Journal of Analytical Toxicology, Vol. 24, March 2000
Analysisof UnderivatizedAmphetaminesand Related
Phenethylamineswith High-PerformanceLiquid
Chromatography-AtmosphericPressureChemical
Ionization MassSpectrometry*
Maciej J. Bogusz, Klaus-Dieter KrLiger, and Rolf-Dieter Maier
Instituteof ForensicMedicine, Aachen Universityof Technology,52057Aachen, Germany
Abstract
I
Amphetamine, methamphetamine, illicit designer phenethylamines
(MDA, MDEA, MDMA, MBDB, and BDMPEA), and other
phenethylamines (benzyl-l-phenylethylamine, cathinone, ephedrine,
fenfluramine, norfenfluramine, phentermine, 1-phenylethylamine,
phenylpropanolamine, and propylhexedrine) were extracted from
serum usinga solid-phase extraction procedure. The extracts were
examined with high-performance liquid chromatographyatmospheric pressurechemical ionization massspectrometry
(LC-APCI-MS). The drugs were separated on ODS column in
acetonitrile/5OmM ammonium formate buffer (pH 3.0) (25:75) as a
mobile phase. Full-scanmass spectra of drugs examined by means of
APCI with collision-induced dissociation showed protonated
molecular ions and fragments typical for particular drugs. LC-APCIMS allowed an unequivocal differentiation of all drugs involved. The
quantitation was performed using selected ion monitoring of
protonated molecular ions and fragments of drugs involved and
their deuterated analogues.The limits of detection ranged from
1 to 5 pg/t serum, and the recoveries ranged from 58 to 96%. A
linear responsewas observed for all drugs in the range from 5 to 500
pg/L. The method was applied for routine determination of
amphetamine, MDMA, MDA, and MDEA in one run. Solid-phase
extraction used assuredsimultaneous isolation of various groups of
basic drugsof forensic interest (opiates, cocaines, phenethylamines,
and benzodiazepines) from biofluids.
Introdudion
Selective and sensitive analysis of amphetamine and related
compounds such as methamphetamine, methylenedioxyamphetamines (e.g., "ecstasy"), and other phenethylamines are
among the most important tasks of the forensic toxicologist.
These drugs particularly ecstasy, were often detected in blood
samples taken from impaired drivers, usuallytogether with other
drugs such as cannabisor ethyl alcohol (1,2). In the 1990s,several
* Dedicated to Prof.Dr.med. Helmut Althoff on the occasion of his 65th birthday.
reports demonstrating that the intake of amphetamines is associated with high risk, especiallywhen consumed in discos and at
rave parties, were published (1,3-7).
Among the analyticalprocedures applied for the forensic analysis of amphetamines, gas chromatography-mass spectrometry
(GC-MS) plays a dominant role. Besides electron impact mode,
the chemical ionization mode (positive and negative ions) was
advocated in order to avoid the misidentification of some overthe-counter sympathomimetic amines with amphetamine or
methamphetamine (8-17).
High-performance liquid chromatography (HPLC) with UV
detection is less suitable for blood analysis because of the low
absorptivity of amphetamines and is mainly applied in the examination of illicit drug samples (18). HPLC with fluorescence
detection showed sufficient sensitivity for detection of
amphetamines in biological fluids (19,20), but the selectivity is
not comparablewith that of MS identification.Among other separation techniques, capillary electrophoresis with diode-array
detection (DAD) (21) and thin-layer chromatography with
Fourier transform infrared detection (22) have been used for the
analysis of forensic samples containing methylenedioxyamphetamines.
The introduction of HPLC coupled with atmospheric pressure
ionization mass spectrometry (LC-API-MS), in either atmospheric pressure chemical ionization (APCI)or electrospray(ESI)
mode, created the possibility of taking advantage of the high
selectivity of MS detection without derivatization, which is
unavoidable in GC-MS procedures. In a previous report (23), we
described a procedure for determination of amphetamines and
related phenethylamines with LC-APCI-MSor DAD.The drugs
were extracted with ether and subjected to derivatization with
phenylisothiocyanatein order to enhance the sensitivityof DAD.
In the meantime, we have developed a common solid-phase
extraction procedure for isolation of basic drugs of abuse, which
has been applied for LC-APCI-MSdetection of opiate agonists,
cocaineand metabolites,flunitrazepamand metabolites,and LSD
in biosamples (24-27).
