Clinical Chemistry 52:9
1728 –1734 (2006)
Drug Monitoring and
Toxicology
Sensitive Gas Chromatography-Mass Spectrometry
Method for Simultaneous Measurement of MDEA,
MDMA, and Metabolites HMA, MDA, and HMMA
in Human Urine
Stephane O. Pirnay, Tsadik T. Abraham, and Marilyn A. Huestis*
Background: A sensitive gas chromatography-mass
spectrometry method was developed and validated for
the simultaneous measurement of MDEA, MDMA, and
its metabolites, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxymethamphetamine
(MDMA or Ecstasy), and its metabolites, 4-hydroxy-3methoxyamphetamine (HMA), 3,4-methylenedioxyamphetamine (MDA), and 4-hydroxy-3-methoxyamphetamine (HMMA) in human urine.
Methods: We hydrolyzed 1 mL urine, fortified with
MDMA-d5, MDA-d5, and MDEA-d6, with 100 ␮L of
concentrated hydrochloric acid at 120 °C for 40 min, then
added 100 ␮L 10 N sodium hydroxide and 3 mL phosphate buffer 0.1 N (pH 6.0) were added to hydrolyzed
urine specimens before solid-phase extraction. After
elution and evaporation, we derivatized extracts with
heptafluorobutyric acid anhydride and analyzed with
gas chromatography-mass spectrometry operated in EIselected ion-monitoring mode.
Results: Limits of quantification were 25 ␮g/L for
MDEA, MDMA, and its metabolites. Calibration curves
were linear to 5000 ␮g/L for MDEA, MDMA, HMA,
MDA, and HMMA, with a minimum r2 > 0.99. At 3
concentrations spanning the linear dynamic range of the
assay, mean overall extraction efficiencies from urine
were >85.5% for all compounds of interest. Intra- and
interassay imprecisions, produced as CV, were <15%
for all drugs at 30, 300, and 3000 ␮g/L.
1
Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan
Shock Drive, Baltimore, MD.
* Address correspondence to this author at: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224; fax
410-550-2971; e-mail [email protected].
Received February 19, 2006; accepted June 8, 2006.
Previously published online at DOI: 10.1373/clinchem.2006.069054
Conclusions: This gas chromatography-mass spectrometry assay provides adequate sensitivity and performance characteristics for the simultaneous quantification of MDEA, MDMA, and its metabolites HMMA,
MDA, and HMA in human urine. The method meets
and exceeds the requirements of the proposed Substance Abuse and Mental Health Services Administration’s guidelines for federal workplace drug testing of
MDEA and MDMA in urine.
© 2006 American Association for Clinical Chemistry
Methylenedioxy derivatives of amphetamines, 3,4-methylenedioxyamphetamine (MDA),3 3,4-methylenedioxyethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA), constitute a major class of central
nervous system stimulants producing well described cardiovascular and behavioral effects (1, 2 ). The US Drug
Enforcement Administration restricted use of these compounds more than 2 decades ago, yet consumption of these
drugs has increased dramatically (3, 4 ). MDMA drug abuse
is especially problematic among young people (5 ). Although
acute intoxications have been widely documented (6, 7 ), the
potential for long-term central nervous system toxicity is the
subject of much controversy (8 ).
MDMA is excreted primarily as unchanged drug in
urine, with additional N-demethylation to MDA, O-dealkylation to 3,4-dihydroxymethamphetamine (HHMA)
and 3,4-dihydroxyamphetamine (HHA), and O-methylation to 4-hydroxy-3-methoxymethamphetamine (HMMA)
and 4-hydroxy-3-methoxyamphetamine (HMA) (see Fig.
3
Nonstandard abbreviations: MDA, 3,4-methylenedioxyamphetamine;
MDEA, 3,4-methylenedioxy-N-ethylamphetamine; MDMA, 3,4-methylenedioxymethamphetamine (Ecstasy); HHMA, 3,4-dihydroxymethamphetamine;
HHA, 3,4-dihydroxyamphetamine; HMMA, 4-hydroxy-3-methoxymethamphetamine; HMA, 4-hydroxy-3-methoxyamphetamine; GC-MS, gas chromatography/mass spectrometry; SPE, solid-phase extraction; LOD, limit of
detection; LOQ, limit of quantification.
