TY - JOUR
AU - , De Leenheer, André P
AB - Abstract Background: The popular designer drugs 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) can be determined in serum, whole blood, and urine, but also in vitreous humor. The latter matrix is interesting when dealing with decomposed bodies in a toxicological setting. 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. The designer drugs 3,4-methylenedioxymethamphetamine (MDMA;1 ecstasy), 3,4-methylenedioxyethylamphetamine (MDEA; eve), and 3,4-methylenedioxyamphetamine (MDA), the latter being a designer drug but also an important metabolite of both MDMA and MDEA, are all methylenedioxyamphetamine derivatives (Fig. 1 ). 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 apparatus The HPLC consisted of a ternary low-pressure gradient pump and an autosampler with a 25-μL loop (both from Kontron Instruments), and was equipped with a solvent degassing module (Shodex). A spectrofluorometric detector (RF-10Axl; Shimadzu) linked to a Kromasystem 2000 data system (Kontron Instruments) was used for data acquisition and storage. 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 × 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) × 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 identity of the internal standard Because we preferred a structurally related analog of MDA, MDMA, and MDEA as internal standard to enhance analytical precision and because such an appropriate compound was not commercially available, we synthesized one ourselves. None of the possible chemical variations in such small molecules as the methylenedioxyamphetamines is beyond the competence of underground chemists, especially considering the rapid evolution of designer drugs, e.g., from MDMA to MDEA and the appearance of, e.g., N-methylbenzodioxazolylbutanamine and benzodioxazolylbutanamine (21)(22). Nevertheless, the use of, e.g., the mono-propyl or mono-butyl analogs of MDA for internal standard purposes had to be explicitly avoided, as did the dimethyl or diethyl analogs, because the latter can be formed inadvertently during poorly controlled synthesis conditions. As a result, it can be expected that these compounds will appear on the illegal market in the near future. Therefore, we chose to synthesize MDMPA, a product that is synthesized through a double, independent alkylation of the nitrogen of MDA, necessitating at least one extra synthesis step. This gives rise to an increasingly complicated, and thus commercially much less attractive, synthesis pathway. 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: 1H NMR (500 MHz, CDCl3, δ) 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 (α)], 1.53 (sextet, 2H, CH3CH2), 2.29–2.35 [m, 4H, N-CH3, C (α)H], 2.43 (app t, 2H, J = 7.5 Hz, CH3CH2CH2), 2.87–2.94 [m, 2H, C (β)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). δ 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. 13C NMR (CDCl3, δ) 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. HRMS-FAB (thioglycerol) of the hydrochloride of MDMPA (m/z): [M+H]+ calculated for C14H21NO2, 236.1651; found, 236.1650. 