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Forensic Science International 177 (2008) 77–84

www.elsevier.com/locate/forsciint

Determination of a newly encountered designer drug

‘‘p-methoxyethylamphetamine’’ and its metabolites
in human urine and blood
Kei Zaitsu a,*, Munehiro Katagi a, Tooru Kamata a, Hiroe Kamata a,
Noriaki Shima a, Hitoshi Tsuchihashi a,
Takeshi Hayashi b, Hisanaga Kuroki b, Ryoji Matoba b
a
Forensic Science Laboratory, Osaka Prefectural Police Headquarters, Japan
b
Department of Legal Medicine, Osaka University Graduate School of Medicine, Japan
Received 29 August 2007; received in revised form 25 October 2007; accepted 1 November 2007
Available online 21 December 2007

Abstract
A newly synthesized designer drug, para-methoxyethylamphetamine (PMEA) was unexpectedly detected in the postmortem specimens of
fatality involving drug intoxication in 2005, Japan. For unequivocal identification, the isomeric discrimination of PMEA and its positional-isomers
was performed by GC/MS with the trifluoroacetylation. In order to prove the intake of PMEA, the characteristic metabolites of PMEA were also
identified by GC/MS analysis of the urine specimen with trifluoroacetylation. As a result, para-methoxyamphetamine, para-hydroxyethylam-
phetamine (POHEA) and para-hydroxyamphetamine were identified as the major metabolites of PMEA. For the quantitative analyses of PMEA
and its three metabolites in body fluids, an automated column-switching LC/MS procedure was developed, and applied to the postmortem blood
and urine specimens. In this fatal case, blood concentration of PMEA was estimated to be 12.2 mg/mL and this level seemed extremely high in
comparison with lethal blood-levels of its analogues, representing acute-intoxication of the victim. Based on the quantitative results, PMEA was
found to be extensively metabolized to POHEA via O-demethylation, partly followed by its conjugation.
# 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: para-Methoxyethylamphetamine; Designer drug; Trifluoroacetylation; GC/MS; LC/MS

1. Introduction Numerous fatalities have been reported to be associated with

para-Methoxyethylamphetamine (PMEA), the N-ethylated
analogue of para-methoxyamphetamine (PMA) is a newly
synthesized designer drug [1], and there has been no literature on
the pharmacological and toxic properties of PMEA. Its parent
compound, PMA is, however, known as a potent drug with
both an amphetamine-like character and a hallucinogenic
potency, which is thought to be five times more potent than
mescaline [2]. Also, para-methoxymethamphetamine (PMMA),
an N-methylated analogue of PMA, is known as a psychoactive
substance with potent toxicity.
* Corresponding author at: Forensic Science Laboratory, Osaka Prefectural
Police Headquarters, 1-3-18, Hommachi, Chuo-ku, Osaka 541-0053, Japan.
Tel.: +81 6 6268 1234; fax: +81 6 6271 8066.
E-mail address: k_zaitsu_fsl_opp@ybb.ne.jp (K. Zaitsu).
the use of PMA and/or PMMA, mainly in Europe [3–11]. These
reports suggest that PMEA would exhibit almost the same
stimulant and psychoactive potency as those of PMA and
PMMA. It was, indeed, described as a potential drug of abuse by
Noggle et al. [12]. However, there has been no report not only on
the abuse of PMEA but also on a fatality involving PMEA.
In 2005, Japan, PMEA was unexpectedly detected in the
postmortem specimens of fatality involving drug intoxication,
and this implied PMEA poisoning.
For indisputable proof of drug intake, the detection of typical
metabolites as well as the parent drug is mandatory. Particularly
in a poisoning case, there is a need to measure the concentrations
of the parent drug and/or its metabolites in blood or urine.
Although there has been an analytical report of PMEA in human
urine [13], no metabolite of PMEA was identified in the report
and the PMEA metabolism in human is currently not clarified.

