We report a case of intoxication by the synthetic cannabinoid MAM-2201 ([1-(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl)-methanone). A 31-year-old man smoked about 300 mg of a herbal blend. He experienced an acute transient psychotic state with agitation, aggression, anxiety, and vomiting associated with a sympathomimetic syndrome. MAM-2201 was detected and quantified in a plasma sample using liquid chromatography-tandem mass spectrometry (LC–MS–MS). The level was 49 ng/ml 1 h after smoking. The use of other drugs was analytically excluded. The presence of MAM-2201 was confirmed in the herbal blend using gas chromatography–mass spectrometry (GC–MS) and LC–high resolution MS. This is the first description of an analytically confirmed intoxication and of the determination of MAM-2201 in human blood plasma.

The opioid 3-methylfentanyl, a designer drug of the fentanyl type, was scheduled by the Controlled Substance Act due to its high potency and abuse potential. To overcome this regulation, isofentanyl, another designer fentanyl, was synthesized in a clandestine laboratory and seized by the German police. The aims of the presented study were to identify the phase I and phase II metabolites of 3-methylfentanyl and isofentanyl in rat urine, to identify the cytochrome P450 (CYP) isoenzymes involved in their initial metabolic steps, and, finally, to test their detectability in urine. Using liquid chromatography (LC)-linear ion trap-mass spectrometry (MS(n)), nine phase I and five phase II metabolites of 3-methylfentanyl and 11 phase I and four phase II metabolites of isofentanyl could be identified. The following metabolic steps could be postulated for both drugs: N-dealkylation followed by hydroxylation of the alkyl and aryl moiety, hydroxylation of the propanamide side chain followed by oxidation to the corresponding carboxylic acid, and, finally, hydroxylation of the benzyl moiety followed by methylation. In addition, N-oxidation of isofentanyl could also be observed. All hydroxy metabolites were partly excreted as glucuronides. Using recombinant human isoenzymes, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were found to be involved in the initial metabolic steps. Our LC-MS(n) screening approach allowed the detection of 0.01 mg/L of 3-methylfentanyl and isofentanyl in spiked urine. However, in urine of rats after the administration of suspected recreational doses, the parent drugs could not be detected, but their common nor metabolite, which should therefore be the target for urine screening.

3-Bromomethcathinone (3-BMC) and 3-Fluoromethcathinone (3-FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so-called bath salts or plant feeders. The aim of the present study was to identify for the first time the 3-BMC and 3-FMC Phase I and II metabolites in rat urine and human liver microsomes using GCMS and LChigh-resolution MS (HR-MS) and to test for their detectability by established urine screening approaches using GCMS or LCMS. Furthermore, the human cytochrome-P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drugdrug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GCMS and by LCHR-MS. The main metabolic steps were N-demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GCMS could be confirmed by LCHR-MS. Furthermore, corresponding Phase II metabolites were identified using the LCHR-MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GCMS and LCMSn after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N-demethylation of 3-BMC and 3-FMC after in vitroin vivo extrapolation. Copyright (C) 2012 John Wiley & Sons, Ltd.

The R- and S-enantiomers of racemic 3,4-methylenedioxymethamphetamine (MDMA) exhibit different dose-concentration curves. In plasma, S-MDMA was eliminated at a higher rate, most likely due to stereoselective metabolism. Similar data were shown in various in vitro experiments. The aim of the present study was the in vivo investigation of stereoselective elimination of MDMA's phase I and phase II metabolites in human urine following controlled oral MDMA administration. Urine samples from 10 participants receiving 1.0 and 1.6 mg/kg MDMA separated by at least one week were analyzed blind by liquid chromatography-high resolution-mass spectrometry and gas chromatography-mass spectrometry after chiral derivatization with S-heptafluorobutyrylprolyl chloride. R/S ratios at C(max) were comparable after low and high doses with ratios >1 for MDMA, free DHMA, and HMMA sulfate, and with ratios <1 for MDA, free HMMA, DHMA sulfate and HMMA glucuronide. In the five days after the high MDMA dose, a median of 21% of all evaluated compounds were excreted as R-stereoisomers and 17% as S-stereoisomers. Significantly greater MDMA, DHMA, and HMMA sulfate R-enantiomers and HMMA and HMMA glucuronide S-stereoisomers were excreted. No significant differences were observed for MDA and DHMA sulfate stereoisomers. Changes in R/S ratios could be observed over time for all analytes, with steady increases in the first 48 h. R/S ratios could help to roughly estimate time of MDMA ingestion and therefore, improve interpretation of MDMA and metabolite urinary concentrations in clinical and forensic toxicology.

