| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Laboratory of Toxicology and
2
Laboratory of Medicinal Chemistry, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium.
3
Laboratory of Forensic Medicine, Ghent University, J.
Kluyskensstraat 29, B-9000 Gent, Belgium.
a Author for correspondence. Fax 32-9-264-81-97; e-mail
Andre.DeLeenheer{at}rug.ac.be
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Methods: After extraction, chromatographic separation was achieved on a narrow-bore C18 column by gradient elution with fluorometric detection; results were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Results: The method was linear over the range of 2–1000 µg/L for whole blood, serum, and vitreous humor, and 0.1–5 mg/L for urine. Extraction recoveries were >70%, imprecision (CV) was 2.5–19%, and analytical recoveries were 95.5–104.4%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.8 and 2 µg/L, respectively, for whole blood, serum, and vitreous humor, and 2.5 µg/L and 0.1 mg/L, respectively, for urine. Excellent correlations between the quantitative LC-fluorescence and LC-MS/MS results were obtained. We found the following concentrations in a thanatochemical distribution study in rabbits: in serum, 5.3–685 µg/L for MDMA and from the LOQ to 14.5 µg/L for 3,4-methylenedioxyamphetamine (MDA); in whole blood, 19.7–710 µg/L for MDMA and from the LOQ to 17.8 µg/L for MDA; in vitreous humor, 12.1–97.8 µg/L for MDMA and from the LOQ to 3.86 µg/L for MDA. In routine toxicological urine samples, concentrations ranged from LOQ to 14.62 mg/L for MDA, from LOQ to 157 mg/L for MDMA, and from LOQ to 32.54 mg/L for MDEA.
Conclusions: The HPLC method described is sensitive, specific, and suitable for the determination of MDMA, MDEA, and MDA in whole blood, serum, vitreous humor, and urine.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
). In the last few decades, these drugs have gained popularity
as recreational drugs and are used chiefly for their agreeable
stimulating effects, especially in gatherings known as "raves"
(1).
|
MDMA and its analogs exert their activities through effects mainly on the serotonin system and, to a lesser extent, on the dopamine system (2). The popularity of the methylenedioxyamphetamine derivatives can be attributed to their psychotropic effects and the so-called entactogenic effects (3) of the drugs.
Although MDMA, MDEA, and MDA generally are regarded as relatively safe recreational drugs, it has become increasingly apparent that their use can be associated with many adverse effects and complications, some of which can lead to a fatal outcome (4)(5)(6). Furthermore, MDMA and MDA have been found to damage serotonin neurons in all experimental animals tested to date, and there are more serious concerns that human users are at risk of serotonin neurotoxicity (7)(8), especially after the repeated use of high doses of the drugs.
The matrices commonly investigated for MDMA and its analogs are blood and urine (9)(10)(11)(12)(13)(14)(15). Nevertheless, in view of its particular physiology, vitreous humor, the material that fills the posterior cavity of the eye, potentially contains drug concentrations showing good correlation with corresponding concentrations in the blood. Moreover, vitreous humor is often the best or only sample available at post mortem, e.g., in cases of severely burned or decomposed bodies, and the lack of any significant metabolic activity in the eye suggests that drug concentrations in the vitreous humor can provide an accurate indication of body drug concentrations (16).
To date, the determination of MDMA, MDEA, and MDA in biological samples has been based mainly on gas chromatography–mass spectrometry (9)(10)(11)(17). Gas chromatography–mass spectrometry provides excellent sensitivity and selectivity, but MDMA and its metabolites require derivatization (9)(10)(11)(17) before gas chromatographic determination to improve their chromatographic properties. HPLC also has been used for the determination of MDMA and its analogs. Unfortunately, the methods using ultraviolet or diode array detection lack sensitivity, with detection limits >5 µg/L (12)(13)(14)(15) regardless of subsequent complicated derivatization steps (14)(15).
Our aim was to develop a simple, rugged but sensitive and specific method for the determination of MDA, MDMA, and MDEA, keeping in mind its intended double use. On the one hand, we wanted a method that could be used in a thanatochemical study for the interpretation of MDMA distribution in a rabbit model to investigate in-depth the possibilities of determining drug concentrations in the vitreous humor as an alternative for blood or serum concentrations. Consequently, the method had to be sensitive and preferably use only small amounts of sample (<0.5 mL). On the other hand, we wanted a method that would also be applicable in our routine toxicological analysis schemes to investigate MDMA, MDEA, or MDA abuse in whole blood, serum, or urine, in which user-friendliness is paramount. To that end, HPLC with fluorescence detection was chosen.
