amines from amphetamine and
methamphetamine in urine by gas chromatography/mass spectrometry. J. Anal. Toxicol. 16:19-27 (1992).
10. C.E. Cook, A.R. Jeffcoat, J.H. Hill, D.E. Pugh, P.A. Patteta,
B.M. Sadler, W. Reid, and A. Perez-Reyes. Pharmacokinetics of
methamphetamine self-administered to human subjects by
smoking S-(+)-methamphetamine hydrochloride. Drug Metab.
Dispos. 21:717-723 (1993).
11. B.D. Paul, M.R. Past, R.M. McKinley, and J.D. Foreman. Amphetamine as an artifact of methamphetamine during periodate
degradation of interfering ephedrine, pseudoephedrine, and
phenylpropanolamine: an improved procedure for accurate quantitation of amphetamine in urine. J. Anal. Toxicol. 18" 331-336
(1994).
12. Y. Yoo, H. Chung, and H. Choi. Urinary methamphetamine concentration following famprofazone administration. J. Anal. Toxicol. 18:265-268 (1994).
13. D.E. Nichols, C.F. Barfknecht, and D.B. Rusterholz. Asymetric
synthesis of psychotomimetic phenylisopropylamines. J. Med.
Chem. 16:480-483 (1973).
14. J.A. Dale, D.L. Dull, and H.S. Mosher. (x-Methoxy-~-trifluoromethylphenylacetic acid, a versatile reagent for the determination of enantiomeric composition of alcohols and amines.
J. Org. Chem. 34:2543-2549 (1969).
15. J.A. Dale and H.S. Mosher. Nuclear magnetic resonance enantiomer reagents. Configurational correlations via nuclear magnetic
resonance chemical shifts of diastereomeric mandelate,
O-methylmandelate, and ~-methoxy-~-trifluoromethylphenylacetate (MTPA) esters. J. Am. Chem. Soc. 95:512-519 (1973).
16. J.W. Westley and B. Halpern. Effect of solute structure on separation of diastereoisomeric esters and amides by gas-liquid chromatography. Anal. Chem. 40:2046-2049 (1968).
17. J. Gal. Stereochemistry of metabolism of amphetamines: use of
(-)-c~-methoxy-c~-(trifluoromethyl)phenylacetyl chloride for GLC
resolution of chiral amines. J. Pharrn. Sci. 66:169-172 (1977).
18. M. Brunnenberg and K.A. Kovar. Stereospecific analysis of ectasylike N-ethyl-3,4-methylenedioxyamphetamine and its metabolites in humans. J. Chromatogr. B 751:9-18 (2001).
19. F.T. Peters, N. Samyn, M. Wahl, T. Kraemer, G.D. Boeck, and
H.H. Maurer. Concentrations and ratios of amphetamine,
methamphetamine, MDA, MDMA, and MDEA enantiomers determined in plasma samples from clinical toxicology and driving
under the influence of drugs cases by GC-NICI-MS. J. Anal. Toxicol. 27:552-558 (2003).
20. J. Broadbent, J.B. Appel, E.K. Michael, and J.H. Ricker. Discriminative stimulus effects of the optical isomers of 3,4-methylenedioxyamphetamine (MDA). Behav. Pharrnacol. 3:443-454
(1992).
21. LE. Baker, J. Broadbent, E.K. Michael, P.K.Mathews, C.A. Metosh,
R.B. Saunders, W.B. West, and J.B. Appel. Assessment of the discriminative stimulus effects of the optical isomers of ecstasy (3,4methylenedioxymethamphetamine; MDMA). Behav. Pharrnacol.
6:263-275 (1995).
22. L.E. Baker, T.B. Virden, M.E. Miller, and C.L. Sullivan. Time course
analysis of the discriminative stimulus effects of the optical isomers of 3,4-methylenedioxymethamphetamine (MDMA). Pharmacol. Biochern. Behav. 58:505-516 (1997).
23. L.E. Baker and M.M. Taylor. Assessmentof the MDA and MDMA
optical isomers in a stimulant-hallucinogen discrimination. Pharmacol. Biochern. Behav. 57:737-748 (1997).
455