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 (Δ lower to overall, Δ 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. Figure 1. Open in new tabDownload slide Structures of the designer drugs and the internal standard. Figure 1. Open in new tabDownload slide Structures of the designer drugs and the internal standard. Figure 2. Open in new tabDownload slide Chromatogram of a blank serum sample enriched with 40 μg/L MDA (peak 1), MDMA (peak 2), and MDEA (peak 3), and 80 μg/L MDMPA (the internal standard; peak 4). Figure 2. Open in new tabDownload slide Chromatogram of a blank serum sample enriched with 40 μg/L MDA (peak 1), MDMA (peak 2), and MDEA (peak 3), and 80 μg/L MDMPA (the internal standard; peak 4). Figure 3. Open in new tabDownload slide MS (top) and MS/MS (bottom) spectra of MDMA. Figure 3. Open in new tabDownload slide MS (top) and MS/MS (bottom) spectra of MDMA. Table 1. Linearity data for MDA, MDMA, and MDEA in whole blood, serum, water (blank for vitreous humor), and urine.1 . Slope2 . CV of slope over 7 days, % . Intercept2 . Sy|x3 . SE of the slope2 . SE of the intercept2 . r23 . Serum MDA 0.013378 5.4 0.0042 0.004208–0.009851 0.001700 0.008161 0.9982–0.9996 MDMA 0.014282 5.1 0.0075 0.003880–0.010908 0.001829 0.008259 0.9979–0.9997 MDEA 0.013992 5.2 0.0127 0.004935–0.011844 0.0001583 0.007545 0.9979–0.9996 Whole blood MDA 0.013161 5.3 0.0144 0.007881–0.010349 0.0002390 0.011477 0.9977–0.9988 MDMA 0.014341 3.8 0.0097 0.004767–0.012172 0.0001947 0.009281 0.9976–0.9996 MDEA 0.014156 4.8 0.0155 0.004911–0.010624 0.0002017 0.009613 0.9980–0.9996 Water MDA 0.014731 4.2 0.0099 0.004224–0.014084 0.0002276 0.010981 0.9970–0.9997 MDMA 0.01496 4.0 0.0000 0.004631–0.012393 0.0002161 0.010365 0.9978–0.9992 MEDA 0.01459 3.6 0.0099 0.004983–0.013898 0.0002177 0.010410 0.9969–0.9997 Urine MDA 1.04728 2.0 −0.0023 0.015309–0.054148 0.0125754 0.008680 0.9986–0.9998 MDMA 1.15503 2.8 −0.0029 0.016010–0.058408 0.0136079 0.010447 0.9986–0.9999 MDEA 1.15330 5.3 −0.0048 0.031770–0.089753 0.0188846 0.011269 0.9971–0.9995 . Slope2 . CV of slope over 7 days, % . Intercept2 . Sy|x3 . SE of the slope2 . SE of the intercept2 . r23 . Serum MDA 0.013378 5.4 0.0042 0.004208–0.009851 0.001700 0.008161 0.9982–0.9996 MDMA 0.014282 5.1 0.0075 0.003880–0.010908 0.001829 0.008259 0.9979–0.9997 MDEA 0.013992 5.2 0.0127 0.004935–0.011844 0.0001583 0.007545 0.9979–0.9996 Whole blood MDA 0.013161 5.3 0.0144 0.007881–0.010349 0.0002390 0.011477 0.9977–0.9988 MDMA 0.014341 3.8 0.0097 0.004767–0.012172 0.0001947 0.009281 0.9976–0.9996 MDEA 0.014156 4.8 0.0155 0.004911–0.010624 0.0002017 0.009613 0.9980–0.9996 Water MDA 0.014731 4.2 0.0099 0.004224–0.014084 0.0002276 0.010981 0.9970–0.9997 MDMA 0.01496 4.0 0.0000 0.004631–0.012393 0.0002161 0.010365 0.9978–0.9992 MEDA 0.01459 3.6 0.0099 0.004983–0.013898 0.0002177 0.010410 0.9969–0.9997 Urine MDA 1.04728 2.0 −0.0023 0.015309–0.054148 0.0125754 0.008680 0.9986–0.9998 MDMA 1.15503 2.8 −0.0029 0.016010–0.058408 0.0136079 0.010447 0.9986–0.9999 MDEA 1.15330 5.3 −0.0048 0.031770–0.089753 0.0188846 0.011269 0.9971–0.9995 1 Concentration ranges: 2-1000 μg/L for whole blood, serum, and water; 0.1–5.0 mg/L for urine. 