When dealing with amphetamine-like structures, it is important to remember that these compounds contain a chiral center. Illegal amphetamine is sometimes synthesized from ephedrine, itself occurring naturally as pure enantiomer, which thus leads to the intake of one enantiomer of amphetamine. However, the methylenedioxyalkylamine designer drugs are virtually always administered as racemic mixtures. Both enantiomers display different pharmacodynamics, e.g., apparently only S-(+)-MDMA is neurotoxic, as well as different pharmacokinetic properties (3)(11)(18). To that end, chiral separations have been found most useful in some pharmacokinetic investigations with respect to these compounds and are routinely performed to confirm the use of drugs of abuse in workplace drug testing. However, in view of our envisaged application, i.e., interpretation of toxicological cases (e.g., overdoses and driving under the influence of stimulants) and a thanatochemical study with emphasis on distribution to the vitreous humor, stereoselective deconvolution was not considered. Exploring the alternative use of the vitreous humor for methylenedioxyamphetamine detection does not necessitate stereoselective data unless perhaps in a final stage when the usefulness of vitreous humor is firmly established but pharmacokinetic fine-tuning is needed.
We report the development of a method for the simultaneous quantitative determination of MDMA, MDEA, and MDA in various biological fluids, based on HPLC analysis, a robust and relatively inexpensive technique that is perfectly suitable for large batch processing, and with detection through the native fluorescence of the methylenedioxylated amphetamines. The method uses liquid-liquid extraction after the addition of an appropriate internal standard (an inadequacy in nearly all published liquid chromatographic methods); we used a suitable MDMA analog, methylenedioxymethylpropylamphetamine (MDMPA), which we synthesized for that purpose. Moreover, supportive data for the fluorescence detection method were obtained for several samples, using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Tagliaro et al. (19) used a LC-fluorescence method (LC-Fl) for the analysis of MDMA and its analogs in hair. However, their method was used exclusively with hair and, unfortunately, was not quantitatively elaborated, e.g., no internal standardization was used. To our knowledge, our report is the first on the quantitative LC-Fl analysis of methylenedioxyamphetamines in blood, vitreous humor, and urine.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
The MS analyses were carried out on a Q-TOF hybrid mass spectrometer (Micromass) equipped with an orthogonal electrospray source (Z-spray); an Alliance 2690 XE separation module (Waters) was integrated with the Q-TOF instrument.
1H and 13C nuclear magnetic resonance (NMR) spectra were obtained with a Bruker WH 500 spectrometer. The CHCl3 solvent signal was used as secondary reference (7.23 and 76.89 ppm for 1H and 13C NMR, respectively). Mass spectra were also obtained by fast atom bombardment high-resolution mass spectrometry (HRMS-FAB) using a Kratos concept 1H mass spectrometer.
reagents and materials
All reagents and chemicals were of analytical grade and were from
Aldrich unless otherwise stated. Solvents were of HPLC grade and were
from Fisher Scientific or Merck. MDA, MDMA, and MDEA pure standards
were from Sigma. Stock solutions of these active substances were
prepared by dissolving 10 mg of the pure compound in 10 mL of methanol.
Appropriate dilution with methanol yielded the working solutions
containing all three compounds. All concentrations of the standards are
expressed as the free base. The stock solutions were stored in the dark
at -20 °C and were stable for at least 1 year. Working solutions
were stored under the same conditions as the stock standards but were
discarded after 6 months.
samples
Whole blood, serum, and vitreous humor samples were obtained
during a thanatochemical distribution study of MDMA in rabbits. Details
of this study are published elsewhere (20). The animals
received intravenous doses containing 1 mg/kg MDMA, and blood samples
were taken at various time intervals (up to 240 min after
administration). In three different subgroups (30, 120, and 240 min
after administration), rabbits were anesthetized with pentobarbital and
decapitated, and both eyes were immediately enucleated. After specific
dissection of the eyes, vitreous humor was obtained. Urine samples were
from urines collected for routine forensic toxicological analysis. All
samples (whole blood, serum, vitreous humor, and urine) were stored at
-30 °C until analysis.