0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2007.11.001
78 K. Zaitsu et al. / Forensic Science International 177 (2008) 77–84
In the present study, we synthesized the expected
metabolites of PMEA on the basis of the previous studies on
the metabolism of PMA and PMMA, and attempted to identify
the metabolites of PMEA in postmortem specimens by gas
chromatography/mass spectrometry (GC/MS). The concentra-
tions of the identified metabolites were also measured by liquid
chromatography/mass spectrometry (LC/MS) with a column-
switching technique, which was developed for simple and rapid
determination of PMEA metabolites in biological fluids.
2. Case history
A 27-year-old male drunk up the unidentified liquid (ca.
20 mL), which had been sold as the ‘‘regal’’ recreational-drug
on the Internet. Approximately 30 min after ingestion, he got
out of shape and vomited. After that, he went into convulsions
and rampage, and finally resulted in a cessation of heartbeat and
breathing. He was immediately taken to a hospital, but
resuscitation was tried unsuccessfully. His rectal temperature
was 39.0 8C at 2 h after death (room temperature: 9 8C).
At Autopsy performed 33 h after death, the external
examination revealed that some bruises and abrasions on the
limbs. From the internal examination, the heart was normal size
and shape, and coronary arteries exhibited mild patchy arterio-
sclerosis. The lungs exhibited widespread marked congestion
and edema. The brain showed severe congestion and moderate
swelling with herniation. Moderate to severe stasis were found in
other organs. The microscopy showed stasis and edema of the
lungs and brain. Apart from mild arteriosclerosis, no other
preexisting pathologic findings could be established.
3. Experimental
3.1. Standards and chemicals
PMEA and its isomers were synthesized by reductive amination of the
corresponding methoxyphenylacetone with sodium cyanoborohydride and
ethylamine hydrochloride [14,15]. PMA was synthesized by reduction of
para-methoxyphenylacetone with sodium cyanoborohydride and ammonium
acetate [14,15]. Para-hydroxyethylamphetamine (POHEA) and para-hydro-
xyamphetamine (POHAP) were, respectively, synthesized by demethylation of
PMEA and PMA with hydrobromic acid [16]. All synthesized standards were
purified as hydrochlorides, and their structures were confirmed by mass
spectrometry, infrared spectroscopy (IR) and proton nuclear magnetic reso-
nance spectroscopy (1H NMR).
The stock standard solutions of these compounds were prepared in distilled
water at 10 mg/mL each. These solutions were stored at 4 8C until analysis and
further diluted to appropriate concentrations immediately prior to the use. The
calibrators for quantitative analysis were prepared by adding appropriately
diluted standard solutions to drug-free urine or blood before the analysis.
Dibenzylamine (DBA), used as an internal standard (I.S.), was purchased from
Wako Pure Chemical Industries (Osaka, Japan). A 2.5 mg/mL aqueous solution
of DBA was prepared by diluting a stock methanol solution (1 mg/mL).
Carbonic buffer solution was prepared by adding the mixture of sodium acid
carbonate (Wako Pure Chemical Industries) and sodium carbonate (Wako Pure
Chemical Industries) (20:80, w/w) to distilled water.
All other chemicals and reagents were purchased from Wako Pure Chemical
Industries (Osaka, Japan). All reagents used were of analytical grade or better
quality. Distilled water and HPLC-grade acetonitrile were used throughout the
experiments. b-Glucuronidase/sulfatase (Helix pomatia, type H-1) was pur-
chased from Aldrich (St. Louis, MO, USA).
3.2. Specimens
The postmortem heart-blood and urine specimens were obtained at the