1728
Clinical Chemistry 52, No. 9, 2006
1 in the Data Supplement that accompanies the online
version of this article at http://www.clinchem.org/
content/vol52/issue9). In determining disposition of xenobiotic compounds in urine, characterization of parent
drug and metabolites is useful because both may contribute to toxicity, and metabolites frequently have longer
terminal elimination half-lives, increasing the window of
drug detection. In support of our controlled human drug
administration studies, we developed and validated a
method to simultaneously measure MDEA, MDMA, and
its most important metabolites.
Gas chromatography/mass spectrometry (GC-MS) is
the most common instrumental technique for analysis of
amphetamines and derivatives (9 –12 ). Derivatization is
required to improve chromatography, sensitivity, and
reproducibility of primary and secondary amines (13, 14 ).
Liquid-liquid (10, 15, 16 ) and solid-phase extraction (SPE)
procedures have been published (17–19 ). Capillary electrophoresis-MS (20, 21 ) and liquid chromatography-MS (22–
25 ) assays also are available. In general, methods have
included a limited number of compounds and a limited
linear range. Our clinical pharmacokinetic research requires
low limits of quantification (LOQ) to determine terminal
elimination half-lives and large dynamic ranges to monitor
initial urinary excretion of amphetamines and metabolites.
The method is suitable for analysis of MDEA, and MDMA
under proposed Substance Abuse and Mental Health Services Administration (SAMHSA) guidelines (26 ).
We report the development of a robust, rapid, sensitive, and relatively inexpensive GC-MS assay for simultaneous quantification of 5 amphetamine analogs. The
method uses a simple and inexpensive acid hydrolysis for
recovery of MDMA-conjugated metabolites and is suitable for pharmacokinetic studies and for analysis of urine
specimens with a large range of drug concentrations in
clinical and forensic toxicology laboratories.
Materials and Methods
reagents
Solvents were HPLC grade from Mallinckrodt Baker.
Ammonium hydroxide, glacial acetic acid, hydrochloric
acid, dibasic potassium phosphate, monobasic potassium
phosphate, sodium acetate, and sodium hydroxide were
American Chemical Society reagent grade, also from
Mallinckrodt Baker. Heptafluorobutyric acid anhydride
was purchased from Pierce Biotechnology, Inc.
SPE columns (Clean Screen ZSDAU020) and vacuum
manifolds were obtained from United Chemical Technologies. Drug-free urine was collected from volunteers and
evaluated by GC-MS to ensure the absence of MDMA and
metabolites.
calibrators and quality control samples
Reference standards for MDA, MDA-d5, HMMA, MDMA,
MDMA-d5, MDEA, and MDEA-d6 were obtained from
Cerilliant and for HMA from Lipomed. All stock solutions
were 1.0 g/L in methanol, except MDEA-d6 stock solu-
1729
tion, which was 100 mg/L in methanol. Intermediate
solutions of 100, 10, 1, and 0.1 mg/L were prepared in
methanol. Working calibrators at 10, 25, 50, 500, 1000,
2500, 5000, and 10 000 ␮g/L were prepared by fortifying
1 mL of blank urine with HMA, MDA, HMMA, MDMA,
and MDEA. A mixture of deuterated internal standards
(50 ␮L of 1000 ␮g/L), MDMA-d5, MDA-d5, and MDEA-d6
was added to each urine specimen before hydrolysis.
acid hydrolysis procedure
We hydrolyzed 1 mL of urine with 100 ␮L of concentrated
hydrochloric acid in a gas chromatograph (GC) oven at
120 °C for 40 min. After specimens cooled to room temperature, 100 ␮L of 10 N sodium hydroxide and 3 mL of
0.1 N phosphate buffer (pH 6.0) were added. Before
application to extraction columns, the pH of samples was
checked and adjusted if needed (pH 5– 6.5). Tubes were
vortex-mixed for 0.5 min and centrifuged at 1850g for 10
min.
extraction and derivatization
SPE columns were conditioned with methanol, deionized
water, and 100 mmol/L phosphate buffer (pH 6.0). Supernatants from acid hydrolysis were applied to conditioned SPE columns under gravity flow. Columns were
washed with deionized water, 1.0 mol/L acetic acid, and
methanol, and dried under vacuum. Analytes were eluted
with methylene chloride:iso-propanol:concentrated ammonium hydroxide; (78:20:2 v/v). We added 50 ␮L of 1%
hydrochloric acid in methanol were added to eluates
before evaporation under nitrogen at 37 °C in a Zymark
Turbovap® LV Evaporator and added 50 ␮L of ethyl
acetate and 50 ␮L of heptafluorobutyric acid anhydride to
dried extracts. Vials were capped and derivatized at 70 °C
for 10 min.