synthesis of the internal standard
MDMPA was prepared as follows. Propyl iodide (245 µL, containing
2.5 equivalents) was added to 195 mg (1.0 mmol) of MDMA
{N-[2-(1,3-benzodioxol-5-yl)-1-methylethyl]-N-methylamine
(IUPAC terminology)} in 5 mL of tetrahydrofuran. Subsequently, the
stirred solution was refluxed for 60 h. Thin-layer chromatographic
analysis (using precoated Merck silica gel F254
plates) with
CH2Cl2-methanol-triethylamine,
(97:3:1 by volume) demonstrated almost complete reaction. The reaction
mixture was concentrated, and the MDMPA was purified by column
chromatography performed on SÜD-Chemie silica gel (0.05–0.2 mm)
with
CH2Cl2-methanol-triethylamine
(100:0:1, then 98:2:1 by volume). The fractions containing pure MDMPA
{N-[2-(1,3-benzodioxol-5-yl)-1-methylethyl]-N-methyl-1-propanamine
(IUPAC terminology)} were collected and evaporated to dryness; the
obtained residue was then co-evaporated twice with toluene (10 mL each
time) to remove all traces of triethylamine. To 158 mg of the base,
obtained as described above, 1.0 mL of a 1.0 mol/L hydrogen chloride
solution in diethyl ether was added. The mixture was evaporated, and
the obtained oily residue was crystallized from ethyl acetate-hexane to
yield 75 mg of the hydrogen chloride salt of MDMPA (diastereoisomeric
mixture). The identity and purity of the product were verified with
1H NMR, 13C NMR, and
HRMS-FAB.
isolation of the compounds
Whole blood, serum, vitreous humor, and urine samples (250 µL)
were extracted with hexane-ethyl acetate (7:3, by volume) after the
addition of 50 µL of the internal standard solution (containing 20 ng
of MDMPA for whole blood, serum, and vitreous humor and 0.25 µg for
urine), dilution with 1 mL of H2O, and adjustment
of the pH with 1 mol/L aqueous
K2CO3 (brought to pH 9.5
with 370 mL/L HCl). Samples were mixed on a rotary mixing device for 10
min and centrifuged at 1200g for 15 min. The organic layer
was transferred to a test tube containing 50 µL of methanolic HCl (5
mol/L acetyl chloride in methanol) and evaporated using a
Turbovap® evaporator at 35 °C under nitrogen.
The residue was dissolved in 100 µL (whole blood, serum, and vitreous
humor) or 250 µL (urine) of HPLC eluant A (see "Chromatography");
25 µL was injected for LC-Fl, whereas for LC-MS/MS only 5 µL was
used.
calibration samples
Calibration curves were prepared in the corresponding blank matrix
except for vitreous humor, for which water was substituted because of
practical unavailability and because it has a high water content
(
98%). Calibrators (2, 10, 20, 40, 100, 400, and 1000 µg/L) were
prepared in serum, whole blood, and water by adding 50 µL of the
appropriate working solution containing MDA, MDMA, and MDEA to a
250-µL aliquot of the sample. For urine, the following concentrations
were obtained: 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mg/L. All calibrators
were all extracted according to the general isolation procedure.
chromatography
Chromatographic separation was achieved on a Hypersil BDS
C18 column (100 x 2.1 mm; 3 µm bead size;
Alltech). The mobile phase was 0.1 mol/L ammonium acetate in 900 mL/L
HPLC-grade water–50 mL/L methanol–50 mL/L acetonitrile (eluant A) or
450 mL/L methanol–450 mL/L acetonitrile–100 mL/L HPLC-grade water
(eluant B). The chromatographic conditions were as follows: 100%
eluant A for 6 min, followed by a linear gradient from 0% to 70%
eluant B within 14 min. After completion of the chromatographic run,
the pump was programmed to return to the initial conditions within 0.5
min, and 8 min of reconditioning time was allowed.
fluorescence detection
The excitation and emission wavelengths of the fluorescence
detector were 288 and 324 nm, respectively (bandwidth was 15 nm for
both slits).
mass spectrometry
Electrospray ionization positive mass spectra (single MS and
product ion scans) were acquired on a Q-TOF mass spectrometer.
