Markus R. Meyer

Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography-mass spectrometry.

In recent years, a new class of designer drugs has appeared on the drugs of abuse market in many countries, namely, the so-called beta-keto (bk) designer drugs such as mephedrone (bk-4-methylmethamphetamine), butylone (bk-MBDB), and methylone (bk-MDMA). The aim of the present study was to identify the metabolites of mephedrone in rat and human urine using GC-MS techniques and to include mephedrone, butylone, and methylone within the authors’ systematic toxicological analysis (STA) procedure. Six phase I metabolites of mephedrone were detected in rat urine and seven in human urine suggesting the following metabolic steps: N-demethylation to the primary amine, reduction of the keto moiety to the respective alcohol, and oxidation of the tolyl moiety to the corresponding alcohols and carboxylic acid. The STA procedure allowed the detection of mephedrone, butylone, methylone, and their metabolites in urine of rats treated with doses corresponding to those reported for abuse of amphetamines. Besides macro-based data evaluation, an automated evaluation using the automated mass spectral deconvolution and identification system was performed. Mephedrone and butylone could be detected also in human urine samples submitted for drug testing. Assuming similar kinetics in humans, the described STA procedure should be suitable for proof of an intake of the bk-designer drugs in human urine.

Drugs of abuse screening in urine as part of a metabolite-based LC-MSn screening concept

Today, immunoassays and several chromatographic methods are in use for drug screening in clinical and forensic toxicology and in doping control. For further proof of the authors’ new metabolite-based liquid chromatography-mass spectrometry (LC-MSn) screening concept, the detectability of drugs of abuse and their metabolites using this screening approach was studied. As previously reported, the corresponding reference library was built up with MS2 and MS3 wideband spectra using a LXQ linear ion trap with electrospray ionization in the positive mode and full scan information-dependent acquisition. In addition to the parent drug spectra recorded in methanolic solution, metabolite spectra were identified after protein precipitation of urine from rats after administration of the corresponding drugs and added to the library. This consists now of data of over 900 parent compounds, including 87 drugs of abuse, and of over 2,300 metabolites and artifacts, among them 436 of drugs of abuse. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs of abuse were determined using spiked human urine, and the resulting data have been acceptable. Using two automatic data evaluation tools (ToxID and SmileMS), the intake of 54 of the studied drugs of abuse could be confirmed in urine samples of drug users after protein precipitation and LC separation. The following drugs classes were covered: stimulants, designer drugs, hallucinogens, (synthetic) cannabinoids, opioids, and selected benzodiazepines. The presented LC-MSn method complements the well-established gas chromatography-mass spectroscopy procedure in the authors’ laboratory.

Ion suppression and enhancement effects of co-eluting analytes in multi-analyte approaches: systematic investigation using ultra-high-performance liquid chromatography/mass spectrometry with atmospheric-pressure chemical ionization or electrospray ionization

In multi-analyte procedures, sufficient separation is important to avoid interferences, particularly when using liquid chromatography/mass spectrometry (LC/MS) because of possible ion suppression or enhancement. However, even using ultra-high-performance LC, baseline separation is not always possible. For development and validation of an LC/MS/MS approach for quantification of 140 antidepressants, benzodiazepines, neuroleptics, beta-blockers, oral antidiabetics, and analytes measured in the context of brain death diagnosis in plasma, the extent of ion suppression or enhancement of co-eluting analytes within and between the drug classes was investigated using atmospheric-pressure chemical ionization (APCI) or electrospray ionization (ESI). Within the drug classes, five analytes showed ion enhancement of over 25% and six analytes ion suppression of over 25% using APCI and 16 analytes ion suppression of over 25% using ESI. Between the drug classes, two analytes showed ion suppression of over 25% using APCI. Using ESI, one analyte showed ion enhancement of over 25% and five analytes ion suppression of over 25%. These effects may influence the drug quantification using calibrators made in presence of overlapping and thus interfering analytes. Ion suppression/enhancement effects induced by co-eluting drugs of different classes present in the patient sample may also lead to false measurements using class-specific calibrators made in absence of overlapping and thus interfering analytes. In conclusion, ion suppression and enhancement tests are essential during method development and validation in LC/MS/MS multi-analyte procedures, with special regards to co-eluting analytes.

Metabolism of designer drugs of abuse: an updated review.