2 Mean value of 7 consecutive days. 3 Range obtained over 7 consecutive days. Open in new tab Table 1. Linearity data for MDA, MDMA, and MDEA in whole blood, serum, water (blank for vitreous humor), and urine.1 . Slope2 . CV of slope over 7 days, % . Intercept2 . Sy|x3 . SE of the slope2 . SE of the intercept2 . r23 . Serum MDA 0.013378 5.4 0.0042 0.004208–0.009851 0.001700 0.008161 0.9982–0.9996 MDMA 0.014282 5.1 0.0075 0.003880–0.010908 0.001829 0.008259 0.9979–0.9997 MDEA 0.013992 5.2 0.0127 0.004935–0.011844 0.0001583 0.007545 0.9979–0.9996 Whole blood MDA 0.013161 5.3 0.0144 0.007881–0.010349 0.0002390 0.011477 0.9977–0.9988 MDMA 0.014341 3.8 0.0097 0.004767–0.012172 0.0001947 0.009281 0.9976–0.9996 MDEA 0.014156 4.8 0.0155 0.004911–0.010624 0.0002017 0.009613 0.9980–0.9996 Water MDA 0.014731 4.2 0.0099 0.004224–0.014084 0.0002276 0.010981 0.9970–0.9997 MDMA 0.01496 4.0 0.0000 0.004631–0.012393 0.0002161 0.010365 0.9978–0.9992 MEDA 0.01459 3.6 0.0099 0.004983–0.013898 0.0002177 0.010410 0.9969–0.9997 Urine MDA 1.04728 2.0 −0.0023 0.015309–0.054148 0.0125754 0.008680 0.9986–0.9998 MDMA 1.15503 2.8 −0.0029 0.016010–0.058408 0.0136079 0.010447 0.9986–0.9999 MDEA 1.15330 5.3 −0.0048 0.031770–0.089753 0.0188846 0.011269 0.9971–0.9995 . Slope2 . CV of slope over 7 days, % . Intercept2 . Sy|x3 . SE of the slope2 . SE of the intercept2 . r23 . Serum MDA 0.013378 5.4 0.0042 0.004208–0.009851 0.001700 0.008161 0.9982–0.9996 MDMA 0.014282 5.1 0.0075 0.003880–0.010908 0.001829 0.008259 0.9979–0.9997 MDEA 0.013992 5.2 0.0127 0.004935–0.011844 0.0001583 0.007545 0.9979–0.9996 Whole blood MDA 0.013161 5.3 0.0144 0.007881–0.010349 0.0002390 0.011477 0.9977–0.9988 MDMA 0.014341 3.8 0.0097 0.004767–0.012172 0.0001947 0.009281 0.9976–0.9996 MDEA 0.014156 4.8 0.0155 0.004911–0.010624 0.0002017 0.009613 0.9980–0.9996 Water MDA 0.014731 4.2 0.0099 0.004224–0.014084 0.0002276 0.010981 0.9970–0.9997 MDMA 0.01496 4.0 0.0000 0.004631–0.012393 0.0002161 0.010365 0.9978–0.9992 MEDA 0.01459 3.6 0.0099 0.004983–0.013898 0.0002177 0.010410 0.9969–0.9997 Urine MDA 1.04728 2.0 −0.0023 0.015309–0.054148 0.0125754 0.008680 0.9986–0.9998 MDMA 1.15503 2.8 −0.0029 0.016010–0.058408 0.0136079 0.010447 0.9986–0.9999 MDEA 1.15330 5.3 −0.0048 0.031770–0.089753 0.0188846 0.011269 0.9971–0.9995 1 Concentration ranges: 2-1000 μg/L for whole blood, serum, and water; 0.1–5.0 mg/L for urine. 2 Mean value of 7 consecutive days. 3 Range obtained over 7 consecutive days. Open in new tab Table 2. Precision data for the analysis of MDA, MDMA, and MDEA in serum, whole blood, water (blank for vitreous humour), and urine. . Matrix . Concentration added . CV, % . . . . . . MDA . MDMA . MDEA . Within-day reproducibility (n = 7)1 Serum 2 μg/L 9.9 12 12 40 μg/L 2.5 4.2 3.4 1000 μg/L 3.6 2.6 4.6 Whole blood 2 μg/L 13 12 12 40 μg/L 6.8 6.8 6.1 1000 μg/L 5.2 5.1 4.5 Water (vitreous humor) 2 μg/L 