Different elimination was reported for the two enantiomers of the designer drug 3,4-methylenedioxyethylamphetamine (MDEA) in vivo. In the present work, the enantioselectivity of glucuronidation and sulfation of the MDEA phase I metabolites 3,4-dihydroxyethylamphetamine (DHEA) and 4-hydroxy-3-methoxyethylamphetamine (HMEA) was investigated. First, glucuronide standards were synthesized using rat liver microsomes. Incubations were performed with recombinant human UDP-glucuronyltransferases (UGT) and pooled human liver microsomes (pHLM) for glucuronidation and using recombinant human sulfotransferases (SULT) and pooled human liver cytosol (pHLC) for sulfation. Product formation experiments were performed by quantification of the phase II metabolites using liquid chromatography-high-resolution mass spectrometry. Additionally, substrate depletion experiments were conducted by gas chromatography-mass spectrometry after chiral derivatization for sulfation. UGT2B7, 2B15, and 2B17 were involved in glucuronidation of HMEA and SULT1A1 and SULT1A3 and SULT1A3 and SULT1E1 in the sulfation of DHEA and HMEA, respectively. SULTs provided much higher affinity, whereas UGTs showed higher capacities. Marked stereoselectivity could be observed for UGT2B15, UGT2B17, and pHLM toward S-HMEA, for SULT1A3 and pHLC toward S-DHEA and for SULT1A3 and pHLC toward R-HMEA. In conclusion, the phase II metabolism might also contribute to the observed different pharmacokinetic properties of MDEA.

This paper reviews analytical approaches published in 2002-2012 for chiral drug analysis and their relevance in research and practice in the field of clinical and forensic toxicology. Separation systems such as gas chromatography, high performance liquid chromatography, capillary electromigration, and supercritical fluid chromatography, all coupled to mass spectrometry, are discussed. Typical applications are reviewed for relevant chiral analytes such as amphetamines and amphetamine-derived designer drugs, methadone, tramadol, psychotropic and other CNS acting drugs, anticoagulants, cardiovascular drugs, and some other drugs. Usefulness of chiral drug analysis in the interpretation of analytical results in clinical and forensic toxicology is discussed as well. (c) 2012 Elsevier B.V. All rights reserved.

3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography-high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography-mass spectrometry with negative-ion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes.

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is excreted in human urine mainly as conjugates of its metabolites 3,4-dihydroxymethamphetamine (DHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA). The glucuronidation kinetics of HMMA showed high capacities, but also high K(m) values, unlikely to be reached after recreational user's doses. Therefore, the aim of the present work was to investigate the sulfation of DHMA and HMMA by human sulfotransferases (SULTs) in pooled human liver cytosol (pHLC). The kinetic data showed deviation from typical Michaelis-Menten kinetics. The overall efficiency for HMMA sulfation was calculated to be 2-10 times higher than for glucuronidation. As the sulfation of both MDMA metabolites showed substrate inhibition effects, their inhibitory potential towards typical sulfation reactions in pHLC was tested. The following substrates for typical sulfation reactions were used: nitrophenol, dopamine, estradiol, and dehydroepi androsten dione. Inhibition was observed towards dopamine sulfation by DHMA and HMMA, but not by MDMA. The 1/V vs. 1/S plots indicated a mixed-type or competitive inhibition model for DHMA and HMMA, respectively. In conclusion, the presented data indicated that sulfation of HMMA should be the major conjugation reaction observed in humans. Furthermore, both, DHMA and HMMA, were identified as inhibitors of dopamine sulfation.

BACKGROUND:3,4-Methylendioxymethamphetamine (MDMA) is excreted inhuman urine as unchanged drug and phase I and II metabolites. Previous urinary excretion studies after controlled oral MDMA administration have been performed only after conjugate cleavage. Therefore, we investigated intact MDMA glucuronide and sulfate metabolite excretion.METHODS:We used LC-high-resolution MS and GC-MS to reanalyze blind urine samples from 10 participants receiving 1.0 or 1.6 mg/kg MDMA orally. We determined median C(max),t(max), first and last detection times, and total urinary recovery; calculated ratios of sulfates and glucuronides; and performed in vitro-in vivo correlations.RESULTS:Phase II metabolites of 3,4-dihydroxymethamphetamine (DHMA),4-hydroxy-3-methoxymethamphetamine (HMMA),3,4-dihydroxyamphetamine (DHA), and 4-hydroxy-3-methoxyamphetamine were identified, although only DHMA sulfates, HMMA sulfate, and HMMA glucuronide had substantial abundance. Good correlation was observed for HMMA measured after acid hydrolysis and the sum of unconjugated HMMA, HMMA glucuronide, and HMMA sulfate (R(2) = 0.87). More than 90% of total DHMA and HMMA were excreted as conjugates. The analyte with the longest detection time was HMMA sulfate. Median HMMA sulfate/glucuronide and DHMA 3-sulfate/4-sulfate ratios for the first 24 h were 2.0 and 5.3, respectively, in accordance with previous in vitro calculations from human liver microsomes and cytosol experiments.CONCLUSIONS:Human MDMA urinary metabolites are primarily sulfates and glucuronides,with sulfates present in higher concentrations than glucuronides. This new knowledge may lead to improvements in urine MDMA and metabolite analysis in clinical and forensic toxicology, particularly for the performance of direct urine analysis.