autopsy described above, and these were stored at 20 8C until analysis. Drug-
free blood and urine were collected from a healthy volunteer (28-year-old
male).
3.3. Sample preparation
3.3.1. Enzymatic hydrolysis
Enzymatic hydrolysis was carried out according to our previous procedure
as follows [17]: to a 500 mL urine specimen, 500 mL of H. pomatia glucur-
onidase/sulfatase solution adjusted to pH 5 with an ammonium acetate buffer
was added at 60 mg/mL. The mixture was then incubated at 37 8C overnight to
hydrolyze conjugates.
3.3.2. Preparation for GC-MS analysis
Extraction with 2 mL of methylene chloride/isopropanol (4:1, v/v) of the
500 mL deconjugated urine sample was carried out at pH 10 using a carbonic
buffer. After centrifuging for 10 min at 1500  g, the organic layer was isolated,
and 10 mL of a methanol/HCl solution (99:1, v/v) were added to the extract in
order to avoid losses of volatile substances. After evaporation to dryness under a
nitrogen stream, the residue was trifluoroacetylated at 60 8C for 20 min using a
200-mL mixture of trifluoroacetic anhydride and ethyl acetate (1:1, v/v). After
evaporation, the final residue was dissolved in 250 mL of ethyl acetate, and a 1-
mL aliquot was manually injected into the GC-MS systems.
3.3.3. Preparation for LC/MS analysis
Both the urine samples before and after hydrolysis were used for LC/MS
analyses, and these were appropriately diluted with drug-free urine if necessary.
To 100 mL each of the blood and the urine samples, 400 mL of a DBA solution
(2.5 mg/mL) was added. After vortex mixing for 15 s, the mixtures were
centrifuged for 10 min at 1500  g, and their supernatants (200 mL) were
isolated. After filtration through a 0.2-mm membrane filter, a 20-mL aliquot
each was automatically injected into the LC-MS systems.
3.3.4. Preparation for LC/MS/MS analysis
A 100-mL urine sample was deproteinized by adding 500-mL methanol.
After centrifuging for 10 min at 1500  g, the supernatant was transferred to
glass tube and evaporated to dryness at 60 8C under nitrogen. The residue was
dissolved in 200 mL of distilled water. After filtration through a 0.2-mm
membrane filter, a 10-mL aliquot was automatically injected into the LC/
MS/MS systems.
3.4. GC/MS analysis
GC/MS analysis was performed on a Shimadzu GC-MS QP2010 (Shi-
madzu, Kyoto, Japan) with a fused-silica capillary column DB-5ms
(30 m  0.32 mm i.d.; film thickness, 0.25 mm; J&W Scientific, Folsom,
CA, USA). The temperature program consisted of an initial temperature of
80 8C held for 1 min, followed by a linear ramp to 300 8C at 10 8C/min. The
split injector temperature was set at 250 8C. The carrier gas was high-purity
helium at a flow rate of 3.0 mL/min. The detector parameters used were as
follows: the interface temperature, 250 8C; ion-source temperature, 200 8C;
ionization mode, electron ionization (EI); ionization voltage, 70 eV; scan time,
0.5 s/scan; scan range, 40–500 Da.
3.5. LC/MS analysis
LC/MS analysis was performed on a Shimadzu LCMS-QP2010 HPLC-
quadrupole mass spectrometer (Shimadzu) equipped with a six-port column-
switching valve and an electrospray ionization (ESI) interface. Based on our
previous studies [18–20], a Shodex MS-pak PK-2A (N-vinylacetamide-contain-
ing copolymer gel, 10 mm  2.0 mm i.d.; Showa Denko, Tokyo, Japan) was
used for on-line extraction, and an L-column ODS semi-micro column (pore
size, 120 Å; 5-mm particles; 150 mm  1.5 mm i.d.; Chemicals Evaluation and
 K. Zaitsu et al. / Forensic Science International 177 (2008) 77–84
Research Institute, Tokyo, Japan) was used as the separation column, respec-
tively.
The mobile phase used for introducing samples to the trap column was