10 10 6.7 40 μg/L 3.3 3.6 3.5 1000 μg/L 2.9 3.1 3.6 Urine 0.1 mg/L 5.3 5.3 5.4 0.5 mg/L 3.9 3.2 4.4 5.0 mg/L 4.4 3.9 4.5 Total reproducibility (n = 7)1 Serum 2 μg/L 16 19 19 40 μg/L 5.4 5.9 6.1 1000 μg/L 5.7 7.1 5.8 Whole blood 2 μg/L 16 17 18 40 μg/L 11 8.9 6.9 1000 μg/L 6.0 4.9 5.2 Water (vitreous humor) 2 μg/L 11 13 10 40 μg/L 7.1 6.7 3.7 1000 μg/L 6.5 4.5 4.5 Urine 0.1 mg/L 4.1 5.1 3.5 0.5 mg/L 4.2 5.4 3.9 5.0 mg/L 4.1 3.8 5.4 . Matrix . Concentration added . CV, % . . . . . . MDA . MDMA . MDEA . Within-day reproducibility (n = 7)1 Serum 2 μg/L 9.9 12 12 40 μg/L 2.5 4.2 3.4 1000 μg/L 3.6 2.6 4.6 Whole blood 2 μg/L 13 12 12 40 μg/L 6.8 6.8 6.1 1000 μg/L 5.2 5.1 4.5 Water (vitreous humor) 2 μg/L 10 10 6.7 40 μg/L 3.3 3.6 3.5 1000 μg/L 2.9 3.1 3.6 Urine 0.1 mg/L 5.3 5.3 5.4 0.5 mg/L 3.9 3.2 4.4 5.0 mg/L 4.4 3.9 4.5 Total reproducibility (n = 7)1 Serum 2 μg/L 16 19 19 40 μg/L 5.4 5.9 6.1 1000 μg/L 5.7 7.1 5.8 Whole blood 2 μg/L 16 17 18 40 μg/L 11 8.9 6.9 1000 μg/L 6.0 4.9 5.2 Water (vitreous humor) 2 μg/L 11 13 10 40 μg/L 7.1 6.7 3.7 1000 μg/L 6.5 4.5 4.5 Urine 0.1 mg/L 4.1 5.1 3.5 0.5 mg/L 4.2 5.4 3.9 5.0 mg/L 4.1 3.8 5.4 1 n, number of determinations. Open in new tab Table 2. Precision data for the analysis of MDA, MDMA, and MDEA in serum, whole blood, water (blank for vitreous humour), and urine. . Matrix . Concentration added . CV, % . . . . . . MDA . MDMA . MDEA . Within-day reproducibility (n = 7)1 Serum 2 μg/L 9.9 12 12 40 μg/L 2.5 4.2 3.4 1000 μg/L 3.6 2.6 4.6 Whole blood 2 μg/L 13 12 12 40 μg/L 6.8 6.8 6.1 1000 μg/L 5.2 5.1 4.5 Water (vitreous humor) 2 μg/L 10 10 6.7 40 μg/L 3.3 3.6 3.5 1000 μg/L 2.9 3.1 3.6 Urine 0.1 mg/L 5.3 5.3 5.4 0.5 mg/L 3.9 3.2 4.4 5.0 mg/L 4.4 3.9 4.5 Total reproducibility (n = 7)1 Serum 2 μg/L 16 19 19 40 μg/L 5.4 5.9 6.1 1000 μg/L 5.7 7.1 5.8 Whole blood 2 μg/L 16 17 18 40 μg/L 11 8.9 6.9 1000 μg/L 6.0 4.9 5.2 Water (vitreous humor) 2 μg/L 11 13 10 40 μg/L 7.1 6.7 3.7 1000 μg/L 6.5 4.5 4.5 Urine 0.1 mg/L 4.1 5.1 3.5 0.5 mg/L 4.2 5.4 3.9 5.0 mg/L 4.1 3.8 5.4 . Matrix . Concentration added . CV, % . . . . . . MDA . MDMA . MDEA . Within-day reproducibility (n = 7)1 Serum 2 μg/L 9.9 12 12 40 μg/L 2.5 4.2 3.4 1000 μg/L 3.6 2.6 4.6 Whole blood 2 μg/L 13 12 12 40 μg/L 6.8 6.8 6.1 1000 μg/L 5.2 5.1 4.5 Water (vitreous humor) 2 μg/L 10 10 6.7 40 μg/L 3.3 3.6 3.5 1000 μg/L 2.9 3.1 3.6 Urine 0.1 mg/L 5.3 5.3 5.4 0.5 mg/L 3.9 3.2 4.4 5.0 mg/L 4.4 3.9 4.5 Total reproducibility (n = 7)1 Serum 2 μg/L 16 19 19 40 μg/L 5.4 5.9 6.1 1000 μg/L 5.7 7.1 5.8 Whole blood 2 μg/L 16 17 18 40 μg/L 11 8.9 6.9 1000 μg/L 6.0 4.9 5.2 Water (vitreous humor) 2 μg/L 11 13 10 40 μg/L 7.1 6.7 3.7 1000 μg/L 6.5 4.5 4.5 Urine 0.1 mg/L 4.1 5.1 3.5 0.5 mg/L 4.2 5.4 3.9 5.0 mg/L 4.1 3.8 5.4 1 n, number of determinations. Open in new tab Table 3. HPLC retention data for compounds evaluated as possible interferents. Compound . Capacity factor (k′) . Morphine 3.01 MDA 6.94 MDMA 7.52 Zopiclone 7.88 MDEA 8.15 MDMPA (internal standard) 9.32 Pentazocine 10.54 Acetaminophen, acetylcodeine, acetylsalicylic