Different pharmacokinetic properties are known for the two enantiomers of the entactogen 3,4-methylendioxy-methamphetamine (MDMA), most likely due to enantioselective metabolism. The aim of the present work was 1) the investigation of the main sulfotransferases (SULT) isoenzymes involved in the sulfation of the main MDMA phase I metabolites 3,4-dihydroxymethamphetamine (DHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA) and 2) the evaluation of a possible enantioselectivity of this phase II metabolic step. Therefore, racemic DHMA and HMMA were incubated with heterologously expressed SULTs, and quantification of the sulfates by liquid chromatography-high-resolution mass spectrometry was conducted. Because separation of DHMA and HMMA sulfate could not be achieved by liquid chromatography, enantioselective kinetic parameters were determined using the substrate-depletion approach with enantioselective quantification of substrate consumption by gas chromatography-negative ion chemical ionization mass spectrometry. SULT1A1 and SULT1A3 catalyzed sulfation of DHMA, and SULT1A3 and SULT1E1 catalyzed sulfation of HMMA. SULT1A1 and SULT1E1 revealed classic Michaelis-Menten kinetics, whereas SULT1A3 kinetics showed deviation from the typical Michaelis-Menten kinetics, resulting in a concentration-dependent self-inhibition. SULT1A3 showed the highest affinity and capacity of the SULT isoforms. Marked enantioselectivity could be observed for S-DHMA sulfation by SULT1A3 and in human liver cytosol, whereas no differences were observed for HMMA sulfation. Finally, comparison of Km and Vmax values calculated using achiral product formation and chiral substrate depletion showed good correlation within 2-fold of each other. In conclusion, preferences for S-enantiomers were observed for DHMA sulfation, but not for HMMA sulfation.

BACKGROUND : 3,4-Methylendioxymethamphetamine (MDMA) is excreted in human urine as unchanged drug and phase I and II metabolites. Previous urinary excretion studies after controlled oral MDMA administration have been performed only after conjugate cleavage. Therefore, we investigated intact MDMA glucuronide and sulfate metabolite excretion. METHODS : We reanalyzed blind urine samples from 10 participants receiving 1.0 or 1.6 mg/kg MDMA orally by liquid chromatography/high-resolution mass spectrometry and gas chromatography/mass spectrometry. We determined median C max , t max , first and last detection times, and total urinary recovery, calculated ratios of sulfates and glucuronides, and performed in vitro–in vivo correlations. RESULTS : Phase II metabolites of 3,4-dihydroxymeth-amphetamine (DHMA), 4-hydroxy-3-methoxymeth-amphetamine (HMMA), 3,4-dihydroxyamphetamine (DHA), and 4-hydroxy-3-methoxyamphetamine (HMA) were identified, although only DHMA sulfates, HMMA sulfate, and HMMA glucuronide had substan-tial abundance. Good correlation was observed for HMMA measured after acid hydrolysis and the sum of unconjugated HMMA, HMMA glucuronide, and HMMA sulfate ( R 2 (cid:1) 0.87). More than 90% of total DHMA and HMMA were excreted as conjugates. The analyte with the longest detection time was HMMA sulfate. Median HMMA

In several cases, fluoxetine, its metabolites, its known artifacts, and supposedly tranylcypromine were detected in urine using the authors' systematic toxicological analysis (STA) procedure based on acid hydrolysis, extraction, and acetylation. As fluoxetine and tranylcypromine are absolutely contraindicated drugs and in none of the cases was tranylcypromine prescribed, the question of whether the detected compound might have been formed by fluoxetine and/or its metabolites arose. Therefore, rat urine taken after dosing with fluoxetine was screened in the same way. In addition, aqueous solutions of fluoxetine, norfluoxetine, tranylcypromine, and a mixture of the latter two drugs were worked-up and analyzed according to the STA and without hydrolysis. In urine specimens obtained from rats dosed with fluoxetine, tranylcypromine was detected as well as in the solution of worked-up norfluoxetine including hydrolysis. Its underlying mass spectrum could be identified by detailed interpretation of the fragmentation patterns as acetylated 3-phenyl-propyl-2-ene-amine. This compound could be postulated as hydrolysis product of norfluoxetine formed by ether cleavage and water elimination. Although this spectrum shows nearly the same fragmentation patterns as that of acetylated tranylcypromine, both compounds could finally be differentiated by their retention indices and by using the positive-ion chemical ionization mode.