This paper reviews the metabolism of new designer drugs of abuse that have emerged on the black market during the last years and is an update of a review published in 2005. The presented review contains data concerning the so-called 2C compounds (phenethylamine type) such as 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B), 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I), 2,5-dimethoxy-4-methyl-beta-phenethylamine (2C-D), 4-ethyl-2,5-dimethoxy-beta-phenethylamine (2C-E), 4-ethylthio-2,5-dimethoxy-beta-phenethylamine (2C-T-2), and 2,5-dimethoxy-4-propylthio-beta-phenethylamine (2C-T-7), beta-keto designer drugs such as 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (butylone, bk-MBDB), 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (ethylone, bk-MDEA), 2-methylamino-1-(3,4-methylene notdioxy notphenyl)propan-1-one (methylone, bk-MDMA), and 2-methylamino-1-p-tolylpropane-1-one (mephedrone, 4-methyl-methcathinone), pyrrolidino notphenones such as 4-methyl-pyrrolidinobutyrophenone (MPBP) and alpha-pyrrolidinovalerophenone (PVP), phencyclidine-derived drugs such as N (1 phenylcyclohexyl) propanamine (PCPr), N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA), N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA), and N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA), tryptamines such as 5-methoxy-N,N-diisopropyl nottryptamine (5-MeO-DIPT), and finally alpha-methylfentanyl (alpha-MF) and 3-methylfentanyl (3-MF). Papers have been considered and reviewed on the identification of in vivo or in vitro human or animal metabolites and the cytochrome P450 or monoamineoxidase isoenzyme-dependent metabolism.

Development of the first metabolite-based LC-MS n urine drug screening procedure-exemplified for antidepressants

In contrast to GC-MS libraries, currently available LC-MS libraries for toxicological detection contain besides parent drugs only some main metabolites limiting their applicability for urine screening. Therefore, a metabolite-based LC-MSn screening procedure was developed and exemplified for antidepressants. The library was built up with MS2 and MS3 wideband spectra using an LXQ linear ion trap with electrospray ionization in the positive mode and full-scan information-dependent acquisition. Pure substance spectra were recorded in methanolic solution and metabolite spectra in urine from rats after administration of the corresponding drugs. After identification, the metabolite spectra were added to the library. Various drugs and metabolites could be sufficiently separated. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs were determined using protein precipitation. Automatic data evaluation was performed using ToxID and SmileMS software. The library consists of over 700 parent compounds including 45 antidepressants, over 1,600 metabolites, and artifacts. Protein precipitation led to sufficient results for sample preparation. ToxID and SmileMS were both suitable for target screening with some pros and cons. In our study, only SmileMS was suitable for untargeted screening being not limited to precursor selection. The LC-MSn method was suitable for urine screening as exemplified for antidepressants. It also allowed detecting unknown compounds based on known fragment structures. As ion suppression can never be excluded, it is advantageous to have several targets per drug. Furthermore, the detection of metabolites confirms the body passage. The presented LC-MSn method complements established GC-MS or LC-MS procedures in the authors’ lab.

THE ROLE OF HUMAN HEPATIC CYTOCHROME P450 ISOZYMES IN THE METABOLISM OF RACEMIC 3, 4-METHYLENEDIOXYETHYLAMPHETAMINE AND ITS SINGLE ENANTIOMERS

The 3,4-methylenedioxy-methamphetamine (MDMA)-related designer drug 3,4-methylenedioxyethylamphetamine (MDEA, Eve) is a chiral compound that is mainly metabolized by N-deethylation and demethylenation during phase I metabolism. The involvement of several cytochrome P450 (P450) isozymes in these metabolic steps has been demonstrated by inhibition assays using human liver microsomes. However, a comprehensive study on the involvement of all relevant human P450s has not been published yet. In addition, the chirality of this drug was not considered in these in vitro studies. The aim of the present work was first to elucidate the contribution of the relevant human P450 isozymes in the demethylenation as well as in the N-dealkylation of racemic MDEA and its single enantiomers and secondly to compare these findings with recently published data concerning the enantioselective metabolism of MDMA. Racemic MDEA and its single enantiomers were incubated using heterologously expressed human P450s, and the corresponding metabolites dihydroxyethylamphetamine and methylenedioxyamphetamine were determined by gas chromatography-mass spectrometry after chiral derivatization with S-heptafluorobutyrylprolyl chloride. The highest contributions to both metabolic steps as calculated from the enzyme kinetic data were obtained for CYP3A4 and CYP2D6 at substrate concentrations corresponding to plasma concentrations of recreational users after intake of racemic MDEA. Both metabolic reactions were found to be enantioselective with a general preference for the S-enantiomers, which was particularly pronounced in the case of CYP2C19. In conclusion, different pharmacokinetic properties of MDEA enantiomers observed in vivo are therefore partially caused by P450-dependent enantioselective metabolism.

