Adherence to Antihypertensive Drugs Assessed by Hyphenated High-Resolution Mass Spectrometry Analysis of Oral Fluids.

Background It is currently unknown if antihypertensive drugs can be monitored in oral fluid (OF) using liquid chromatography coupled to high-resolution mass spectrometry. Methods and Results We assessed adherence using liquid chromatography coupled to high-resolution mass spectrometry in OF, plasma, and urine of 56 consecutive patients with hypertension referred to a tertiary hypertension unit. Of these patients, 59% were completely adherent (all drugs detectable in urine), whereas 29% and 13% were partially adherent (1 drug not detectable in urine) or nonadherent (>1 drug not detectable in urine), respectively. Adherent patients were on fewer antihypertensive drugs (P=0.001), had fewer daily drug doses (P=0.012), and had lower 24-hour ambulatory systolic (P=0.012) and diastolic (P=0.009) blood pressures than nonadherent or partially adherent patients. Most drugs were detected in urine compared with plasma and OF (181 versus 119 versus 88; P=0.001). Compared with urine and plasma, detection rates of angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and diuretics were lower in OF. There was no difference in the frequency of detecting ß blockers (P=1.0) and calcium channel blockers (P=0.063) when comparing OF with urine. There was no difference in the number of calcium channel blockers (P=0.727), ß blockers (P=1.000), thiazide diuretics (P=0.125), and α-2 agonists (P=0.125) identified between OF and plasma. Conclusions This study shows the feasibility of drug adherence testing for several antihypertensive drugs, especially those without acidic components, in OF, with a similar recovery compared with plasma. Therefore, drug adherence testing in OF should be further explored as a noninvasive approach, which can easily be performed in an "out-of-office" setting.

Studies on the in vitro and in vivo metabolism of the synthetic opioids U-51754, U-47931E, and methoxyacetylfentanyl using hyphenated high-resolution mass spectrometry.

New Synthetic Opioids (NSOs) are one class of New Psychoactive Substances (NPS) enjoying increasing popularity in Europe. Data on their toxicological or metabolic properties have not yet been published for most of them. In this context, the metabolic fate of three NSOs, namely, trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzenacetamide (U-51754), trans-4-bromo-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47931E), and 2-methoxy-N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl] acetamide (methoxyacetylfentanyl), was elucidated by liquid chromatography high-resolution mass spectrometry after pooled human S9 fraction (phS9) incubations and in rat urine after oral administration. The following major reactions were observed: demethylation of the amine moiety for U-51754 and U-47931E, N-hydroxylation of the hexyl ring, and combinations thereof. N-dealkylation, O-demethylation, and hydroxylation at the alkyl part for methoxyacetylfentanyl. Except for U-47931E, parent compounds could only be found in trace amounts in rat urine. Therefore, urinary markers should preferably be metabolites, namely, the N-demethyl-hydroxy and the hydroxy metabolite for U-51754, the N-demethylated metabolite for U-47931E, and the N-dealkylated metabolite as well as the O-demethylated one for methoxyacetylfentanyl. In general, metabolite formation was comparable in vitro and in vivo, but fewer metabolites, particularly those after multiple reaction steps and phase II conjugates, were found in phS9. These results were consistent with those of comparable compounds obtained from human liver microsomes, human hepatocytes, and/or human case studies.

Toxicokinetics and analytical toxicology of the abused opioid U-48800 - in vitro metabolism, metabolic stability, isozyme mapping, and plasma protein binding.