After the samples cooled, 25 ␮L of 1% hydrochloric
acid in acetonitrile were added, and derivatized extracts
were evaporated to dryness. Extracts were reconstituted
with 75 ␮L of heptane, vortex-mixed, centrifuged at 1850g
for 5 min, and transferred to autosampler vials.
gc-ms analysis
GC/MS analysis was performed with an Agilent 6890 GC
interfaced to an Agilent 5973 mass-selective detector. The
GC was equipped with a DB-35MS capillary column
(15 m ⫻ 0.32 mm, internal diameter ⫻ 0.25 ␮m film
thickness). Helium was used as carrier gas at a constant
pressure of 2.0 psi; 1 ␮L of derivatized urinary extract was
injected (split ratio 10:1). Injection port and transfer line
temperatures were 250 °C and 280 °C, respectively. The
initial column temperature of 70 °C was held for 2 min,
followed by temperature ramps of 20 °C/min to 160 °C,
hold for 2 min; 15 °C/min to 200 °C, with a 1-min postrun
hold at 300 °C. Total separation run time was 12.2 min.
The ion source was maintained at 230 °C and quadrupole
at 150 °C. Selected ion monitoring mode was used with a
dwell time of 10 ms. Three ions for each analyte and 2 for
1730
Pirnay et al.: Sensitive GC-MS to Measure MDMA and Metabolites
each deuterated internal standard were monitored (quantification ions are italic): HMA-d0, m/z 240, 360, 333;
MDA-d5, m/z 167, 244; MDA-d0, m/z 162, 135, 375;
HMMA-d0, m/z 360, 254, 210; MDMA-d5, m/z 258, 213;
MDMA – d0, 254, 210, 162, MDEA-d0, m/z 268, 240, 162;
and MDEA-d6, m/z 274, 244.
Choice of monitored ions for MDMA, MDA, and
MDEA were reported previously by Stout et al. (27 ).
Moreover, HMA and HMMA were derivatized on both
amine and hydroxyl functional groups corresponding to
240 m/z, 360 m/z, and 333 m/z for HMA, and 254 m/z, 360
m/z, and 210 m/z for HMMA.
Deuterated HMA and HMMA are not yet commercially available; therefore, MDA-d5 was chosen as the
internal standard for HMA, MDA, and HMMA.
data analysis
Calibration with internal standardization was accomplished by linear regression analysis curve fitting with a
weighting factor of 1/x. Peak area ratios of target analytes
and internal standards were calculated by MSD Chemstation software (v D.00.00). Working calibration standards
were prepared daily at 10, 25, 50, 500, 1000, 2500, 5000,
and 10 000 ␮g/L.
validation experiments and acceptance
criteria
Method linearity was investigated by evaluation of the
regression line and produced by the squared correlation
coefficient (r2). Six calibrators were included in each curve
and were required to meet all qualitative identification
and quantification criteria. Each calibrator was calculated
against the linear regression curve from 6 calibrators;
quantitative accuracy was required within 20% of target.
Linearity was achieved with a minimal r2of 0.99.
Method sensitivity was evaluated by determining limit
of detection (LOD) and LOQ for each compound. LOD
was defined as lowest concentration with ion signal-tonoise ratio (determined by peak height) ⱖ3:1 with satisfactory chromatography (peak shape and resolution),
acceptable retention time (⬃2% of average calibrator
retention time), and qualifier ion ratios within 20% of the
average of 6 calibrators. LOQ was defined as the lowest
concentration that met LOD criteria with measured concentration within 20% of target concentration in 6 replicates.