The purpose of the present paper was to use the same isolation
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
77
Journal of Analytical Toxicology, Vol. 24, March 2000
ramine were supplied by Servier (Orleans, France). 2S-Cathinone
by Radian (Austin, TX) and N-benzyl-l-phenylethylamine (BEA)
were supplied by Fluka (Buchs, Switzerland). N-Methyl-l-(3,4methylenedioxyphenyl)-2-butanamine(MBDB)and 4-bromo-2,5dimethoxyphenethylamine (BDMPEA)were purified from street
drug samples.
Deuterated internal drug standards. Amphetamine-dn,
methamphetamine-dl0, MDEA-ds,and MDMA-d7were obtained
from High Standards Products (Inglewood,CA).
Serum validation standards. Serum samples were obtained
from localblood bank were prescreened for absence of drugs with
immunoassay (CEDIATM).Samplesshowing no reaction (zero) to
amphetamines were spiked individuallywith the listed drugs to
the concentrations of 5, 50, 100, and 500 IJg/L.The concentration
of appropriate internal standard was always 50 1Jg/L.The control
serum BTMFS-plus from Medichem (Steinenbronn, Germany),
containing amphetamine (56 1Jg/L), MDMA (121 IJg/L), and
MDEA(81 IJg/L)was used for precision and accuracy studies. This
serum was also spiked with MDAto the concentration of 50 IJg/L.
procedure for the determination of amphetamines and related
phenethylamines with LC-APCI-MSwithout any derivatization.
The use of a standardized extraction procedure for simultaneous
isolation of potentially all basic drugs of abuse may save laboratory time and (usually limited) sample. It must be added that in
April 1998, German Parliament issued a law stating that the mere
presence of any defined drug of abuse in the blood of a driver is
equivalent to impairment. The actual list of drugs included
tetrahydrocannabinol (THC), morphine, benzoylecgonine,
amphetamine, methylenedioxymethamphetamine (MDMA),and
methylenedioxyethylamphetamine (MDEA).The following concentrations of drugs were suggested as unequivocal evidence for
their presence in blood: THC, 2 IJg/L; morphine, 20 IJg/L;
amphetamine, MDMA,and MDEA,50 IJg/L;and benzoylecgonine,
150 IJg/L (28). In the available literature only some studies were
found, which concerned examination of amphetamines with
LC-MS in biological fluids. Verweij and Lipman (29) compared
the sensitivity of detection of MDMA, methylenedioxyamphetamine (MDA),and MDA(pure drugs) using thermospray-MS,
LC-APCI-MS,and LC-ESI-MS.Goffet al. (30) determined MDMA
and MDAwith LC-ESI-MSin blood and liver taken from rabbits
after administration of MDMA,and in maggots fed the rabbit tissues. To our knowledge, no systematic study on all forensically
relevant phenethylamines with LC-API-MShas been performed.
Isolation procedure
The method, already applied for several other basic drugs
(24-27), was used with minor modification. Solid-phase extraction (SPE) cartridges (200-rag Bond Elut C18) were supplied by
VarianAnalytichem(Harbor City,CA).The cartridges were rinsed
with 1 mL methanol, 1 mL H20, and 2 mL of 0.01M ammonium
carbonate buffer (pH 9.3) before use. Spiked serum samples or
Experimental
forensic blood samples were centrifuged 5 rain at 14,000 xg, and
0.2-mL to 1-mL volumes of supernatant were vortex mixed with
2 mL of 0.01M ammonium carbonate buffer (pH 9.3) and with
Reagents
appropriate internal standards (listed in Table I). After 10 min
Drug standards. Amphetamine, methamphetamine, MDMA,
centrifugation at 5000 x g, 2 mL of clear supernatant was applied
MDA, MDEA, ephedrine, 1-phenylethylamine, phenylpropanon the SPE cartridge and slowlypassed through (approximately5
olamine, phentermine, and propylhexedrinewere obtained from
rain). The SPE cartridge was rinsed with 2 mL of 0.01M ammoSigma-Aldrich (St. Louis, MO). Fenfluramine and norfenflunium carbonate buffer (pH 9.3) and vacuum dried
for 5 rain. The retained drugs were eluted with 0.5
Table I. Measured Ions and Retention Times of Substances Involved*
mL methanol/0.5Macetic acid (9:1) under gravity
Rt
force to standard, capped Eppendorf 1-mL
Flow rate
Substance
Ions measured
IS
(mL/min)
(min:s) polypropylene tubes. After addition of 10 1JL of 1
mmol HCI, the eluates were dried under nitrogen,
3:39
Amphetamine
147, 136, 119, 91
A-d11f
0.3
reconstituted in 100 1JL of HPLC mobile phase,
4:20
MDMA
199, 194, 163, 133
MDMA-ds
0.3
and centrifuged 4 rain at 14,000 x g. Clear super3:50
MDA
180, 163,147
A-du
0.3
natant (5-20 IJL)was injected into LC-MS. The
5:16
MDEA
215, 208, 163
MDEA-d7
0.3
only
modification in this procedure was that the
5:52
MBDB
215, 208, 177, 147
MDEA-d7
0.3
amount
of HC1 in the drying phase was doubled;
6:50
BDMPEA
260, 262,245, 215
MDEA-d7
0.4
during
the
pilot experiments it was stated that the
4:13
Methamphetamine
160, 150, 119, 91
MA-dl0
0.3
use
of
10
tJL
rather than 5 IJL HC1 increased the
4:25
Phentermine
160, 150, 133, 91
MA-dl0
0.3
recovery by about 20%.