The cytochrome P450 isoform CYP3A7 (wildtype) is the major form of CYP in human fetal liver. Since it is not exclusively expressed in the fetus but also in a significant number of adults, CYP3A7 has been moving into the focus of investigation on adverse drug reactions and interindividual differences in drug metabolism in the last few years. In addition, CYP3A7 is overexpressed in hepatocellular carcinoma (HCC), where it contributes to the elimination of drugs. We here report the development of a convenient and reliable whole-cell system for testing CYP3A7 activity using recombinant fission yeast. As expected, catalytic properties of wild type CYP3A7.1 and its polymorphic form CYP3A7.2 towards DHEA and testosterone resembled those reported previously. Interestingly, both isoforms of CYP3A7 did not metabolize the anti-cancer drug sorafenib (which is approved for the treatment of HCC), while CYP3A4 produced the N-oxide in our system, as expected. This finding suggests that CYP3A7 activity does not influence the effectiveness of this anti-cancer drug against HCC. Furthermore, CYP3A7-expressing fission yeast cells specifically converted a luciferin-derivate (luciferin-PFBE) to a luminescent product and this activity can conveniently be monitored by spectrometry, which allowed the determination of IC50-values for the broad-range P450 inhibitors econazole and miconazole, respectively. We believe that these new tools for a fast and easy investigation of substrates and inhibitors of human CYP3A7 will contribute to the gain of important insights for drug metabolism, efficacy and safety.

The growing need for the characterization of cytochrome P450 (P450) metabolites often necessitates their synthesis up to Gram-scale. This task may in principle be achieved by using various techniques including chemical synthesis, the use of laboratory animals, in vitro P450 systems or microbial biotransformation. However, these approaches are in many instances unfavorable due to low yields, laborious purification, costs of cofactors, or the formation of non-physiologic metabolites. The fission yeast Schizosaccharomyces pombe has previously been shown by others and us to be very well suited for the heterologous expression of human P450s. In this study, we demonstrate whole-cell biotransformation reactions carried out with fission yeast strains that coexpress human cytochrome P450 reductase (CPR) and one of the following P450 isoforms: CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4, respectively. These strains could successfully convert their respective standard substrates but showed different responses with respect to incubation pH, the presence of glucose, and temperature, respectively. In addition, the preparative of synthesis of 2.8 g of 4′-hydroxydiclofenac was achieved by whole-cell biotransformation of diclofenac using a CPR-CYP2C9 coexpressing fission yeast strain.

Standards of drug metabolites are required for drug metabolism studies as a basis for toxicological risk assessment with respect to drug interactions and pharmacogenetic polymorphisms. They are further needed as reference compounds in analytical toxicology. However, metabolite standards are often not commercially available, particularly in the case of new designer drugs. As an alternative to often cumbersome chemical synthesis, human cytochrome P450 (CYP) isoenzymes heterologously expressed in the fission yeast Schizosaccharomyces pombe can be used for the biotechnological synthesis of drug metabolites. In the present study this concept was applied to the synthesis of N-(1-phenylcyclohexyl)-2-hydroxyethanamine (PCHEA), the common O-dealkyl metabolite of the designer drugs N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA) and N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA). After adding 250μmol PCEEA·HCl (62mg), a 1l culture of CAD65 (S. pombe strain co-expressing human CYP reductase and CYP2B6) was fermented over 65h (pH 8, 30°C) and centrifuged. PCHEA and remaining parent drug were isolated from the supernatant by solid-phase extraction (SPE). The eluate was evaporated to dryness and reconstituted in HPLC solvent. Aliquots were separated by semi-preparative HPLC. From the respective fraction, PCHEA was extracted by liquid–liquid extraction and precipitated as hydrochloric salt. Approximately 80% of PCEEA was converted to PCHEA. The final yield of PCHEA·HCl was 9mg (35μmol). Its identity was confirmed by GC–MS, 1H NMR and 13C NMR. The product purity, as determined by HPLC-UV, was 95%.