Conditions, optimized using flow injection of standard solutions, were
as follows: electrospray ionization capillary voltage, 600 V; cone
voltage, 17 V (MDA) and 22 V (all other compounds); source temperature,
120 °C. The electrospray ionization gas was nitrogen. For LC-MS/MS
product ion analysis, the quadrupole was set to pass precursor ions of
the selected mass to the hexapole collision cell (using argon as
collision gas for collision-induced dissociation), and product ion
spectra were acquired with the time-of-flight (TOF) analyzer. The
resolution of the quadrupole mass filter was set such that the peak
width was 1.2 mass units at half height, and the collision energy was
optimized for each compound (14 eV for MDA and 18 eV for the other
compounds). All TOF measurements were performed at high resolution
settings (5000 full width at half-maximum at mass 1500), and the TOF
analyzer was "scanned" at m/z 50–850 with a 1-s
integration time.
method validation
Linearity.
Separate calibrations curves were
prepared over a concentration range of 2–1000 µg/L for MDA, MDMA,
and MDEA in whole blood, serum, or vitreous humor. For each curve,
seven different concentrations (2, 10, 20, 40, 100, 400, and 1000
µg/L) were used. In urine, six different concentrations were used
over a concentration range of 0.1–5 mg/L (0.1, 0.2, 0.5, 1.0, 2.0, and
5.0 mg/L). Peak-area ratios between the compounds and the internal
standard were plotted against the concentration of each compound. We
used weighted linear regression (1/x) in an effort to
account for data heteroscedasticity.
Extraction recovery.
Three individual extracts and three
replicates of the compounds, prepared directly in the eluant, were
injected onto the column. The assay recovery for each compound was
determined as follows: (mean peak area of the extract/mean peak area
direct injection) x 100.
Precision.
Precision was evaluated by analyzing blank samples
to which the tested compounds had been added at three different
concentrations. For within-day reproducibility, seven replicates were
analyzed on the same day; for total reproducibility, seven replicates
were analyzed on separate days.
Analytical recovery.
To determine the analytical recovery, two
positive control samples (in any matrix) were independently prepared
using different standard solutions and volumetric materials other than
those used to prepare calibration samples. The positive controls were
extracted and analyzed with each batch of samples, and their
quantitative results were related to the exact concentrations added.
Interference.
To investigate the selectivity of the method,
several drugs, dissolved in eluant at a comparatively high
concentration (4 mg/L), were injected into the HPLC system. These
solutions were chromatographed under the same conditions as the samples
and detected with the least specific detection system used in this
study, i.e., fluorescence detection. When retention behavior and
fluorescence detection produced potential interference with one of the
investigated compounds or the internal standard, the compounds were
added to serum, extracted, and re-injected.
Limits of detection and of quantification.
The limit of
detection (LOD) and the limit of quantification (LOQ) were determined
by analyzing decreasing concentrations of the compounds added to the
blank matrices. The LOD was established as the lowest concentration
that produced a response three times the background noise. The LOQ was
defined as the lowest concentration that could be quantified with an
imprecision <25% and was established as the lowest point of the
calibration graph. For urine, however, the calibration range was
shifted upward in view of the physiological concentrations in urine,
and the LOQ moved accordingly.
safety considerations
The method demands no specific safety considerations. General
guidelines for work with organic solvents, acids, and alkalines were
followed. For waste disposal in general but for acetonitrile in
particular, governmental and institutional environmental guidelines
were followed.
All animal work was performed in accordance with established guidelines for animal care. The study protocol was approved by the Ethics Committee on Animal Research of the Ghent University Hospital (request numbers ECP 98/1 and ECP 99/9).