2:58
Cathinone
150, 147, 132
A-d~
0.3
Phenylethylamine
Propylhexedrine
Ephedrine
Phenylpropanolamine
Fenfluramine
Noffenfluramine
BEA
147,122, 105
204, 156, 157
160, 166, 148
152, 147, 134
232, 212, 159
21Z 204, 187, 159
212, 204, 108, 105
A-du
NF
MA-dm
A-du
BEA
BEA
NF
0.3
0.8
0.3
0.3
0.8
0.8
0.8
* Protonated molecular ions are given in bold characters, the ions of internal standards in italics.
t Abbreviations: A, amphetamine, MA; methamphetamine; NF, noffenfluramine; BEA,
benzyl-1 -phenylethylamine.
78
3:07
3:52
3:06
2:46
7:02
4:22
4:33
HPLC
The separations were performed in isocratic
conditions on Superspher 100 RP 18 column (125
mm x 3-ram i.d., 4-tJm particle size, E. Merck,
Darmstadt, Germany).The mixture of acetonitrile
and 50raM pH 3.0 ammonium formate buffer
(25:75) was used as a mobile phase. The flow rates
and the retention times observed for particular
compounds are listed in Table I.
Journal of Analytical Toxicology, Vol. 24, March 2000
119
loa
150
A
91
B
).041"
It+
,o
- -
/--\
//-c"2tc"-c
3
l?"'i"§
0
|O"
N, H2
I"~"~
tO"
9l/
119 /
J.SO
136
~o
[
119
136
40
so
91
|0
n . . . . , ....
m/z
163
C
',,-'~
/
194
58
133
\
105
so
. ....
i
tS0
163
,.
D
o"~k )2-'-CH'--CH-!-NH,
80
L-o2-~"
i ....
300
n'~
L
t..^/~'
..
,~4
leo
0
40'
180
40'
20"
~135
loo
~.so
loo
....
2~o
, ....
so
u . . . . . . . . . . .
$oo
1,~o
n'Vz
z~
|$0
n'V'z
H 3 H- +
200
E
260/262
H3CQ
~oo
F
80
2O8
243/24s !
163
OCH~
40
260/2G2
40
182
10'
72
"9[ I
i ....
, ........
, ....
*TJ "T
i-',*
.
200
=Io. . . . . . . . .
~to
rn/z
208
O
G
i
....
.o
~
cIH~,~
4O
so
. . . . . . . . .
~do ........
132
"
11
33
30
72
]
H
H+
177
4o
10
H
F~C'c~H2
"
H+
150
II
2O8
135
I
..... iJ.
rn/z
~Hz--CH3
0
2 H"
"'
I~o .
m/z
i
. . . . . . . .
,dO . . . . . . . . .
=i,~
SO
100
" '
. . . .
'
. . . .
.,"
. . . . .
"
" " l~o
rn/z
Figure 1. Mass spectra and proposed fragmentation patterns of amphetamine (A), methamphetamine (B), MDMA (C), MDA (D), MDEA (E), BDMPEA (F), MBDB (G),
and cathinone (H).
79
Journal of Analytical Toxicology, Vol. 24, March 2000
100, and 500 IJg/L of a particular drug and stored at -20~ until
extraction. Additional accuracy and precision standards, which
contained amphetamine, MDMA,MDA,and MDEA,were examined three times on different days. Analytical recovery for
amphetamine, MDMA,MDA, and MDEAwas tested on three different days at the concentration levels of 5, 50, and 100 Vg/Land
was defined as percent peak areas of corresponding amounts of a
given drug spiked postextraction to blank serum extract. The
recovery of other drugs was determined in the same manner on
two different days. The stability of extracted drugs was tested with
three samples of extracted BTMF S-plus serum. Reconstituted
extracts were frozen again after analysis, stored at -20~ and
reexamined 2-3 more times within 8 weeks. These extracts were
stored, as usual, in capped Eppendorf tubes.