acid, alprazolam, amfepramone, amitriptyline, amphetamine, apomorphine, atropine, benzoylecgonine, bromazepam, brotizolam, caffeine, camazepam, carbamazepine, citalopram, clonazepam, clotiazepam, cloxazolam, cocaine, codeine, desipramine, diacetylmorphine, diazepam, dihydrocodeine, diphenhydramine, disulfiram, dothiepin, doxepin, ephedrine, ethyl loflazepate, fenfluramine, fentanyl, flunitrazepam, fluoxetine, flurazepam, halazepam, haloperidol, hexobarbital, hydromorphone, imipramine, ketazolam, lidocaine, loprazolam, lorazepam, lormetazepam, loxapine, medazepam, meprobamate, methadone, methamphetamine, methaqualone, methotrimeprazine, N-methylephedrine, mianserin, midazolam, 6-monoacetylmorphine, nicotine, nitrazepam, nordazepam, norpseudoephedrine, oxazepam, oxycodone, papaverine, phenobarbital, phenytoin, piracetam, propoxyphene, quinine, secobarbital, strychnine, temazepam, theobromine, theophylline, tilidine, triazolam ND1 Compound . Capacity factor (k′) . Morphine 3.01 MDA 6.94 MDMA 7.52 Zopiclone 7.88 MDEA 8.15 MDMPA (internal standard) 9.32 Pentazocine 10.54 Acetaminophen, acetylcodeine, acetylsalicylic acid, alprazolam, amfepramone, amitriptyline, amphetamine, apomorphine, atropine, benzoylecgonine, bromazepam, brotizolam, caffeine, camazepam, carbamazepine, citalopram, clonazepam, clotiazepam, cloxazolam, cocaine, codeine, desipramine, diacetylmorphine, diazepam, dihydrocodeine, diphenhydramine, disulfiram, dothiepin, doxepin, ephedrine, ethyl loflazepate, fenfluramine, fentanyl, flunitrazepam, fluoxetine, flurazepam, halazepam, haloperidol, hexobarbital, hydromorphone, imipramine, ketazolam, lidocaine, loprazolam, lorazepam, lormetazepam, loxapine, medazepam, meprobamate, methadone, methamphetamine, methaqualone, methotrimeprazine, N-methylephedrine, mianserin, midazolam, 6-monoacetylmorphine, nicotine, nitrazepam, nordazepam, norpseudoephedrine, oxazepam, oxycodone, papaverine, phenobarbital, phenytoin, piracetam, propoxyphene, quinine, secobarbital, strychnine, temazepam, theobromine, theophylline, tilidine, triazolam ND1 1 ND, not detected. Open in new tab Table 3. HPLC retention data for compounds evaluated as possible interferents. Compound . Capacity factor (k′) . Morphine 3.01 MDA 6.94 MDMA 7.52 Zopiclone 7.88 MDEA 8.15 MDMPA (internal standard) 9.32 Pentazocine 10.54 Acetaminophen, acetylcodeine, acetylsalicylic acid, alprazolam, amfepramone, amitriptyline, amphetamine, apomorphine, atropine, benzoylecgonine, bromazepam, brotizolam, caffeine, camazepam, carbamazepine, citalopram, clonazepam, clotiazepam, cloxazolam, cocaine, codeine, desipramine, diacetylmorphine, diazepam, dihydrocodeine, diphenhydramine, disulfiram, dothiepin, doxepin, ephedrine, ethyl loflazepate, fenfluramine, fentanyl, flunitrazepam, fluoxetine, flurazepam, halazepam, haloperidol, hexobarbital, hydromorphone, imipramine, ketazolam, lidocaine, loprazolam, lorazepam, lormetazepam, loxapine, medazepam, meprobamate, methadone, methamphetamine, methaqualone, methotrimeprazine, N-methylephedrine, mianserin, midazolam, 