5 mM ammonium acetate (0.5 mL/min). The trapped analytes were eluted and
chromatographed by gradient elution with mobile phases A (10 mM formic
acid–acetonitrile; 95:5, v/v) and B (10 mM formic acid–acetonitrile; 70:30, v/
v) at a flow rate of 0.2 mL/min (0–4 min, B: 0%; 4–25 min, B: 0–100%; 25–
30 min, B: 100–0%; 30–45 min, B: 0%). Both LC separation and on-line
extraction were carried out at 30 8C, and the entire flow of the eluate was
introduced to the ESI interface. ESI-MS was performed in the positive mode.
Quantitative analyses were carried out in triplicate in the selected-ion
monitoring (SIM) mode. The other operating parameters were as follows:
nebulizer nitrogen gas flow-rate, 1.5 L/min; curved desolvation line (CDL)
voltage, 25 V; Q-array bias; 10 V; CDL temperature, 250 8C; ion-source
temperature, 200 8C.
3.6. LC/MS/MS analysis
LC/MS/MS analysis was performed on a Quattro LC (Waters, Milford,
MA, USA) system equipped with an Agilent 1100 pump (Agilent Technol-
ogies, Palo Alto, CA, USA). The same chromatographic conditions proposed
in our previous study were used for the direct analysis of the conjugates [21]:
an L-column ODS semi-micro column (as shown earlier) was used as the
separation column, and the analytes were chromatographed by linear-gradient
elution with 10 mM ammonium formate buffer (pH 3.5) and methanol (5–
40%, v/v). Argon was used as the collision gas, and its energy was set at
15 eV. The ions of m/z 356 and m/z 260, corresponding to the protonated
molecules of glucuronide and sulfate of POHEA, respectively, were selected
as precursor ions. The other parameters of the tandem MS detector were as
follows: the type of ionization, ESI-positive; scan mode, product ion scan;
scan range, m/z 40–360 for glucuronide, and m/z 40–280 for sulfate; cone
voltage, 20 V; capillary voltage, 2.0 kV; ion-source temperature, 280 8C;
nebuliser nitrogen-gas flow rate, 90 L/h.
Fig. 1. Mass chromatograms and EI mass spectra of free-base (A) and trifluoroacetyl-derivative (B), respectively, obtained from the postmortem urine by GC/MS
analysis. An arrow on the chromatograms points out the target peak.
79
4. Results and discussion
4.1. Identification of p-methoxyethylamphetamine
The extract from the urine specimen after enzymatic
hydrolysis was subjected to GC/MS analysis. As shown in
Fig. 1, a large peak was observed on the TIC, and the EI mass
spectrum for the peak presented a predominant ion at m/z 72,
and other less intense ions at m/z 44 and 121. Based on our
previous study, the characteristic ions at m/z 72 and 121 would
correspond to immonium ions generated by an a-cleavage of
the amine bond and methoxy-substituted tropylium cations
generated by an a-cleavage of the benzyl bond, respectively.
Therefore, methoxyethylamphetamines or methoxydimethy-
lamphetamines were proposed as possible candidates [14].
Also, the fragment ion at m/z 44 in the mass spectra must be due
to an olefin loss reaction of the immonium ion on losing an
ethyl group at the nitrogen [22]. These results suggested the
chemical structure of methoxyethylamphetamine.
For further structural determination, trifluoroacetylation of
the extract was carried out, followed by GC/MS. The peak of
the trifluoroacetyl (TFA)-derivative of the analyte was
observed, representing the methoxyethylamphetamine struc-
ture (the obtained mass spectrum is shown in Fig. 1). However,
there are three positional-isomers (ortho-, meta- and para-)
among the methoxyethylamphetamines, and it is necessary to
determine the position of the methoxy-moiety on the benzene
ring. Our previous study on PMA and PMMA revealed that the
80 K. Zaitsu et al. / Forensic Science International 177 (2008) 77–84