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
The identity and purity of the synthesized internal standard, MDMPA, were confirmed on the basis of 1H NMR, 13C NMR, and HRMS-FAB. The following results were obtained:
) of the free base of MDMPA: 0.90 (t, 3H, J = 7.4
Hz, propyl CH3), 0.93 [t, 3H, J
= 6.6 Hz, CH3-C (
)], 1.53 (sextet, 2H,
CH3CH2), 2.29–2.35
[m, 4H, N-CH3, C ()H], 2.43 (app t, 2H,
J = 7.5 Hz,
CH3CH2CH2),
2.87–2.94 [m, 2H, C (ß)H2], 5.91 (s, 2H,
H-2'), 6.61 (d, J = 7.9 Hz, H-6), 6.67 (d,
J = 1.1 Hz, H-2), 6.72 (d, J = 7.9 Hz,
H-5). represents the chemical shift (in ppm), t indicates a
triplet, m indicates a multiplet, app t indicates an apparent triplet,
s indicates a singlet, and d indicates a doublet. ) of the
hydrochloride of MDMPA: 11.14, 11.35; 11.26, 12.84; 17.76, 17.87;
34.71, 37.02; 36.68, 37.66; 53.25, 55.81; 61.67, 62.50; 100.94, 100.98;
108.34, 108.40; 109.30, 109.42; 122.24, 122.34; 129.12, 129.42; 146.63,
146.70; 147.80, 147.86. On the basis of the above results, we concluded that the internal standard was the intended compound, MDMPA, and that it was of a high purity. The use of the synthesized internal standard in the analytical method demonstrated that MDMPA behaves similarly to MDA, MDMA, and MDEA throughout the whole analytical procedure and shows similar recoveries to the other compounds from the investigated biological fluids.
isolation of the compounds
Our goal was to develop an analytical method that is convenient
for the processing of large batches of samples such as those generated
in pharmacokinetic studies. Therefore, an efficient, simple,
inexpensive, and robust single-step extraction procedure was developed
for MDA, MDMA, MDEA, and the internal standard. The recovery results
indicated that the analyzed compounds can be effectively extracted with
hexane-ethyl acetate (70:30 by volume) at pH 9.5 from the various
biological matrices. In fact, the absolute recovery of the compounds
was nearly unaffected when the extraction pH varied between 9 and 10 or
when the ethyl acetate concentration varied between 250 and 350 mL/L.
Consequently, we concluded that neither the pH nor the ethyl acetate
concentration was very critical with respect to robustness.
During our experimental work we found that, analogously to amphetamine and methamphetamine (23), up to 50% of the free MDA and MDMA base could get lost when the organic phase, containing the compounds, was evaporated to dryness under a stream of nitrogen. Surprisingly, this is a problem that other published analytical methods have failed to mention (9)(15). Therefore, we decided to convert the amines to their corresponding hydrochloric acid salts before drying to ensure nonvolatility of the drugs. We also avoided overdrying for long periods (>0.5 h). As a result of these procedural adaptations, recovery and reproducibility of the assay were much improved.
chromatography
With the HPLC conditions described, MDA, MDMA, and MDEA were well
resolved (resolution >1.5) and eluted in symmetric peaks. Attempts to
use isocratic conditions were unsatisfactory because optimal resolution
of the three investigated compounds, which are secondary amines,
produced retention times that were too long for the internal standard
MDMPA, a tertiary amine, influencing its retention behavior. The
retention times of MDA, MDMA, MDEA, and the internal standard were
13.1, 14.1, 15.1, and 17.1 min, respectively, yielding capacity factors
(k') of 6.9, 7.5, 8.2, and 9.5 (Fig. 2
). Our chromatographic analysis clearly combines optimal
resolution and an acceptable run time. An important advantage of our
gradient approach is the possibility of multiple sequential injections
without the risk of interfering, late-eluting peaks.
|
The use of narrow-bore HPLC, on the other hand, offered two benefits inherent to reducing the HPLC column diameter, i.e., a reduction in solvent usage and a net increase in detection limits because of enhanced mass sensitivity.
fluorescence detection
The ring-substituted amphetamines, dissolved in the
chromatographic eluant mixture, exhibited a good native fluorescence
with an excitation maximum at 288 nm. When the compounds were
irradiated at 288 nm, an emission maximum at 324 nm was recorded. No
spectral differences were found between the various compounds. The use
of fluorescence detection provided greater selectivity compared with
conventional ultraviolet detection (as shown in preliminary
experiments) and allowed the use of a simple sample pretreatment
without the risk of interfering endogenous compounds, even for
whole-blood samples.
mass spectrometry
We achieved a simple and straightforward methodological crossover
between LC-Fl detection and LC-MS/MS. The latter was of great
importance to validate the results obtained with fluorescence detection
and to give complementary confirmation of the presence of MDA, MDMA,
and MDEA, if necessary. The same column and chromatographic conditions
were used on both systems, and the samples, after one common
extraction, could be injected on both systems.