APCI-MS
A SSQ 7000 single quadrupole instrument (Finnigan MAT,San
Jose, CA) equipped with an APCI source was used in positive ionization mode. The following APCI inlet conditions were applied:
sheath gas (nitrogen) pressure 70 psi, auxiliary gas (nitrogen) 20
mL/min, heated vaporizer temperature 450~ heated capillary
temperature 190~ corona current 5 t~A.In order to establish the
appropriate selected ion monitoring (SIM) conditions, the fullscan mass spectra of substances involved were taken in the range
of 50 to 300 amu at octapole offset values of 10 V. For this examination, the loop injections of pure drugs dissolved in the mobile
phase (50 ng/5 I~L),without HPLC separation, were applied.
On the basis of observed mass spectra, the procedures were
written for SIM detection of particular drugs. Table I shows the
ions monitored in SIM procedures for particular drugs and for
respective internal standards.
Results and Discussion
Validation
The validation was performed in duplicate on three different
days, using serum standards spiked to the concentrations of 5, 50,
Table II. Validation Data for Examined Drugs
LOD*
(pg/L)
Drug
Amphetamine
MDMA
MDA
MDEA
MBDB
BDMPEA
Methamphetamine
Phentermine
Cathinone
Phenethylamine
Propylhexedrine
Ephedrine
Phenylpropanolamine
Fenfluramine
Norfenfluramine
[3EA
2.0
1.O
2.0
1,0
1,0
2.0
1.0
1.0
5.0
2.0
2,0
1.0
1.0
1.0
1.0
2.0
tinearity
r
y = 10.117x- 2.308
y = 4,079x + 18,931
y = 26.305x + 0.775
y= 34.029x- 14.688
y = 5.367x + 1.294
y = 2.249x + 3.243
y = 5.252x- 3.628
y = 35.751x + 1.642
y = 0.1095x + 8.24
y = 51.648x + 1.680
y = 59,861x + 0.401
y = 1t ,976x- 3.205
y = 10.126x- 2.969
y = 8.037x + 4.881
y = 3,756x + 6.129
y = 0.11l x - 0.663
1.000
0,9986
0.9990
1.0000
1.0000
0.9999
0,9999
1.0000
0.999]
0.9999
1.0000
1,0000
1.0000
0.9995
0.9987
1.0000
APCI massspectra
Figures 1 and 2 show the mass spectra of 16
examined phenethylamines. In applied conditions,
several fragments were observed beside the protonated molecular ion for all drugs but propylRecoveryf
(%)
hexedrine. Observed fragmentation patterns
allowed an unequivocal differentiation between
86 • 6
drugs with identical molecular mass, such as
90 • 4
MDEAand MBDBor methamphetamine, phenter96 • 5
mine, and cathinone. Also, sympathomimetic
87 • 5
phenethylamines used in over-the-counter prepa87 • 6
rations showed quite different mass spectra than
86 • 7
amphetamine
or metharnphetamine. Figure 3
82 • 2
shows
the
mass
spectra of deuterated internal stan96 + 3
dards:
amphetamine-du,
rnethamphetamine-dl0,
88 • 5
MDMA-ds,and MDEA-dT.
58 _* 6
86 •
58 •
63 •
96 •
90 •
79 •
3
6
7
2
3
1
* Defined as signal-to-noise ratio of 3.
* Defined as percent peak area of corresponding amounts of nonextracted drugs added to blank serum extract
and injected into LC-MS. Determined in duplicate at the concentration levels of 5, 50, and 100 pg/L on
three different days (for amphetamine, MDMA, MDA, and MDEA) or on two different days (for other drugs).
Table III.
LC-APCI-MS results
Applied chromatographic conditions ensured
fast elution of examined drugs and chromatographic peaks of acceptable symmetry. The analysis time in each case did not exceed 8 min. For
most of the drugs, a basic separation was achieved.