6-monoacetylmorphine, nicotine, nitrazepam, nordazepam, norpseudoephedrine, oxazepam, oxycodone, papaverine, phenobarbital, phenytoin, piracetam, propoxyphene, quinine, secobarbital, strychnine, temazepam, theobromine, theophylline, tilidine, triazolam ND1 Compound . Capacity factor (k′) . Morphine 3.01 MDA 6.94 MDMA 7.52 Zopiclone 7.88 MDEA 8.15 MDMPA (internal standard) 9.32 Pentazocine 10.54 Acetaminophen, acetylcodeine, acetylsalicylic acid, alprazolam, amfepramone, amitriptyline, amphetamine, apomorphine, atropine, benzoylecgonine, bromazepam, brotizolam, caffeine, camazepam, carbamazepine, citalopram, clonazepam, clotiazepam, cloxazolam, cocaine, codeine, desipramine, diacetylmorphine, diazepam, dihydrocodeine, diphenhydramine, disulfiram, dothiepin, doxepin, ephedrine, ethyl loflazepate, fenfluramine, fentanyl, flunitrazepam, fluoxetine, flurazepam, halazepam, haloperidol, hexobarbital, hydromorphone, imipramine, ketazolam, lidocaine, loprazolam, lorazepam, lormetazepam, loxapine, medazepam, meprobamate, methadone, methamphetamine, methaqualone, methotrimeprazine, N-methylephedrine, mianserin, midazolam, 6-monoacetylmorphine, nicotine, nitrazepam, nordazepam, norpseudoephedrine, oxazepam, oxycodone, papaverine, phenobarbital, phenytoin, piracetam, propoxyphene, quinine, secobarbital, strychnine, temazepam, theobromine, theophylline, tilidine, triazolam ND1 1 ND, not detected. Open in new tab Figure 4. Open in new tabDownload slide Chromatogram of an extract from a rabbit vitreous humor sample containing 38.2 μg/L MDMA (peak 2), MDA below the LOQ (peak 1), and 80 μg/L MDMPA (the internal standard; peak 4). Figure 4. Open in new tabDownload slide Chromatogram of an extract from a rabbit vitreous humor sample containing 38.2 μg/L MDMA (peak 2), MDA below the LOQ (peak 1), and 80 μg/L MDMPA (the internal standard; peak 4). Figure 5. Open in new tabDownload slide Chromatogram of a blank whole-blood sample containing only 80 μg/L MDMPA (the internal standard; peak 4). Figure 5. Open in new tabDownload slide Chromatogram of a blank whole-blood sample containing only 80 μg/L MDMPA (the internal standard; peak 4). Figure 6. Open in new tabDownload slide Chromatogram of a positive forensic toxicological urine sample containing 1.89 mg/L MDA (peak 1), 12.62 mg/L MDMA (peak 2), and 5.44 mg/L MDEA (peak 3). Peak 4, MDMPA. Figure 6. Open in new tabDownload slide Chromatogram of a positive forensic toxicological urine sample containing 1.89 mg/L MDA (peak 1), 12.62 mg/L MDMA (peak 2), and 5.44 mg/L MDEA (peak 3). Peak 4, MDMPA. Figure 7. Open in new tabDownload slide Plasma concentration–time curve and vitreous humor concentrations in a rabbit after a single intravenous administration of 1 mg/kg MDMA. Figure 7. Open in new tabDownload slide Plasma concentration–time curve and vitreous humor concentrations in a rabbit after a single intravenous administration of 1 mg/kg MDMA. We thank F. Piessens for practical assistance with validation of the method. 