Due to the risk of new synthetic opioids (NSOs) for human health, the knowledge of their toxicokinetic characteristics is important for clinical and forensic toxicology. U-48800 is an NSO structurally non-related to classical opioids such as morphine or fentanyl and offered for abuse. As toxicokinetic data of U-48800 is not currently available, the aims of this study were to identify the in vitro metabolites of U-48800 in pooled human liver S9 fraction (pS9), to map the isozymes involved in the initial metabolic steps, and to determine further toxicokinetic data such as metabolic stability, including the in vitro half-life (t1/2 ), and the intrinsic (CLint ) and hepatic clearance (CLh ). Furthermore, drug detectability studies in rat urine should be done using hyphenated mass spectrometry. In total, 13 phase I metabolites and one phase II metabolite were identified. N-Dealkylation, hydroxylation, and their combinations were the predominant metabolic reactions. The isozymes CYP2C19 and CYP3A4 were mainly involved in these initial steps. CYP2C19 poor metabolizers may suffer from an increased U-48800 toxicity. The in vitro t1/2 and CLint could be rated as moderate, compared to structural related compounds. After administration of an assumed consumer dose to rats, the unchanged parent compound was found only in very low abundance but three metabolites were detected additionally. Due to species differences, metabolites found in rats might be different from those in humans. However, phase I metabolites found in rat urine, the parent compound, and additionally the N-demethyl metabolite should be used as main targets in toxicological urine screening approaches.

Phenethylamine-derived new psychoactive substances 2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY: Investigations on their metabolic fate including isoenzyme activities and their toxicological detectability in urine screenings.

Psychoactive substances of the 2C-series are phenethylamine-based designer drugs that can induce psychostimulant and hallucinogenic effects. The so-called 2C-FLY series contains rigidified methoxy groups integrated in a 2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran core. The aim of the presented work was to investigate the in vivo and in vitro metabolic fate including isoenzyme activities and toxicological detectability of the three new psychoactive substances (NPS) 2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY to allow clinical and forensic toxicologists the identification of these novel compounds. Rat urine, after oral administration, and pooled human liver S9 fraction (pS9) incubations were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). By performing activity screenings, the human isoenzymes involved were identified and toxicological detectability in rat urine investigated using standard urine screening approaches (SUSAs) based on gas chromatography (GC)-MS, LC-MSn , and LC-HRMS/MS. In total, 32 metabolites were tentatively identified. Main metabolic steps consisted of hydroxylation and N-acetylation. Phase I metabolic reactions were catalyzed by CYP2D6, 3A4, and FMO3 and N-acetylation by NAT1 and NAT2. Methoxyamine was used as a trapping agent for detection of the deaminated metabolite formed by MAO-A and B. Interindividual differences in the metabolism of the 2C-FLY drugs could be caused by polymorphisms of enzymes involved or drug-drug interactions. All three SUSAs were shown to be suitable to detect an intake of these NPS but common metabolites of 2C-E-FLY and 2C-EF-FLY have to be considered during interpretation of analytical findings.

Development and application of a LC-HRMS/MS method for analyzing antihypertensive drugs in oral fluid for monitoring drug adherence.

Nonadherence to antihypertensive drugs therapy is known to be a serious issue in hypertension treatment. Liquid chromatography (LC) coupled to mass spectrometry (MS) was shown to allow the assessment of such nonadherence in blood and urine sample. However, their sampling may represent a logistical challenge and are often not favored by the patients. We questioned whether oral fluid (OF) might be an easier accessible alternative matrix for adherence monitoring of cardiovascular drugs (CD). A qualitative method for adherence monitoring of 78 commonly prescribed cardiovascular drugs in OF using LC high-resolution MS (LC-HRMS/MS) was therefore developed, validated, and used to study the presence of antihypertensive medication in OF. Selectivity, ion suppression and enhancement due coeluting analytes, carry over, limits of detection (LOD), limits of identification (LOI), recovery (RE), matrix effects (ME), and process efficiency (PE) were investigated. For demonstrating applicability, over 50 OF samples were investigated and data were compared to findings in blood and urine. Selectivity in OF was given for all compounds via their MS2 spectra and no total suppression of signals could be observed. Determined LOI in OF for ten analytes was higher than the given therapeutic plasma concentration. Furthermore, RE, ME, and PE were in acceptable ranges for more than 65% of the compounds. In total, 208 prescriptions of CD to 57 patients were analyzed and demonstrated the suitability of for adherence monitoring in principle. OF was comparable to plasma regarding the drug categories and the frequencies of hits, except for acidic compounds but more hits could be found in urine samples. A analytical method using OF as analytical matrix was successfully developed. Application showed that it might be a suitable alternative for adherence monitoring of selected drugs in the future, particularly those having no acidic function.