The MS and MS/MS (low energy collision-induced dissociation with argon)
spectra of MDMA are shown in Fig. 3
. The MS spectra obtained were dominated by an intense
[M+H]+ peak (m/z 180.1 for MDA,
m/z 194.1 for MDMA, m/z 208.1 for MDEA, and
m/z 236.1 for MDMPA) and essentially a single major fragment
ion at m/z 163.1, the same for all compounds. This ion was
also the base peak in the product ion spectra, which were almost
identical for the four compounds.
|
High-quality mass spectral data were obtained, not only for standards but for calibration samples and, more importantly, for the biological samples investigated with both techniques. None of the peaks in LC-Fl was erroneously identified, substantiating its value, and no peaks containing impurities were observed.
During our mass spectrometric experiment, we not only confirmed the identities of the different compounds, we also found an excellent correlation between the quantitative results obtained with LC-Fl and LC-MS/MS. The aspects of quantification, using the Q-TOF instrument, have been validated extensively and recently were published elsewhere (24).
method validation
Linearity.
The calibration curves were linear over the
specified ranges (2–1000 µg/L for whole blood, serum, and vitreous
humor and 0.1–5.0 mg/L for urine) for all three compounds in all four
analyzed matrices. Weighted linear regression (1/x) revealed
a correlation coefficient of 0.9969 or higher for the relationship
between peak-area ratio (compound/internal standard) and the
corresponding calibration concentrations. The y-intercept
for each calibration curve was essentially zero (the 95% confidence
interval of the constant included zero in all cases). Linearity data
are given in Table 1
. The method shows good linearity over a broad concentration
range comparable to (9)(12) or better than
(13) other published methods, and the linearity data
indicate a good day-to-day match for the various calibration curves.
The validity of the extended calibration range was demonstrated by
comparing the slopes from the lowest to the middle and from the middle
to the highest points of the calibration range with the overall slope.
The results were as follows (n = 7): MDA in serum (
lower to
overall, higher to overall), 1.64%, 0.26%; MDMA in serum, 0.82%,
0.66%; MDEA in serum, 0.68%, 0.25%; MDA in whole blood, 3.37%,
0.26%; MDMA in whole blood, 4.38%, 0.44%; MDEA in whole blood,
2.65%, 0.08%; MDA in water, 3.36%, 0.75%; MDMA in water, 4.75%,
2.37%; MDEA in water, 2.10%, 4.54%; MDA in urine, 1.85%, 0.73%;
MDMA in urine, 0.24%, 0.10%; MDEA in urine, 2.78%, 0.92%. In all
cases, the deviation was <5%, which indicates that the distribution
of the standards was acceptable. For sample concentrations exceeding
the calibration curve, the samples were diluted and reextracted.
|
Extraction recovery.
The extraction recovery was determined at
all calibration points in the four different matrices. The results
obtained showed a high, reproducible, and fully
concentration-independent recovery for all four compounds in all three
analyzed matrices. Recoveries were >70% in all cases.
Precision.
Table 2
shows the within-day and total reproducibility data obtained
(n = 7) for the different concentrations tested in the four
matrices. The CVs were 2.5–19%. This indicates that the precision was
good over the studied concentration range and meets with our objective
of a routinely applicable analysis for MDA, MDMA, and MDEA in serum,
whole blood, vitreous humor, and urine.
|
Analytical recovery.
For validation purposes, the analytical
recovery for the three compounds was also determined in the four
investigated matrices at two separate concentrations (5 and 550 µg/L
for whole blood, serum, and vitreous humor and 0.15 and 2.5 mg/L for
urine). The values obtained were between 95.5% and 104.4%, indicating
excellent analytical recovery, even in the lowest concentration range.
Interference study.