Two pairs of drugs were not completely resolved:
amphetamine/MDAand methamphetamine/phentermine. In the case of amphetamine and MDA,a
Validation Data for Amphetamine, MDMA, MDA, and MDEA
Amphetamine
Accuracy Precision
Recovery (%)
(%CV)
BTMF serum
5pg/L
50 pg/L
100 pg/L
87+4
84+5
86 • 7
100
102
98
96
4
11
4
6
MDMA
Accuracy Precision
Recovery
86+4
94•
90 • 4
MDA
Accuracy Precision
(%)
(%CV)
Recovery
(%)
(%CV)
110
103
101
97
3
10
3
4
97•
98+7
92 • 2
112
106
100
95
4
12
5
7
* Recovery (mean • SD from three determinations); BTMF = amphetamine 56 pg/L, MDMA ]21 pg/L, MDA 50 pg/L, MDEA 81 pg/L.
8O
MDEA
Accuracy Precision
Recovery
89_+6
84•
88 + 2
(%)
112
96
103
95
(%CV)
9
6
5
5
lournal of Analytical Toxicology, Vol. 24, March 2000
140
134
A
OH
H +
B
I+OI
H +
l0
'CH3
zs6 CH3
H--?H--NH+
CH3
141
152
166
40
152
t
. . . .
p0 . . . .
,
. . . .
+++ . . . .
.
9 ,
+
9 . . . .
SO
I
. . . .
+ "
200
m ~
156
150
C
H +
CH~
(,
133
l@"
H+
D
lg41
,)--CHz~[-C--CH3
150
I11 +
40"
91
10"
m/z
m/z
105
E
X00
J-'+
CHz--CH+ NHz
II0
122
105~
..
159~ H
II0"
F3C
122
40
i0
40
40
20
;III
232
|CH3 H +
2~2
1S9
/
. . . .
"
. . . .
l~o
"
"
"
. . . . .I ~ o. . .
'
"
"
2omf"
-
-
"
. . . .
3so
so
.
.
.
.
.
.
.
.
.
1oo
' .
.
.
.
.
.
.
}d 0 .
lJlO
'
H3C--CH-~-NHH~-CH2
G
~00 '
l
i ....
SO
.
.
.
.
jl,
/ ~
H
~?H3 H +
I0"
|0
J
.
204
105
212
.
F3C/--
204 159~7|
212
gl
41
187
|O
9 ....
s
101
" " " " ....
[11~0
Figure 2. Mass spectra and propsed fragmentation patterns of ephedrine (A), phenylpropanolamine (B), propylhexedrine (CL phentermine (D), phenethylamine (E),
fenfluramine (F), BEA(G), and noffenfluramine (H)+
81
Journal of Analytical Toxicology, Vol. 24, March 2000
simultaneous quantitation of both drugs is possible because these
drugs have completely different mass spectra. The mass spectra of
methamphetamine and phentermine show differences only in one
fragment. Therefore, for simultaneous quantitation, these drugs
have to be chromatographically separated. This is possible using a
mobile phase containing 15% acetonitrile and 85% ammonium
formate buffer. The validation results are given in the Tables II and
III. For the majority of drugs, the absolute recovery of some
80-90% was noted, and the limits of detection ranged from 1.0 to
5.0 pg/L. Only in the cases of ephedrine, phenylpropanolamine,
and 1-phenethylamine, the recovery was around 60%. All drugs
showed linear response in the measured concentration range of 5
to 500 pg/L serum. Extracted drugs, stored in a refrigerator, were
stable over at least 8 weeks (Table IV).
The method was applied for the routine determination of
amphetamine, MDMA,MDA,and MDEA in forensic drug samples
in one chromatographic run. For this purpose, the SIM proceTable IV. Concentrations of Drugs in Serum Extract Stored
at-20~ and Analyzed Several Times*
Day 10
A (56 pg/L)
MDMA (121 pg/L)
MDA (50 pg/L)
MDEA (81 i~g/L)
97
101
98
104
Day 22
Day 55
97+8
91 _+4
98 _+2
99 -+ 7
100+6
101 + 5
102 _ 3
__.8
+7
_+3
• 4
* The results are presentedas percent of initial value (mean _ SD from three
samples).
dure was written to monitor relevant ions (119 and 136 for
amphetamine, 147 for amphetamine-du, 163 for MDA, MDMA,
and MDEA, 180 for MDA, 194 for MDMA, 199 for MDMA-dg,208
for MDEA,and 215 for MDEA-d?).Figure 4 shows the mass chromatograms of blank serum extract, serum spiked with drug standards, and casework blood serum containing amphetamine,
MDMA,and MDA,respectively. In the first phase of method implementation, over 40 forensic samples were examined in parallel
with the present method and with the LC-APCI-MS method used
previously (23). Virtually identical results were obtained with
both procedures.