1 " Nonstandard abbreviations: MDMA, 3,4-methylenedioxymethamphetamine; MDEA, 3,4-methylenedioxyethylamphetamine; MDA, 3,4-methylenedioxyamphetamine; MDMPA, methylenedioxymethylpropylamphetamine; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LC-Fl, LC-fluorescence; TOF, time of flight; NMR, nuclear magnetic resonance; HRMS-FAB, fast atom bombardment high-resolution mass spectrometry; LOD, limit of detection; and LOQ, limit of quantification. References 1 Wills S. Drugs of abuse, 1st ed 1997 : 61 -77 The Pharmaceutical Press London. . 2 Robbins TW, Everitt BJ. Drug addiction: bad habits add up. Nature 1999 ; 398 : 567 -570. Crossref Search ADS PubMed 3 Nichols DE. Differences between the mechanism of action of MDMA, MBDB, and the classic hallucinogens. Identification of a new therapeutic class: entactogens. J Psychoactive Drugs 1986 ; 18 : 305 -313. Crossref Search ADS PubMed 4 Fineschi V, Masti A. Fatal poisoning by MDMA (ecstasy) and MDEA: a case report. Int J Legal Med 1996 ; 108 : 272 -275. Crossref Search ADS PubMed 5 Iwersen S, Schmoldt A. Two very different fatal cases associated with the use of methylenedioxyethylamphetamine (MDEA): Eve as deadly as Adam. Clin Toxicol 1996 ; 34 : 241 -244. 6 Randall T. Ecstasy-fueled ‘Rave’ parties become dances of death for English youths. JAMA 1992 ; 268 : 1505 -1506. Crossref Search ADS PubMed 7 McCann UD, Szabo Z, Scheffel U, Dannals RF, Ricaurte GA. Positron emission tomographic evidence of toxic effect of MDMA (“Ecstasy”) on brain serotonin neurons in human beings. Lancet 1998 ; 352 : 1433 -1437. Crossref Search ADS PubMed 8 Morris K. Ecstasy users face consequences of neurotoxicity. Lancet 1998 ; 353 : 1913 . 9 Ensslin HK, Kovar KA, Maurer HH. Toxicological detection of the designer drug 3,4-methylenedioxyethylamphetamine (MDE, “Eve”) and its metabolites in urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay. J Chromatogr B 1996 ; 683 : 189 -197. Crossref Search ADS 10 Kunsman GW, Levine B, Kuhlman JJ, Jones RL, Hughes RO, Fujiyama CI, Smith ML. MDA-MDMA concentrations in urine specimens. J Anal Toxicol 1996 ; 20 : 517 -521. Crossref Search ADS PubMed 11 Lim HK, Su Z, Foltz RL. Stereoselective disposition: enantioselective quantitation of 3,4-(methylenedioxy)methamphetamine and three of its metabolites by gas chromatography/electron capture negative chemical ionization mass spectrometry. Biol Mass Spectrom 1993 ; 22 : 403 -411. Crossref Search ADS PubMed 12 Helmlin HJ, Bracher K, Bourquin D, Vonlanthen D, Brenneisen R, Styk J. Analysis of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in plasma and urine by HPLC-DAD and GC-MS. J Anal Toxicol 1996 ; 20 : 432 -440. Crossref Search ADS PubMed 13 Helmlin HJ, Brenneisen R. Determination of psychotropic phenylalkylamine derivatives in biological matrices by high-performance liquid chromatography with photodiode-array detection. J Chromatogr 1992 ; 593 : 87 -94. Crossref Search ADS PubMed 14 Bogusz MJ, Kala M, Maier RD. Determination of phenylisothiocyanate derivatives of amphetamine and its analogues in biological fluids by HPLC-APCI-MS or DAD. J Anal Toxicol 1997 ; 21 : 59 -69. Crossref Search