Recovery for each analyte was assessed by adding
internal standard to 1 set of low, medium, and high
control solutions (30, 300, and 3000 ␮g/L) before solid
phase extraction and to a second set after extraction, but
before evaporation. Samples were derivatized and analyzed. Another set was prepared with undiluted analyte
and internal standard solutions that were evaporated,
derivatized, and analyzed. Recovery (percentage) was
calculated by comparing the peak area ratios of analyte to
internal standard for extracted and unextracted samples.
Validation samples at 3 concentrations (30, 300, and
3000 ␮g/L) were prepared from stock solutions and
analyzed (n ⫽ 6) with independent calibration curves on
6 assays. Method recovery, produced as a percentage, was
calculated by comparing mean calculated concentrations
of HMA, MDA, HMMA, MDMA, and MDEA in validation samples to target concentrations. Interassay precision
was assessed by calculating percent relative standard
deviation (%RSD) of 6 replicates over 5 independently
calibrated assays (n ⫽ 30). Intraassay precision was assessed by calculation of %RSD from one run (n ⫽ 6).
Intraassay data are presented for the day with greatest
variability. Intraassay and interassay precision were calculated at 3 concentrations across the assay’s dynamic
range.
To evaluate interference and method selectivity, 8
blank (no analyte or IS added) urine specimens were
evaluated for coeluting chromatographic peaks that might
interfere with detection of analytes of interest or deuterated internal standards. Internal standard materials also
were tested for the presence of native analyte ions.
To assess possible interference from other commonly
available drugs, low concentration quality control (QC)
samples were enriched individually to contain 10 000
␮g/L of 6-acetylmorphine, acetylsalicylic acid, anhydroecgonine methyl estermesylate, benzoylecgonine, brompheniramine, caffeine, cannabinol, clonidine, chlorpheniramine, diphenhydramine, dextromethorphan, ecgonine
ethyl ester, ecgonine methyl ester, ephedrine, fenfluramine,
GHB, hydrocodone, hydromorphone, 11-hydroxy-tetrahydrocannabinol, ibuprofen, m-hydroxybenzoylecgonine,
m-hydroxycocaine, methadone, morphine, nicotine, norcocaine, norcocaethylene, norpseudoephedrine, oxymorphine,
p-hydroxybenzoylecgonine, p-hydroxycocaine, p-methoxyamphetamine, p-methoxymethamphetamine, pentazocine,
phentermine, phenylpropanolamine, and pseudoephedrine.
Moreover, to evaluate interference with high concentrations
of ephedrine, pseudoephedrine, and propanolamine, low
concentration QC samples were enriched individually to
contain 1 g/L of ephedrine, pseudoephedrine, and propanolamine. Acceptance criteria required adequate resolution
and peak shape and quantification within 20% of target
concentrations.
Several studies were performed to determine stability
of HMA, MDA, HMMA, MDMA, and MDEA under
different conditions. Temperature stability studies were
performed on 4 sets of control urine samples enriched at
30, 300, and 3000 ␮g/L for each analyte. One set of control
samples (n ⫽ 6) was subjected to 3 freeze/thaw cycles.
The 2nd set of samples (n ⫽ 6) was maintained at room
temperature for 14 h before analysis, the 3rd set (n ⫽ 6)
was refrigerated for 72 h before analysis, and the 4th set
(n ⫽ 3) was maintained at room temperature for 72 h after
derivatization on the autosampler. Concentrations of analytes in these samples were calculated and compared with
freshly prepared control samples.
Clinical Chemistry 52, No. 9, 2006
1731
clinical specimens
method validation
A within-subject, placebo-controlled, double-blind, randomized MDMA administration study was approved by
the National Institute on Drug Abuse Institutional Review
Board, and participants provided written informed consent. Individuals with a history of MDMA use, substantiated with a positive biological test, received 1.6 mg/kg
oral MDMA and resided on the secure clinical research
unit. Urine specimen was collected 1.2 h after MDMA
administration and was stored at ⫺20 °C until analysis.
Linearity. Regression analysis of calibration data achieved
satisfactory linearity over the concentration range investigated. Squared correlation coefficients (r2) were ⬎0.99,
indicating a linear relationship from 25–5000 ␮g/L for
HMA, MDA, HMMA, MDMA, and MDEA. Slopes, intercepts, and squared coefficients of correlation are presented in Table 1.