Hence, the LC-APCI-MSmethod enabled the selective and sensitive determination of all forensically relevant phenethylamines
without any defivatization procedure. The "in source" fragmentation, provided by a single quadrupole instrument, was sufficient for
the positive identification of all drugs examined. It seems that the
single quadrupole LC-MS is a real alternative to much more expensive triple quadrupole LC-MS--MS instruments. The solid-phase
extraction procedure allowed the isolation of all relevant
amphetamines, as well all other basic drugs of abuse, in one run.
This is of practical importance, particularly in Germany. The extension of road traffic law regulation in this country confronted the
toxicologists working in this country with the need for a rapid and
selective method for the drug mentioned (i.e., morphine, benzoylecgonine, amphetamine, MDMA, MDEA, and THC). Among
German authors coping with this problem, Weinmann et al. (31)
developed a common solid-phase extraction method with consecutive simultaneous GC-MS quantitation of amphetamine, benzoylecgonine, morphine, and codeine in serum samples. The limit
130
,.o
199
1,?
A
D
,o
D
_~
D
'~
1oo
D
o-~l
D] CD
3j
eo -
C--C-'~NH2
I
II
D
D | It+
io
B
CH3
))---c--c-l-s.--c~
199
130
*D
D
D
Io
147
2o
~o
.il
*+00 .........
5"0
, . . . . . . .
l .
17S
. . . . . . . . .
, .
300
. . . . . . . .
. . . . . . . .
, .
:125
.
.
.
.
.
.
l~o ....
,
350
' ....
.,' ....
' ....... 1;0
m/z
m/z
D
160
100
C
D
D
I10
Lo>=,, ,1
00"
I
I
CH3
~ 1
I. m
o"-tL )k--c, --C-tN"--cr~-cr~
100
165
-CH 3
40
g \o
160
D
215
~165
#0"
40
215
40
\ 12 9
30
20
so
i, ': )'+ J
. . . . . . . . . .
' ....
'7~
.
1~o
m~
Figure
82
56
.
,
.
.
.
.
i ....
:oo
) ....
:Jo
so
135
loo
" ~ 9 ~
"
"1~o
"
. . . . . . ,;o.. J
mb/z
3. Mass spectra and proposed fragmentation patterns of amphetamine-du (A), MDMA-d5 (B), methamphetamine-dt0 (C), and MDEA-d7 (D).
)SO
Journal of Analytical Toxicology,Vol. 24, March 2000
of quantitation for amphetamine as a pentafluoropropionyl derivative was about 2 pg/L. Kderstein and Sticht (32) also used GC-MS
with perfluorobutyryl derivatization for confirmative determination of opiates, cocaine, benzoylecgonine, cannabinoids, and
amphetamines (amphetamine, MDMA,MDA,MDEA)in the blood
of suspected drivers. Separate extraction procedures were used for
particular groups of drugs. The present report shows an alternative
option for the simultaneous isolation of all basic drugs mentioned
in the legal regulation with consecutive LC-APCIMS assay without derivatization. It must be added
i" A
that the extracts of drugs not subjected to defivatization were very stable when frozen, and practically
r
no changes were observed in the concentrations of
r
drugs involved over eight weeks of storage. The
same was observed previously for other drugs of
abuse (24-27).
j m/z 13S
]
0
:00: 4t ::V':
0
3:3?
/~A~,
~
t m/z 1~3
o
ot
ot
r
r
r
r
4:20
'~
....
ot '''~
O-
B
9 " ' ....
V
I ....
SO
' " " ~
I ....
100
l/S 136
' ....
I ....
150
' ....
3:40
100 1
[B
A ^
3:37
a,'z .~
ZO
r
r
t'-..
3:49
~
100 -~ ,m/: 1q;3
0 i ai/z 18o
__
1oo t
0 JIV'Z194
ZOO t
0 m/'Z 199
100 t
0 a~/Z 208
4:20
S:14
3:49
r
r
4:20
4:1S
5:14
,oo t
0
,oo t
II~z 21S
....
, ....
, ....
, . .=. , ....
5:13
, ....
, . . .__~'M~A-a~|...~
0
SO
100
:oo t w , ~j.,
150
,:4o
A
r c
3:40
3:37
Ik"~
_
1000 tW/" 180
100 t l / "
194
0
100 t l / "
199
o
~
~
----
. . . . . . . . .
n
50
,-,-a--.
3:49
j~DA
--
,
.
"
"/"~T"
~
100
. . . . . . .
r
r
4:20
~,,MDMA
I
4:18
/ ~M' D M A ' d I
r
"
"~
150
. . . . . . . . .