Tools for studying the metabolism of new psychoactive substances for toxicological screening purposes - A comparative study using pooled human liver S9, HepaRG cells, and zebrafish larvae.

New psychoactive substances (NPS) are an emerging topic amongst abused compounds. New varieties appear constantly on the market, without any knowledge about their toxicodynamic and/or -kinetic properties and knowledge of their metabolism is crucial for the development of analytical methods employed for their detection. Controlled human studies would of course be best suited but due to ethical reasons and lack of preclinical safety data, they are usually not available. Often, in vitro models are used to evaluate similarities to human in vivo hepatic phase I and II metabolism and systems explored include primary human hepatocytes, pooled human S9 fraction, and HepaRG, a human hepatic cell line. All these in vitro models have considerable limitations and drug distribution, reabsorption, enterohepatic circulation, and renal elimination cannot be studied. In the recent years, zebrafish (Danio rerio) larvae (embryos) were discussed as a potential in vivo model to overcome these limitations. To date, no studies demonstrating its suitability for studying NPS metabolism in the context of analytical toxicology are available. The aim of this study was to elucidate whether zebrafish larvae can serve as a surrogate for human hepatic metabolism of NPS to develop toxicological screening procedures. Here, we used methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate (7'N-5F-ADB), a new synthetic cannabinoid, whose human metabolism was recently described in the literature, as a model compound to evaluate zebrafish larvae as a new tool for metabolism studies. Different conditions for zebrafish larvae and HepaRG protocols were tested. As zebrafish larvae and HepaRG cell incubations provided the highest number of metabolites and the most authentic spectrum of human metabolites. The most suitable larvae protocol was the incubation via medium and the analysis of the extracted zebrafish larvae. The zebrafish larvae model might be a promising preclinical surrogate for human hepatic metabolism of NPS.

In vitro metabolic fate of nine LSD-based new psychoactive substances and their analytical detectability in different urinary screening procedures.

The market of new psychoactive substances (NPS) is characterized by a high turnover and thus provides several challenges for analytical toxicology. The analysis of urine samples often requires detailed knowledge about metabolism given that parent compounds either may be present only in small amounts or may not even be excreted. Hence, knowledge of the metabolism of NPS is a prerequisite for the development of reliable analytical methods. The main aim of this work was to elucidate for the first time the pooled human liver S9 fraction metabolism of the nine d-lysergic acid diethylamide (LSD) derivatives 1-acetyl-LSD (ALD-52), 1-propionyl-LSD (1P-LSD), 1-butyryl-LSD (1B-LSD), N6-ethyl-nor-LSD (ETH-LAD), 1-propionyl-N6-ethyl-nor-LSD (1P-ETH-LAD), N6-allyl-nor-LSD (AL-LAD), N-ethyl-N-cyclopropyl lysergamide (ECPLA), (2'S,4'S)-lysergic acid 2,4-dimethylazetidide (LSZ), and lysergic acid morpholide (LSM-775) by means of liquid chromatography coupled to high-resolution tandem mass spectrometry. Identification of the monooxygenase enzymes involved in the initial metabolic steps was performed using recombinant human enzymes and their contribution confirmed by inhibition experiments. Overall, N-dealkylation and hydroxylation, as well as combinations of these steps predominantly catalyzed by CYP1A2 and CYP3A4, were found. For ALD-52, 1P-LSD, and 1B-LSD, deacylation to LSD was observed. The obtained mass spectral data of all metabolites are essential for reliable analytical detection particularly in urinalysis and for differentiation of the LSD-like compounds as biotransformations also led to structurally identical metabolites. However, in urine of rats after the administration of expected recreational doses and using standard urine screening approaches, parent drugs or metabolites could not be detected.

In vitro glucuronidation of designer benzodiazepines by human UDP-glucuronyltransferases.