Because our method should be suitable for
routine use in a toxicological laboratory, in addition to its use for
thanatochemical distribution studies, selectivity considerations,
especially those pertaining to prescription drugs and drugs of abuse,
are of prime importance. Table 3
shows the capacity factors (k', or mass distribution
ratio) for a list of drugs evaluated as potential interferents. As can
be seen, all but three drugs were not detectable either because of the
specificity of the fluorescence detector or because they were not
retained on the column. Morphine, pentazocine, and zopiclone were
detected, and because of their basic nature, they were also extracted
with the liquid-liquid extraction used in our method. However, they
were chromatographically separated from the compounds of interest and
consequently were easily distinguished from the compounds of interest:
MDA, MDMA, and MDEA. With reference to detection, LC-MS/MS is highly
specific. Use of the Q-TOF mass spectrometer makes selectivity an
almost inherent aspect of the data collection.
|
LOD and LOQ.
The LOD, as defined in Materials and
Methods, was 0.8 µg/L for all three compounds in whole blood,
serum, and vitreous humor and 2.5 µg/L in urine. The LOQ, the lowest
point of the calibration graph, was 2 µg/L in whole blood, serum, and
vitreous humor and 0.1 mg/L in urine. As can be seen from Table 2
, this
concentration can be measured with acceptable reproducibility in whole
blood, serum, water (blank for vitreous humor), and urine.
analysis of samples
As seen in the chromatograms of a serum sample (Fig. 2
)
supplemented with 40 µg/L MDA, MDMA, and MDEA; a positive vitreous
humor sample (Fig. 4
) containing 38.2 µg/L MDMA and MDA below the LOQ; a negative
whole-blood sample (Fig. 5
); and a positive forensic toxicological urine sample (resulting
from the combined intake of MDMA and MDEA; Fig. 6
) containing 1.89 mg/L MDA, 12.62 mg/L MDMA, and 5.44 mg/L MDEA,
there were no interfering peaks from endogenous compounds in any of the
investigated matrices.
|
|
|
The usefulness of our method was substantiated by analysis of >200
whole-blood and serum samples and 60 vitreous humor samples in a
thanatochemical distribution study in rabbits. We found the following
concentrations in the different matrices: in serum, 5.3–685 µg/L for
MDMA and from below the LOQ to 14.5 µg/L for MDA; in whole blood,
19.7–710 µg/L for MDMA and from below the LOQ to 17.8 µg/L for
MDA; in vitreous humor, 12.1–97.8 µg/L for MDMA and from below the
LOQ to 3.86 µg/L for MDA. We did not find any MDEA in the rabbit
samples because we infused only MDMA. Fig. 7
shows a representative plasma concentration–time curve
resulting from the intravenous administration of 1 mg/kg MDMA to a
rabbit, illustrating the concentrations found in the pharmacokinetic
study. The concentrations found in routine forensic toxicological urine
samples (n = 35) were between the LOQ and 14.62 mg/L for MDA, the
LOQ and 157.1 mg/L for MDMA, and the LOQ and 32.54 mg/L for MDEA.
|
We were able to confirm both the qualitative (no erroneous identification of MDA or MDMA) and quantitative results (the values obtained by both methods were the same for the same samples) of the LC-Fl method with LC-MS/MS for a representative number of the samples (24).
A large amount of pharmacokinetic data has been generated to date. The scope of the study as well as length considerations have made these data and their interpretation the subject of a upcoming article.
In conclusion, we have established a reliable analytical method for MDA, MDMA, and MDEA quantification in whole blood, serum, vitreous humor, and urine that uses LC-Fl detection, liquid-liquid extraction, and internal standardization with a structurally related analog of the designer drugs, i.e., MDMPA. A simple methodologic crossover between LC-Fl detection and LC-MS/MS allowed successful validation of the results obtained by LC-Fl and enables complementary identification of analytes on the basis of their unique MS/MS spectra, generally considered vital in forensic applications. We have also demonstrated the practical applicability of the LC-Fl-based procedure by analyzing of a large number of samples in a pharmacokinetic and thanatochemical investigation, as well as some forensic samples in our routine toxicological practice.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  S. O. Pirnay, T. T. Abraham, and M. A. Huestis Sensitive Gas Chromatography-Mass Spectrometry Method for Simultaneous Measurement of MDEA, MDMA, and Metabolites HMA, MDA, and HMMA in Human Urine Clin. Chem., September 1, 2006; 52(9): 1728 - 1734. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