Figure4. SIM masschromatogramsof blank serumextractspikedwith amphetamine-dinMDMA-ds,
and MDEA-d7(A),of serum spikedwith amphetamine,MDMA, MDA, and MDEAto the concentration 50 IJg/L(B), andof forensic blood serumsample(C).The following concentrationswerefound in
this sample:amphetamine300 pg/L, MDMA 159 FJg/L,and MDA 18 tJg/L.
Conclusions
Solid-phase extraction procedure may be
applied for simultaneous isolation of psychoactive
phenethylamines, as well as for other alkaline
drugs of abuse of forensic importance.
LC-APCI-MS (single quadrupole) allowed the
differentiation of underivatized amphetamines
and other forensically relevant phenethylamines
with specificity and sensitivity sufficient for
forensic blood examination.
References
1. J.G. Omtzigt, C.J. Vermaase, and P.G. Zweipfenning. Deaths associated with amphetamine,
3,4-methylenedioxymethamphetamine (MDMA),
3,4-methylenedioxyethamphetamine (MDEA) or
3,4-methylenedioxyamphet- amine (MDA) abuse.
Proceedings of the 1994 TIAFT/SOFT Meeting, V.
Spiehler, Ed. Newport Beach, CA, 1995.
2. M.R. Moeller and M. Hartung. Ecstasyand related
substances--serum levels in impaired drivers.
J. Anal ToxicoL 21:591 (1997).
3. J.A. Henry, K.J.Jeffreys, and S. Dawling. Toxicity
and deathsfrom 3,4-methylenedioxymethamphetamine ("ecstasy"). Lancet 340:384-387 (1992).
4. A.R.W. Forrest, J.H. Galloway, I.D. Marsh, G.A.
Strachan, and J.C. Clark. A fatal overdose with 3,4methylenedioxyamphetamine derivatives. Forensic
5ci. Int. 64:57-59 (1994).
5. C.M. Milroy, J.C. Clark, and A.R.W. Forrest.
Pathology of deaths associated with "ecstasy" and
"eve" misuse.J. Clin. PathoL 49:149-153 (1996).
6. V. Fineschi and A. Masti. Fatal poisoning by
MDMA (ecstasy) and MDEA: a case report. Int.
J. Leg. Med. 108:272-275 (1996)9
7. W. Weinmann and M. Bohnert. Lethal monointoxication by overdosage of MDEA. Forensic 5ci. Int.
91:90-101 (1998).
8. R.W. Taylor, S.D. Le, S. Philip, and N.C. Jain.
Simultaneous identification of amphetamine,
methamphetamine using solid-phase extraction
and gas chromatography/nitrogen phosphorus
detection or gas chromatography/mass spectrometry. J. AnaL Toxicol. 13:293-295 (1989).
83
Journal of Analytical Toxicology,Vol. 24, March 2000
9. P. Lillsunde and T. Korte. Determination of ring-and N-substituted
amphetamines as heptafluorobutyryl derivatives. Forensic Sci. Int.
49:205-213 (1991).
10. C.R. Clark, J. DeRuiter, A. Valaer, and F.T. Noggle. Gas chromatographic-mass spectrometric and liquid chromatographic analysis of
designer butanamines related to MDMA. J. Chromatogr. Sci. 33:
328-337 (1995).
11. E. Meyer, J.F. Van Bocxlaer, W.E. Lambert, L. Thienpont, and A.P.
Leenheer. Identification of o~-phenethylamine in judicial samples.
J. Anal. Toxicol. 20:116-120 (1996).
12. F. Centini, A. Masti, and I. Barni Comparini. Quantitative and qualitative analysis of MDMA, MDEA, MA and amphetamine in urine by
head-space/solid phase microextraction (SMPE) and GC/MS.
Forensic Sci. Int. 83:161-166 (1996).
13. R. Kronstrand. Identification of N-methyl-l-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in urine from drug users.
J. Anal. Toxicol. 20" 512-516 (1996).
14. M. Yashiki, T. Kojima, T. Miyazaki, N. Nagasdawa, Y. Iwasaki, and
K. Hara. Detection of amphetamines in urine using head space-solid
phase microextraction and chemical ionization selected ion monitoring. Forensic Sci. Int. 76:169-177 (1995).
15. M.S. Kaufmann, A.C. Hatzis, and J.G. Stuart. Negative-ion chemical
ionization of amphetamine derivatives. J. Mass Spectrom. 31"
913-929 (1996).
16. A. Dasgupta and A.P. Hart. Distinguishing amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine from other
sympathomimetic amines after rapid derivatization with propyl chloroformate and analysis by gas chromatography-chemical ionization
mass spectrometry. J. Forensic Sci. 42:106-110 (1997).