Fig. 2. EI mass spectra of trifluoroacetyl-derivatives of the authentic standards for methoxy-substituted isomers. Abbreviations: OMEA-TFA, trifluoroacetyl-
derivative of ortho-methoxyethylamphetamine; MMEA-TFA, trifluoroacetyl-derivative of meta-methoxyethylamphetamine; PMEA-TFA, trifluoroacetyl-derivative
of para-methoxyethylamphetamine.

trifluoroacetylation of methoxy-isomers including PMA and 4.2. Determination of PMEA metabolites

PMMA proved to be effective for their mass spectral
differentiation [14].
Thus, the authentic standards of ortho-, meta- and para-
methoxyethylamphetamine were synthesized and subjected to
GC/MS determination with the TFA-derivatization. Fig. 2
shows the mass spectra of the TFA-derivatives of the three
authentic standards. As shown in Fig. 2, significant differences
in ion intensities, especially at m/z 121 and 148, were observed
between PMEA and the others, and the compound was
identified as PMEA.
For unequivocal proof of the PMEA intake, determination of
the urinary metabolites of PMEA was carried out. No report has
appeared on the analysis of PMEA metabolites in human
biological specimens, as mentioned earlier. According to the
previous studies on the metabolism of PMA, PMMA and PMEA
[23–27], POHAP, POHMA and PMA were expected as the main
metabolites of PMEA in human urine. The extract from the urine
specimen was then analyzed by GC/MS after TFA-derivatiza-
tion. As shown in Fig. 3, three unknown compounds were