Results
chromatography
As shown in Fig. 1, the retention times of internal standards, MDA-d5, MDMA-d5, and MDEA-d6 added into
blank urine were 8.74, 10.18, and 10.34 min, respectively.
Retention times of HMA, MDA, HMMA, MDMA, and
MDEA were 7.94, 8.78, 9.09, 10.22, and 10.39 min, respectively. Total GC-MS analysis time was 12.2 min per
sample. Selected ion monitoring chromatograms of target
ions of HMA, MDA, HMMA, MDMA, and MDEA in
urine are depicted in Fig. 1. To document method applicability, a urine specimen collected 1.2 h after controlled
administration of 1.6 mg/kg oral MDMA was analyzed
(Fig. 2). Quantification of this urine specimen revealed
total drug concentrations of 1462.4 ␮g/L MDMA, 29.3
␮g/L HMA, 46.7 ␮g/L MDA, and 4261.8 ␮g/L HMMA.
Sensitivity. LODs were established by extraction of urine
samples containing decreasing concentrations of HMA,
MDA, HMMA, MDMA, and MDEA. The LOD was 10
␮g/L, and LOQ was 25 ␮g/L for all analytes (Table 1).
Extraction recovery. Mean extraction efficiencies of HMA,
MDA, HMMA, MDMA, and MDEA were calculated from
6 replicates of 30, 300, and 3000 ␮g/L QC samples.
Recoveries for all analytes at all concentrations were
⬎85.5%.
Precision and recovery. To evaluate precision and recovery,
we analyzed QC samples containing 30, 300, and 3000
␮g/L of HMA, MDA, HMMA, MDMA, and MDEA.
Results are summarized in Tables 2 and 3. To determine
intraassay recovery and precision, we performed 6 replicate analyses at each concentration. Within-run CVs for all
compounds were ⬍6%. Recovery, calculated as the percentage of target concentration, was 90%–117% (Table 2).
Fig. 1. Extracted ion chromatograms of the 50 ␮g/L HMA* (240 m/z, peak 1), MDA-d5 (167 m/z, peak 2), MDA (162 m/z, peak 3), HMMA (360
m/z, peak 4), MDMA-d5 (258 m/z, peak 5), MDMA (254 m/z, peak 6), MDEA-d6 (274 m/z, peak 7), and MDEA (268 m/z, peak 8) urine calibrator.
1732
Pirnay et al.: Sensitive GC-MS to Measure MDMA and Metabolites
Fig. 2. Extracted ion chromatograms
containing 29.3 ␮g/L HMA* (240
m/z, peak 1), 50 ␮g/L MDA-d5 (167
m/z, peak 2), 46.7 ␮g/L MDA (162
m/z, peak 3), 4261.8 ␮g/L HMMA
(360 m/z, peak 4), 50 ␮g/L MDMA-d5
(258 m/z, peak 5), 1462.4 ␮g/L
MDMA (254 m/z, peak 6), and 50
␮g/L MDEA-d6 (274 m/z, peak 7), in a
urine specimen collected 1.2 h after
1.6 mg/kg oral administration of
MDMA to a research participant.
Interassay precision and recovery, determined at the
same concentrations, for 6 batches and 6 replicates per
batch, were 3.5%–12.9% and 90%–112% respectively, for
all analytes at all concentrations. Interassay precision is
shown in Table 3.
Specificity. Eight blank urine specimens from different
individuals were analyzed to evaluate chromatographic
interference. No interference peaks were detected. The
absence of analyte ions in blank urines fortified with
internal standards demonstrated that internal standards
did not contain relevant amounts of native drug.
Interference study. No interference was detected in the
drugs tested up to 10 000 ␮g/L with HMA, MDA,
HMMA, MDMA, and MDEA. Moreover no interference
was detected with ephedrine, pseudoephedrine, and propanolamine up to 1 g/L.