17. P. Dallakian, H. Budzikiewicz, and H. Brzezinka. Detection and
quantitation of amphetamine and methamphetamine: electron
impact and chemical ionization with ammonia~omparative investigation on Shimadzu QP 5000 GC-MS system. J. Anal. Toxicol. 20:
255-261 (1996).
18. F. Sadeghuipour, C. Giroud, L. Rivier, and J.-L.Veuthey. Rapid determination of amphetamines by high-performance liquid chromatography with UV detection. J. Chromatogr. A 761 : 71-78 (1997).
19. E.Sadeghuipour and J.-L. Veuthey. Sensitiveand selective determination of methylenedioxylated amphetamines by high-performance
liquid chromatography with fluorimetric detection. J. Chromato~a A
787:137-143 (1997).
20. O.A. Dirbashi, N. Kuroda, S. Akiyama, and K. Nakashima. High-performance liquid chromatography of methamphetamine and its
related compounds in human urine following derivatization with fluorescein isothiocyanate. J. Chromatogr. B 695:251-258 (1997).
21. P. Esseiva, E. Lock, O. Gueniat, and M.D. Cole. Identification and
quantification of amphetamine and analogues by capillary zone
electrophoresis. Sci. Justice 37:113-119 (1997).
22. W. Pisternick, K.A. Kovar, and H. Ensslin. High-performance thinlayer chromatographic determination of N-ethyl-3,4-methylene-
84
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
dioxyamphetamine and its major metabolites in urine and comparison with high-performance liquid chromatography. J. Chromatogr. B
688:63-69 (1997).
M.J. Bogusz, M. Kala, and R.-D. Maier. Determination of
phenylisothiocyanate derivatives of amphetamine and its analogues
in biological fluids by HPLC-APCI-MS or DAD. J. Anal. ToxicoL 21:
59-69 (1997).
M.J. Bogusz, R.-D. Maier, and S. Driessen. Morphine, morphine-3glucuronide, morphine-6-glucuronide and 6-monoacetylmorphine
determined by means of atmospheric pressurechemical ionizationmass spectrometry-liquid chromatography in body fluids of heroin
victims. J. Anal. ToxicoL 21:346-355 (1997).
M.J. Bogusz, R.-D. Maier, M. Erkens, and S. Driessen. Determination
of morphine and its 3- and 6-glucuronides, codeine, codeine-6-glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography-atmospheric pressure chemical ionization mass
spectrometry. J. Chromatogr. B 703:115-127 (1997).
M.J. Bogusz, R.-D. Maier, K.D. KrCiger,and U. Kohls. Determination
of common drugs of abuse in body fluids using one isolation procedure and liquid chromatograph-atmospheric pressurechemical ionization mass spectrometry. J. Anal. Toxicol. 22:549-558 (1998).
M.J. Bogusz, R.-D. Maier, K.D. Kriiger, and W. Fr~chtnicht.
Determination of flunitrazepam and its metabolites in blood by
means of liquid chromatography-atmospheric pressure chemical
ionization mass spectrometry. J. Chromatogr. B 713:361-369
(1998).
Anderung des w 24a des Strassenverkehrsgesetzesund Bericht der
Grenzwertkommision. Toxichem und Krimtech 65:70-71 (1998).
A.M.A. Verweij and P.J.L.Lipman. Comparison of massspectrometric
ionization techniques for the analysis of phenethylamines.
J. Chromatogr. Sci. 34:379-382 (1996).
M.L. Goff, M i . Miller, J.D. Paulson, W.D. Lord, E. Richards, and A.I.
Omori. Effects of 3,4-methylenedioxymethamphetamine in decomposing tissues on the development of Parascophaga ruficornis
(Diptera:Sarcophagidae) and detection of the drug in postmortem
blood, liver tissue, larvae and puparia. J. Forensic Sci. 42:276-280
(1997).
W. Weinmann, M. Renz, C. Pelz, P. Brauchle, S. Vogt, and S. Pollak.
A micro-analytical method for simultaneously quantifying benzoylecgonine, amphetamine, codeine and morphine in blood serum
by means of GC/MS. Blutalkoho135:195-203 (1998).
H. K~ferstein and G. Sticht. Results of immunochemical and chromatographic investigations of blood samples in suspected drug
abuse. 1. Report:opiates, cocaine, cannabinoids and amphetamines.
Rechtsmedizin 8:173-177 (1998).
Manuscript received March 4, 1999;
revision received April 28, 1999.