ADS PubMed 15 Tedeschi L, Frison G, Castasgna F, Giorgetti R, Ferrara SD. Simultaneous identification of amphetamine and its derivatives in urine using HPLC-UV. Int J Legal Med 1993 ; 105 : 265 -269. Crossref Search ADS PubMed 16 Logan BK, Stafford DT. Direct analysis of anticonvulsant drugs in vitreous humour by HPLC using a column switching technique. Forensic Sci Int 1989 ; 41 : 125 -134. Crossref Search ADS PubMed 17 Kintz P, Cirimele V, Tracqui A, Mangin P. Simultaneous determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphtamine in human hair by gas chromatography mass spectrometry. J Chromatogr B 1995 ; 670 : 162 -166. Crossref Search ADS 18 Nichols DE, Oberlender R. Structure activity relationships of MDMA-like substances. In: Asghar K, De Souza E, eds. NIDA research monograph 94. Rockville MD: National Institute on Drug Abuse, 1989:1–12.. 19 Tagliaro F, De Battisti Z, Groppi A, Nakahara Y, Scarcella D, Valentini R, et al. High sensitivity simultaneous determination in hair of the major constituents of ecstasy (3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxyethylamphetamine) by high-performance liquid chromatography with direct fluorescence detection. J Chromatogr B 1999 ; 723 : 195 -202. Crossref Search ADS 20 De Letter EA, De Paepe P, Clauwaert KM, Belpaire FM, Lambert WE, Van Bocxlaer JF, Piette MH. Is vitreous humour useful for the interpretation of 3,4-methylenedioxy-methamphetamine (MDMA) blood levels? Int J Legal Med 2000; in press.. 21 Furnari C, Ottaviano V, Rosati F, Tondi V. Identification of 3,4-methylenedioxyamphetamine analogs encountered in clandestine tablets. Forensic Sci Int 1998 ; 92 : 49 -58. Crossref Search ADS PubMed 22 Maurer HH. On the metabolism and the toxicological analysis of methylenedioxyphenylalkylamine designer drugs by gas chromatography-mass spectrometry. Ther Drug Monit 1996 ; 18 : 465 -470. Crossref Search ADS PubMed 23 Cheung S, Nolte H, Otton SV, Tyndale RF, Wu PH, Sellers EM. Simultaneous gas chromatographic determination of methamphetamine, amphetamine and their p-hydroxylated metabolites in plasma and urine. J Chromatogr B 1997 ; 690 : 77 -87. Crossref Search ADS 24 Clauwaert KM, Van Bocxlaer JF, Major HJ, Claereboudt JA, Lambert WE, Van den Eeckhout EM, et al. Investigation of the quantitative properties of the quadrupole orthogonal acceleration time-of-flight mass spectrometer with electrospray ionisation using 3,4-methylenedioxymethamphetamine. Rapid Commun Mass Spectrom 1999 ; 13 : 1540 -1545. Crossref Search ADS PubMed © 2000 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Determination of the Designer Drugs 3,4-Methylenedioxymethamphetamine, 3,4-Methylenedioxyethylamphetamine, and 3,4-Methylenedioxyamphetamine with HPLC and Fluorescence Detection in Whole Blood, Serum, Vitreous Humor, and Urine
JF - Clinical Chemistry
DO - 10.1093/clinchem/46.12.1968
DA - 2000-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/determination-of-the-designer-drugs-3-4-methylenedioxymethamphetamine-IjSPKfo0rC
SP - 1968
VL - 46
IS - 12
DP - DeepDyve
ER -