Fig. 3. Mass chromatograms obtained from the urine extract by GC/MS analysis after trifluoroacetyl-derivatization. Abbreviations: POHAP-(TFA)2, di-
trifluoroacetyl-derivative of para-hydroxyamphetamine; PMA-TFA, trifluoroacetyl-derivative of para-methoxyamphetamine; POHEA-(TFA)2, di-trifluoroacetyl-
derivative of para-hydroxyethylamphetamine; PMEA-TFA, trifluoroacetyl-derivative of para-methoxyethylamphetamine. EI mass spectra obtained from the urine
extract (upper part), and their fragmentation pattern (lower part). Abbreviations: TFA, trifluoroacetyl-group; POHAP-(TFA)2, di-trifluoroacetyl-derivative of para-
hydroxyamphetamine; PMA-TFA, trifluoroacetyl-derivative of para-methoxyamphetamine; POHEA-(TFA)2, di-trifluoroacetyl-derivative of para-hydroxyethy-
lamphetamine.
 K. Zaitsu et al. / Forensic Science International 177 (2008) 77–84
Fig. 4. LC/MS profiles obtained from a spiked urine sample at 1 mg/mL each in
the selected ion monitoring mode. (The concentration of I.S. corresponds to
0.5 mg/mL.) Abbreviations: POHAP, para-hydroxyamphetamine; POHEA,
para-hydroxyethylamphetamine; PMA, para-methoxyamphetamine; PMEA,
para-methoxyethylamphetamine; I.S., internal standard (dibenzylamine).
detected, which were most likely to be the tentative metabolites,
POHAP, POHEA and PMA, described above. To confirm the
above-mentioned tentative identification, the synthesized-
authentic standards of POHEA, POHAP and PMAwere analyzed
by GC/MS with the trifluoroacetylation, and the analytical data
were compared. Both mass spectra and retention times for the
three analytes obtained from the urine specimen were identical to
those of the authentic standards. Thus, the three compounds were
identified as POHEA, POHAP and PMA.
4.3. Method development for simultaneous determination
by column-switching LC-MS
For the simultaneous determination of PMEA and its three
metabolites in blood and urine, an automated column-switching
LC/MS procedure was employed. This technique provides an
efficient and effective analytical methodology, which enables a
simultaneous detection of both a parent drug and its metabolites
without a tedious sample pretreatment. Our previous studies
have shown that a hydrophilic polymer precolumn gives high
sensitivity in analyses of amphetamines and their para-
hydroxylated metabolites [19].
In order to evaluate the extraction efficiencies of the
precolumn, the analytes were then trapped only with the
precolumn, and its eluate with 5 mM ammonium acetate
(0.5 mL/min) was analyzed by LC/MS. As a result, no analyte
was detected in the eluate, and it proved that sufficient retention
of analytes was achieved with the precolumn (data not shown).
Table 1
Validation data of the established on-line extraction LC/MS procedure for spiked blood samples (N = 3)
POHAP
Linearitya 0.996
b
Accuracy and precision (mg/mL  CV%) 0.51  2.8
1.10  2.4
10.6  3.6
Recovery (%)c 44
d
LOD (pg) 600
a
Linearity of calibration curve constructed in the range of 0.1–20 mg/mL.
b
Estimated by analyzing the samples spiked at 0.5, 1 and 10 mg/mL each.
c
Evaluated at 1 mg/mL each.
d
Estimated as the absolute amount showing signal-to-noise (S/N) ratio greater than 3.
81
To pursue the best LC separations and MS responses of the
analytes, mobile phase systems were optimized on the basis of
our previous studies [19,20]: The proposal condition was shown
in Section 3. Fig. 4 shows the extracted mass chromatograms in
the SIM mode obtained from a blood sample, which was spiked
with PMEA and its metabolites at 1 mg/mL each, by the
optimized method. No chromatographic or spectral inter-
ference by blood and urine components was observed for each
analyte, and excellent peak separation of each analyte was
achieved.
The optimized LC/MS methodology was then validated.
Calibration curves constructed by the I.S. method showed good
linearity over the ranges from 0.1 to 20 mg/mL. The limit of
detection (LOD) was estimated as the absolute amount showing
the signal-to-noise (S/N) ratio greater than 3, and the limit of
quantitation (LOQ) was defined at the lower limit of the
established calibration curves (0.1 mg/mL) for all the analytes.
The on-line extraction recoveries were determined by
comparing the peak areas from the spiked samples and those
from the reference aqueous solutions at the same concentration
(1 mg/mL each). The accuracy and precision were evaluated by
triplicate analyses of spiked samples at 0.5, 1 and 10 mg/mL
each for all the analytes. The validation data for blood and urine
are summarized in Tables 1 and 2, respectively.
As listed in Tables 1 and 2, the recoveries obtained from
blood were around 50%, while those obtained from urine
specimens were about 90%. Such lower recoveries observed in
blood samples might be mainly due to binding of the analytes to
protein. However, linearity with excellent correlation coeffi-
cients (r2 > 0.99) was achieved in the investigated range, even
for the blood samples. Therefore, the present methodology was
found to be effective for quantitative analyses of PMEA and its
metabolites.
4.4. Quantitative analysis of PMEA and its metabolites
The validated method was applied to the postmortem blood
and urine specimens, and especially the urinary levels of PMEA
and its metabolites were compared between specimens before
and after hydrolysis. The quantitative results are listed in
Table 3. Although there is no report dealing with the fatal
PMEA blood-level, it has been reported that the lethal blood-
POHEA PMA PMEA
0.990 0.997 0.992
0.49  4.3 0.51  1.1 0.51  2.7
1.03  1.1 0.98  2.2 0.98  1.6
9.95  5.1 10.4  2.5 9.84  4.0
46 55 62
100 400 50
82 K. Zaitsu et al. / Forensic Science International 177 (2008) 77–84