Systematic investigation of ion suppression and enhancement effects of fourteen stable-isotope-labeled internal standards by their native analogues using atmospheric-pressure chemical ionization and electrospray ionization and the relevance for multi-analyte liquid chromatographic/mass spectrometric procedures.

In clinical and forensic toxicology, multi-analyte procedures are very useful to quantify drugs and poisons of different classes in one run. For liquid chromatographic/tandem mass spectrometric (LC/MS/MS) multi-analyte procedures, often only a limited number of stable-isotope-labeled internal standards (SIL-ISs) are available. If an SIL-IS is used for quantification of other analytes, it must be excluded that the co-eluting native analyte influences its ionization. Therefore, the effect of ion suppression and enhancement of fourteen SIL-ISs caused by their native analogues has been studied. It could be shown that the native analyte concentration influenced the extent of ion suppression and enhancement effects leading to more suppression with increasing analyte concentration especially when electrospray ionization (ESI) was used. Using atmospheric-pressure chemical ionization (APCI), methanolic solution showed mainly enhancement effects, whereas no ion suppression and enhancement effect, with one exception, occurred when plasma extracts were used under these conditions. Such differences were not observed using ESI. With ESI, eleven SIL-ISs showed relevant suppression effects, but only one analyte showed suppression effects when APCI was used. The presented study showed that ion suppression and enhancement tests using matrix-based samples of different sources are essential for the selection of ISs, particularly if used for several analytes to avoid incorrect quantification. In conclusion, only SIL-ISs should be selected for which no suppression and enhancement effects can be observed. If not enough ISs are free of ionization interferences, a different ionization technique should be considered.

Current applications of high-resolution mass spectrometry in drug metabolism studies

This paper reviews high-resolution mass spectrometry (HRMS) approaches published in 2007–2011 for the elucidation of drug metabolism with a focus on new therapeutics, new drugs of abuse, and doping agents using time-of-flight, single-stage Orbitrap, ion trap Orbitrap, and other Fourier transform MS-based techniques. The present review provides an overview of metabolite-generating systems and assays used, sample preparation techniques, ionization and fragmentation techniques, as well as data mining strategies and software tools which were used in the reviewed papers. Furthermore, HRMS-specific topics such as demand for a certain resolution or a specific mass accuracy are discussed in detail and corresponding recommendations are given. Finally, the advantages and limitations of these methods are discussed.

Fast and simple procedure for liquid–liquid extraction of 136 analytes from different drug classes for development of a liquid chromatographic-tandem mass spectrometric quantification method in human blood plasma

In clinical and forensic toxicology, different extraction procedures as well as analytical methods are used to monitor different drug classes of interest in biosamples. Multi-analyte procedures are preferable because they make the analytical strategy much simpler and cheaper and allow monitoring of analytes of different drug classes in one single body sample. For development of such a multi-analyte liquid chromatography-tandem mass spectrometry approach, a rapid and simple method for the extraction of 136 analytes from the following drug classes has been established: antidepressants, neuroleptics, benzodiazepines, beta-blockers, oral antidiabetics, and analytes relevant in the context of brain death diagnosis. Recovery, matrix effects, and process efficiency were tested at two concentrations using six different lots of blank plasma. The recovery results obtained using absolute peak areas were compared with those calculated using area ratios analyte/internal standard. The recoveries ranged from 8% to 84% for antidepressants, from 10% to 79% for neuroleptics, from 60% to 81% for benzodiazepines, from 1% to 71% for beta-blockers, from 10% to 73% for antidiabetics, and from 60% to 86% for analytes relevant in the context of brain death diagnosis. With the exception of 52 analytes at low concentration and 37 at high concentration, all compounds showed recoveries with acceptable variability with less than 15% and 20% coefficients of variation. Recovery results obtained by comparing peak area ratios were nearly the same, but 35 analytes at low concentration and 17 at high concentration lay above the acceptance criteria. Matrix effects with more than 25% were observed for 18 analytes. The results were acceptable for 119 analytes at high concentrations.

2-Methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques

Abstract2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling “research chemicals.” The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors’ well-established gas chromatography–mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MSn) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography–high-resolution linear ion trap mass spectrometry (LC-HR-MSn) and the phase II metabolites by LC-HR-MSn. The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user’s dose, 2-MPA and its metabolites were identified in rat urine using the authors’ GC-MS and the LC-MSn screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.