Investigation of sample storage/stability. Stability of HMA,
MDA, HMMA, MDMA, and MDEA at low-, medium-,
and high-QC concentrations (n ⫽ 6) was evaluated under
several conditions; after 3 freeze/thaw cycles, 72 h at 4 °C,
and 14 h at room temperature (see Table 1 in the online
Data Supplement). Quantification of all QC samples was
acceptable (within 15% of target) for all stability challenges. Studies also were conducted to evaluate stability
of analytes after storage at 72 h at ambient temperature
on the autosampler. Concentrations of samples analyzed
immediately and after 72 h differed by ⬍20% (see Table 1
in the online Data Supplement).
Discussion
selection of deuterated internal standards
MDMA-d5, MDA-d5, and MDEA-d6 were included as
internal standards for their respective d0-compounds.
Deuterated analogs are not commercially available for
HMA and HMMA. MDA-d5 and MDMA-d5 were evaluated as internal standards for these analytes because of
their similar chemical structure (see Fig. 1 in the online
Data Supplement). MDA-d5 has a primary amine function
(similar to HMA), and MDMA-d5 has a secondary amine
function (similar to HMMA). Both compounds produced
equivalent quantitative recovery and limits of quantification for HMA and HMMA. Because of the fact that
MDA-d5 has a retention time (8.86 min) closer to HMA
(8.04 min) and HMMA (9.20 min) than MDMA-d5 (10.28
min) (Fig. 1), MDA d5 was selected as the internal
standard for HMA and HMMA.
isolation of the compounds
Our goal was to develop an analytical method that is
robust and highly sensitive to effectively characterize
terminal elimination half-lives. In addition, it was important to monitor total metabolite excretion and thus, the
value of the hydrolysis step to liberate glucuronide conjugates. This required optimization of hydrolysis before
extraction. Furthermore, as addressed by Clauwaert et al.
(16 ), up to 50% of free MDA and MDMA may be lost
during extraction when the organic phase containing the
compounds is evaporated to dryness under a stream of
Table 1. LOD, LOQ and calibration curves for HMA, MDA, HMMA, MDMA, and MDEA in urine.a
Analyte
Internal Standard,
50 ␮g/L
HMA
MDA
HMMA
MDMA
MDEA
d5–MDA
d5–MDA
d5–MDA
d5–MDMA
d6–MDEA
a
b
n ⫽ 5.
Data are mean (SD).
LOD
(␮g/L)
LOQ
(␮g/L)
Equationb
r2
10
10
10
10
10
25
25
25
25
25
Y ⫽ 0.0705 (0.00693) X–0.7628 (0.2236)
Y ⫽ 0.0421 (0.00408) X–0.2595 (0.1875)
Y ⫽ 0.1605 (0.2513) X–0.6274 (0.15356)
Y ⫽ 0.0475 (0.1115) X–0.1429 (0.09786)
Y ⫽ 0.0270 (0.0007) X–0.1632 (0.0752)
0.998
0.998
0.999
0.999
0.999
Clinical Chemistry 52, No. 9, 2006
Table 2. Intraassay precision and recovery for
determination of HMA, MDA, HMMA, MDMA, and MDEA in
urine.a
Analyte
HMA
MDA
HMMA
MDMA
MDEA
a
b
Expected
Concentration,
␮g/L
Observed
Concentration,
␮g/Lb
Precision,
%
Recovery,
%
30
300
3000
30
300
3000
30
300
3000
30
300
3000
30
300
3000
33.4 (0.7)
286.6 ( (16.7)
3243.1 ( (145.4)
33.9 ( (0.6)
273.8 ( (9.9)
3044.6 (105.3)
33.2 (1.1)
280.8 (15.9)
3253.1 (168.9)
35.1 (1.0)
270.0 (9.8)
3122.5 (120.9)
33.5 (0.7)
273.4 (10.0)
3118.8 (126.3)
2.1
5.8
4.5
1.8
3.6
3.5
3.1
5.6
5.2
2.8
3.6
3.9
2.2
3.6
4.0
111.3
95.5
108.1
113.0
91.3
101.5
110.7
93.6
108.4
117.0
90.0
104.1
111.7
91.1
104.0
n ⫽ 5.
Data are mean (SD).
nitrogen. Thus, we converted the amines to their chloride
salts before dry-down steps to decrease volatility. Also,
extracts and derivatized extracts were promptly removed
from the Turbovap when evaporation was complete.