Multiple new psychoactive substances (NPS) are released into the recreational drug market each year. One NPS drug class that has become more common in recent years is that of the benzodiazepines (designer benzodiazepines, DBZ). Several metabolism studies have been performed to improve their bioanalytical detection via the best target. These studies have shown the presence of parent glucuronides and, as polymorphisms have been noted for the catalyzing enzymes (UDP-glucuronyltransferases) responsible for glucuronide conjugation reactions, it is important to keep this in mind when interpreting DBZ cases in clinical and/or forensic toxicology. Therefore, the aim of this study was to determine the UDP-glucuronyltransferases (UGTs) responsible for parent compound conjugation of nine DBZ to facilitate interpretation of related cases. Clonazolam, deschloroetizolam, etizolam, flubromazolam, flunitrazolam, metizolam, nifoxipam, nitrazolam, and pyrazolam were incubated with pooled human liver microsomes (pHLM) or 13 different human UGTs. The samples were analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Glucuronide conjugates of flunitrazolam and nifoxipam were only detected in pHLM, suggesting that these reactions are performed by dimer complexes of several UGTs or complexes between UGTs and other metabolizing enzymes contained in pHLM. Nitrazolam or pyrazolam glucuronides were not detected. Glucuronidation of clonazolam, deschloroetizolam, etizolam, flubromazolam, and metizolam was catalyzed exclusively by UGT1A4. The conjugation of the majority of the DBZ was performed by the UGT isoform 1A4 for which polymorphisms have been described. This underlines the importance of taking glucuronidation polymorphism into consideration when interpreting intoxication cases.

Metabolic fate of the new synthetic cannabinoid 7'N-5F-ADB in rat, human, and pooled human S9 studied by means of hyphenated high-resolution mass spectrometry.

New psychoactive substances (NPS) are an important issue in clinical/forensic toxicology. 7'N-5F-ADB, a synthetic cannabinoid derived from 5F-ADB, appeared recently on the market. Up to now, no data about its mass spectral fragmentation pattern, metabolism, and thus suitable targets for toxicological urine screenings have been available. Therefore, the aim of this study was to elucidate the metabolic fate of 7'N-5F-ADB in rat, human, and pooled human S9 (pS9). The main human urinary excretion products, which can be used as targets for toxicological screening procedures, were identified by Orbitrap (OT)-based liquid chromatography-high resolution-tandem mass spectrometry (LC-HRMS/MS). In addition, possible differentiation of 7'N-5F-ADB and 5F-ADB via LC-HRMS/MS was studied. Using the in vivo and in vitro models for metabolism studies, 36 metabolites were tentatively identified. 7'N-5F-ABD was extensively metabolized in rat and human with minor species differences observed. The unchanged parent compound could be found in human urine but metabolites were far more abundant. The most abundant ones were the hydrolyzed ester (M5), the hydrolyzed ester in combination with hydroxylation of the tertiary butyl part (M11), and the hydrolyzed ester in addition to glucuronidation (M30). Besides the parent compound, these metabolites should be used as targets for urine-based toxicological screening procedures. Two urine-paired human plasma samples contained mainly the parent compound (c = 205 µg/L, 157 µg/L) and, at a higher abundance, the compound after ester hydrolysis (M5). In pS9 incubations, the parent compound, M5, and M30 were detectable among others. Furthermore, a differentiation of both compounds was possible due to different retention times and fragmentation patterns.

Cytotoxicity of new psychoactive substances and other drugs of abuse studied in human HepG2 cells using an adopted high content screening assay.