Table 2
Validation data of the established on-line extraction LC/MS procedure for spiked urine samples (N = 3)
POHAP POHEA PMA PMEA
a
Linearity 0.999 0.994 0.999 0.998
Accuracy and precisionb (mg/mL  CV%) 0.50  3.5 0.49  3.0 0.51  4.5 0.52  4.7
0.98  1.7 0.97  2.8 1.05  2.7 0.99  5.3
10.4  4.4 9.75  3.7 9.78  4.9 9.64  3.3
Recovery (%) c 79 95 100 99
d
LOD (pg) 250 50 100 20
a
Linearity of calibration curve constructed in the range of 0.1–20 mg/mL.
b
Estimated by analyzing the samples spiked at 0.5, 1 and 10 mg/mL each.
c
Evaluated at 1 mg/mL each.
d
Estimated as the absolute amount showing signal-to-noise (S/N) ratio greater than 3.

Table 3 heart blood might be about twice or so as high as that in

The concentrations of PMEA and its metabolites in blood and urine (mg/mL) peripheral blood. Therefore, the present heart-blood PMEA
Blood Urine level (12.2 mg/mL) seemed extremely high in comparison with
Before hydrolysis After Hydrolysis the peripheral lethal levels of PMA or MA, representing acute-
intoxication of the victim.
POHAP <0.1 0.59 1.53
POHEA 1.07 93.8 106
As listed in Table 3, POHEA was the most abundant among
PMA 0.51 1.18 1.19 the identified metabolites, and its urinary level was about 80–
PMEA 12.2 94.7 96.2 160 times higher than those of the others. This suggests that
PMEA is extensively metabolized mainly by O-demethylation
into POHEA in phase 1 metabolism. A previous report on in
levels of PMA and methamphetamine (MA), the analogues of vitro PMEA metabolism has shown that PMEA was principally
PMEA, ranged over 0.2–4.9 (average, 2.0 mg/mL) and 5–7 mg/ metabolized into POHEA via CYP2D6-mediated metabolism
mL, respectively [3,28]. [24], and our results were in good agreement with theirs.
These reference data were obtained from peripheral blood Also, a slight increase in the urinary POHEA concentration
samples, and site-dependent concentration differences have was observed after hydrolysis, compared with that before
been well known for amphetamine analogues [6,27,29,30]; hydrolysis. This suggests the existence of POHEA conjugates
Barnhart et al. have been reported that postmortem heart/ in the urine specimen. Direct analysis of POHEA conjugates in
femoral blood-concentration ratios of MA and amphetamine the urine specimen by LC/MS/MS was then carried out
averaged 2.1 (range, 1.2–5.0) and 2.4 (range, 1.2–5.8), according to our previously published procedure for urinary
respectively [29]. Martin have been reported that only slight POHMA conjugates (see Section 3).
differences between heart and peripheral blood-concentration Fig. 5 shows the product ion spectra derived from precursor
in PMA fatal cases were observed [6]. These data suggests that ions at m/z 356 and 260, which correspond to the protonated
the concentration of amphetamine analogues in the postmortem molecules of the glucuronide and sulfate of POHEA,

Fig. 5. Product ion spectra produced from precursor ions at m/z 355.9 and 259.9, respectively, and schematic diagrams for their fragmentation pattern. Abbreviations:
POHEA-glu, glucuronide of POHEA; POHEA-sul, sulfate of POHEA.
 K. Zaitsu et al. / Forensic Science International 177 (2008) 77–84
respectively. In either of the spectra, some characteristic
fragment ions representing the glucuronide and sulfate of
POHEA were observed. In addition, the hydrolysis of the urine
specimens resulted in the disappearance of the peaks
corresponding to the conjugates.
These results revealed that POHEA was partly excreted as its
conjugates in the urine, though it was impossible to exactly
measure their urinary concentrations because of the unavail-
ability of authentic standards for the conjugates. However,
based on our previous studies on the glucuronide and sulfate of
POHMA, the several-fold higher ion intensity of sulfate than
that of glucuronide revealed that the sulfation would be much
superior to glucuronidation in the metabolism of POHEA
[17,31].
Also, our previous study on MA metabolism in humans
demonstrated that an average of 80% of POHMA was excreted
as its sulfate in urine [17]. On the basis of the structural
similarities between POHMA and POHEA, POHEA was
mostly expected to be excreted as its conjugates, especially the
sulfate. In this study, however, the increase in the concentration
of free-form POHEA after hydrolysis was equivalent to about
13%. This increasing-rate was significantly low in comparison
with that in the case of urinary POHMA in MA abusers.
It is well known that an increase in the substrate (for
example, derived from overdosing) readily results in drying up
the coenzyme 3-phosphadenosine-5-phosphosulfate (PAPS),
which is indispensable for sulfation, and it leads to saturation of
the sulfation pathway in the metabolism of phenols [32]. Thus,
in the present study, an extremely high concentration of the
substrate POHEA may be responsible for saturation of the
sulfation of POHEA. However, there is a need for further
investigation on the metabolism of PMEA, especially the
conjugation of POHEA.
5. Conclusion
This is the first report on the analyses of the new designer
drug PMEA along with its metabolites in human biological
samples. By using GC/MS analysis of the postmortem urine
and blood specimens, POHEA, PMA and POHAP were
identified as the metabolites of PMEA. Reliable simultaneous
determination of PMEA and its metabolites was also achieved
using our established column-switching LC-MS technique.
Blood concentration of PMEA (12.2 mg/mL) was extremely
high in comparison with lethal blood-levels of MA and PMA,
and it would cause acute-death of the victim in this fatal case.
Among the identified urinary metabolites, POHEA was the
most abundant, and this suggests the extensive metabolism of
PMEA to POHEA via O-demethylation. Since a slight increase
in the POHEA concentration was observed after hydrolysis of a
urine sample, POHEA was expected to be partly excreted as its
conjugates. Although the direct analyses of POHEA con-
jugates by LC/MS/MS suggested that most of the conjugates
would be sulfate, further studies on the PMEA metabolism
would be indispensable, especially on the conjugation of
POHEA.
83
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