Assay recovery and reproducibility were improved as a
result of these procedural adaptations.
chromatography
With the GC conditions described, HMA, MDA, HMMA,
MDMA, and MDEA were well resolved and eluted with
Table 3. Interassay precision and recovery for
determination of HMA, MDA, HMMA, MDMA, and MDEA in
urine.a
Analyte
HMA
MDA
HMMA
MDMA
MDEA
a
b
Expected
Concentration,
␮g/L
Observed
Concentration,
␮g/Lb
Precision,
%
Recovery,
%
30
300
3000
30
300
3000
30
300
3000
30
300
3000
30
300
3000
31.8 (3.6)
285.7 (13.2)
3047.9 (234.8)
31.1 (3.0)
283.3 (11.4)
2931.3 (126.5)
33.6 (2.0)
270.9 (13.5)
2894.9 (264.8)
31.1 (4.0)
270.5 (12.0)
2939.3 (193.5)
31.2 (2.4)
275.8 (9.8)
2949.7 (181.8)
11.2
4.6
7.7
9.6
4.0
4.3
6.1
5.0
9.1
12.9
4.4
6.6
7.8
3.5
6.2
106.0
95.2
101.6
103.7
94.4
97.7
112.0
90.3
96.5
103.7
90.2
98.0
104.0
91.9
98.3
n ⫽ 5.
Data are mean (SD).
1733
symmetric peaks in ⬍12 min. Attempts to use a different
chromatographic column (HP 5MS 30 m ⫻ 0.25 ␮m ⫻ 0.25
mm) were unsatisfactory because of longer retention
times and lack of resolution between MDMA and MDEA.
A split ratio of 10:1 was selected for injection onto the
GC-MS. Splitless injection and a split ratio of 1:5 improved LOQs, but column overload and poorer peak
resolution occurred at higher analyte concentrations. The
linear dynamic range of the assay was much greater with
the 10:1 split injection (Table 1).
method validation
The purpose of this investigation was to develop a specific
and sensitive assay for the simultaneous determination of
HMA, MDA, HMMA, MDMA, and MDEA. Three previously published studies investigated the recovery of
HMA and HMMA from urine after hydrolysis (12, 28, 29 ).
Our validated hydrolysis is faster and logistically easier
than autoclave hydrolysis (12 ) and notably more costeffective than enzyme hydrolysis (28, 29 ). Moreover, our
validated SPE procedure followed by optimized derivatization yielded higher average recoveries, especially for
HMA, than previously published methods.
In addition, the other methods for HMA and HMMA
showed higher LOQ and smaller dynamic ranges (28, 29 ).
Although Helmlin et al. investigated HMA and HMMA in
combination with MDMA, no validation data were presented (12 ). Other studies by Hensley et al. (30 ) showed a
wide dynamic range (5–1000 ␮g/L) for MDMA, MDA,
and MDEA but did not investigate MDMA metabolites,
HMA, and HMMA.
Intra- and interassay results also were satisfactory (CVs
⬍15%). Quantification of HMA, MDA, HMMA, MDMA,
and MDEA was acceptable (within 20% of target) for all
stability challenges. Our study demonstrated no considerable loss of MDEA, MDMA, or metabolites, HMA,
MDA, and HMMA in urine at any of the investigated
temperatures and times. The same results were observed
by Clauwaert et al. (31 ), who performed extended studies
of stability of MDA, MDMA, and MDEA and who furthermore demonstrated stability of these drugs at ⫺20, 4,
or 20 °C in urine for 21 weeks.
A robust, rapid, sensitive, and relatively inexpensive
gas chromatography-mass spectrometry assay for simultaneous quantification of urinary MDEA, MDMA, MDA,
HMMA, and HMA is presented. The method is suitable
for pharmacokinetic studies, specimen batches with various drug concentrations, and for identification and quantification of MDEA, MDMA, and MDA in postmortem,
emergency, and workplace toxicology.
This research was supported by the Intramural Research
Program of the National Institute on Drug Abuse, National Institutes of Health. We thank Ross Lowe for
assistance with validation of the method and David
Darwin and Deborah Price for technical assistance.
1734
Pirnay et al.: Sensitive GC-MS to Measure MDMA and Metabolites
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