New psychoactive substances (NPS) are still an emerging issue in clinical and forensic toxicology. Information about their cytotoxic potential is limited or even unavailable before distribution and thus their intake can be of high risk for consumers. The aim of the presented study was to develop a strategy to identify cytotoxic potential of NPS based on a high content screening assay (HCSA) using HepG2 cell line and four fluorescent dyes, namely Hoechst33342, TMRM, CAL-520, and TOTO-3. The HCSA was optimized to work without an automated analyzer by using the model compounds fluvastatin, paracetamol, propranolol, and simvastatin. The following parameters were monitored: stained nuclei as a measure for cell count as well as nuclear size and nuclear intensity (all Hoechst33342), mitochondrial membrane potential (TMRM), cytosolic calcium level (CAL-520), and plasma membrane integrity (TOTO-3). The present study showed strong cytotoxic potential for the NPS 5F-PB-22 and MDAI, moderate effects for MDMA, MDPV, methylone, cathinone, 4-MEC, and mephedrone, and no toxic effects for methamphetamine. To assess the metabolic suitability of HepG2 cells under the chosen conditions, cell culture supernatants were analyzed by liquid chromatography-high resolution-tandem mass spectrometry. Metabolites were merely detected for lipophilic drugs such as 5F-PB-22 and MDPV and in addition with a much lower abundance in comparison to the parent compound but the study only allowed a qualitative look for metabolites and the used liver cell line might not ideal when considering metabolism.

Different in vitro and in vivo tools for elucidating the human metabolism of alpha-cathinone-derived drugs of abuse.

In vitro and in vivo experiments are widely used for studying the metabolism of new psychoactive substances (NPS). The availability of such data is required for toxicological risk assessments and development of urine screening approaches. This study investigated the in vitro metabolism of the 5 pyrrolidinophenone-derived NPS alpha-pyrrolidinobutyrophenone (alpha-PBP), alpha-pyrrolidinopentiothiophenone (alpha-PVT), alpha-pyrrolidinohexanophenone (alpha-PHP), alpha-pyrrolidinoenanthophenone (alpha-PEP, PV8), and alpha-pyrrolidinooctanophenone (alpha-POP, PV9). First, they were incubated with pooled human liver microsomes (pHLM) or pooled human liver S9 fraction (pS9) for identification of the main phase I and II metabolites. All substances formed hydroxy metabolites and lactams. Longer alkyl chains resulted in keto group and carboxylic acid formation. Comparing these results with published data obtained using pHLM, primary human hepatocytes (PHH), and authentic human urine samples, PHH provided the most extensive metabolism. Second, enzyme kinetic studies showed that the initial metabolic steps were formed by cytochrome P450 isoforms (CYP) CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 resulting in pyrrolidine, thiophene or alkyl hydroxy metabolites depending on the length of the alkyl chain. The kinetic parameters indicated an increasing affinity of the CYP enzymes with increase of the length of the alkyl chain. These parameters were then used to calculate the contribution of a single CYP enzyme to the in vivo hepatic clearance. CYP2C19 and CYP2D6 were mainly involved in the case of alpha-PBP and CYP1A2, CYP2C9 and CYP2C19 in the case of alpha-PVT, alpha-PHP, alpha-PEP, and alpha-POP.

New psychoactive substances: Studies on the metabolism of XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam using human liver preparations in comparison to primary human hepatocytes, and human urine.

New psychoactive substances (NPS) are an increasing problem in clinical and forensic toxicology. The knowledge of their metabolism is important for toxicological risk assessment and for developing toxicological urine screenings. Considering the huge numbers of NPS annually appearing on the market, metabolism studies should be realized in a fast, simple, cost efficient, and reliable way. Primary human hepatocytes (PHH) were recommended to be the gold standard for in vitro metabolism studies as they are expected to contain natural enzyme clusters, co-substrates, and drug transporters. In addition, they were already successfully used for metabolism studies of NPS. However, they also have disadvantages such as high costs and limited applicability without special equipment. The aims of the present study were therefore first to investigate exemplarily the phase I and phase II metabolism of six NPS (XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam) from different drug classes using pooled human S9 fraction (pS9) or pooled human liver microsomes combined with cytosol (pHLM/pHLC) after addition of the co-substrates for the main metabolic phase I and II reactions. Second to compare results to published data generated using primary human hepatocytes and human urine samples. Results of the incubations with pS9 or pHLM/pHLC were comparable in number and abundance of metabolites. Formation of metabolites, particularly after multi-step reactions needed a longer incubation time. However, incubations using human liver preparations resulted in a lower number of total detected metabolites compared to PHH, but they were still able to allow the identification of the main human urinary excretion products. Human liver preparations and particularly the pooled S9 fraction could be shown to be a sufficient and more cost-efficient alternative in context of metabolism studies also for developing toxicological urine screenings. It might be recommended to use the slightly cheaper pS9 fraction instead of a pHLM/pHLC combination. As formation of some metabolites needed a long incubation time, two sampling points at 60 and 360min should be recommended.

Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-MAPB, and 5-API as examples.

Metabolism studies play an important role in clinical and forensic toxicology. Because of potential species differences in metabolism, human samples are best suitable for elucidating metabolism. However, in the case of new psychoactive substances (NPS), human samples of controlled studies are not available. Primary human hepatocytes have been described as gold standard for in vitro metabolism studies, but there are some disadvantages such as high costs, limited availability, and variability of metabolic enzymes. Therefore, the aim of our study was to investigate and compare the metabolism of six methylenedioxy derivatives (MDMA, MDBD, butylone, MDPPP, MDPV, MDPB) and two bioisosteric analogues (5-MAPB, 5-API) using pooled human liver microsomes (pHLM) combined with cytosol (pHLC) or pooled human liver S9 fraction (pS9) all after addition of co-substrates for six phase I and II reactions. In addition, HepaRG and HepG2 cell lines were used. Results of the different in vitro tools were compared to each other, to corresponding published data, and to metabolites identified in human urine after consumption of MDMA, MDPV, or 5-MAPB. Incubations with pHLM plus pHLC showed similar results as pS9. A more cost efficient model for prediction of targets for toxicological screening procedures in human urine should be identified. As expected, the incubations with HepaRG provided better results than those with HepG2 concerning number and signal abundance of the metabolites. Due to easy handling without special equipment, incubations with pooled liver preparations should be the most suitable alternative to find targets for toxicological screening procedures for methylenedioxy derivatives and bioisosteric analogues.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

Methylenedioxy designer drugs: mass spectrometric characterization of their glutathione conjugates by means of liquid chromatography-high-resolution mass spectrometry/mass spectrometry and studies on their glutathionyl transferase inhibition potency.

Methylenedioxy designer drugs of abuse such as 3,4-methylenedioxymethamphetamine (MDMA) can be selectively toxic to serotonergic neurons and glutathione (GSH) adducts have been implicated in its neurotoxicity. The catecholic demethylenyl metabolites of MDMA, 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine, are metabolically oxidized to the corresponding ortho-quinones, which are highly reactive intermediates. These intermediates can then be conjugated with GSH preventing cellular damage. Furthermore, glutathionyl transferase (GST) activity was described to be irreversibly inhibited by the catechols dopamine, α-methyldopa and their GSH conjugates. Therefore, the aims of the present work were the detection and characterization of GSH conjugates of ten methylenedioxy drugs of abuse and their phase I metabolites as well as to assess their inhibition potency on GST activity. The substrates were incubated using human placental GST with or without preincubation by cytochrome P450 enzymes preparations. GST inhibition was tested using chlorodinitrobenzene GSH conjugation as marker reaction. GSH conjugates were analyzed and characterized using LC-high-resolution-MS/MS. For confirmation of postulated fragmentation patterns, formation of GSH conjugates of selected deuterated analogs (deuterated analogue approach, DAA) of the investigated drugs was explored. For the methylenedioxy amphetamines the following steps could be identified: conjugation of the parent compounds at position 2, 5, 6, of the demethylenyl metabolites at position 2 and 5, and of the further deaminated demethylenyl metabolites at position 2. For the ß-keto-phenylalkylamine and pyrrolidinophenone, conjugation of the demethylenyl metabolites and of the deaminated demethylenyl metabolites at position 2 could be identified. The DAA allowed the differentiation of the 2 and 5/6 isomers by confirmation of the postulated mass spectral fragments. Finally, the tested drugs and phase I metabolites showed no inhibition potency on GST activity.