New designer drug 4'-methyl-alpha-pyrrolidinohexanophenone: studies on its metabolism and toxicological detection in urine using gas chromatography-mass spectrometry

R,S-4'-Methyl-alpha-pyrrolidinohexanophenone (MPHP) is a new designer drug which has appeared on the illicit drug market. The aim of this study was to identify the MPHP metabolites using solid-phase extraction, ethylation or acetylation, as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and GC-MS. Analysis of urine samples of rats treated with MPHP revealed that MPHP was completely metabolized by hydroxylation of the tolyl methyl group followed by dehydrogenation to the corresponding carboxylic acid, hydroxylation of the side chain, hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam and/or reduction of the keto group. The carboxy and/or hydroxy groups were found to be only partly conjugated. Based on these data, MPHP could be detected in urine via its metabolites by GC-MS using mass chromatography for screening and library search for identification.     

New designer drug p-methoxymethamphetamine: studies on its metabolism and toxicological detection in urine using gas chromatography-mass spectrometry

Studies are described on the metabolism and the toxicological analysis of the new designer drug rac-p-methoxymethamphetamine (PMMA) in rat urine using gas chromatography-mass spectrometry (GC-MS). The identified metabolites indicated that PMMA was extensively metabolized mainly by O-demethylation to pholedrine and to a minor extent to p-methoxyamphetamine (PMA), 1-hydroxypholedrine diastereomers (one being oxilofrine), 4'-hydroxy-3'-methoxymethamphetamine and 4'-hydroxy-3'-methoxyamphetamine. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of PMMA in rat urine after a dose corresponding to that of drug users. Therefore, this procedure should be suitable for detection of PMMA intake in human urine via its metabolites. However, it must be considered that pholedrine and oxilofrine are also in therapeutic use. Differentiation of PMMA, PMA and/or pholedrine intake is discussed.     

Metabolism of the new designer drug alpha-pyrrolidinopropiophenone (PPP) and the toxicological detection of PPP and 4'-methyl-alpha-pyrrolidinopropiophenone (MPPP) studied in rat urine using gas chromatography-mass spectrometry

R,S-alpha-pyrrolidinopropiophenone (PPP) is a new designer drug with assumed amphetamine-like effects which has appeared on the illicit drug market. The aim of this study was to identify the PPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and gas chromatography-mass spectrometry (GC-MS). Analysis of urine samples of rats treated with PPP revealed that PPP was extensively metabolized by hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam, hydroxylation of the aromatic ring in position 4' or double dealkylation of the pyrrolidine ring to the corresponding primary amine (cathinone) partly followed by reduction of the keto group to the corresponding secondary alcohol (norephedrines). As cathinone and the norephedrine diastereomers are also formed after intake of other drugs of abuse or medicaments, special attention must be paid to the detection of the unequivocal metabolite 2"-oxo-PPP as an unambiguous proof for the intake of PPP. The hydroxy groups were found to be partly conjugated. Based on these data, PPP could be detected in urine via its metabolites by full-scan GC-MS using mass chromatography for screening and library search for identification by comparison of the spectra with reference spectra. The same toxicological detection procedure can be applied to other designer drugs of the pyrrolidinophenone type, like MOPPP, MDPPP, MPHP, and MPPP. The detection of the latter will also be presented here.     

Studies on the metabolism and toxicological detection of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using gas chromatography-mass spectrometry

The aim of the presented study was to identify the metabolites of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using GC-MS techniques. After enzymatic hydrolysis, extraction and various derivatizations, seven metabolites of MPBP could be identified suggesting the following metabolic steps: oxidation of the 4'-methyl group to the corresponding alcohol and further oxidation to the respective carboxy compound, hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or reduction of the keto group to the 1-dihydro compound. A previously published GC-MS-based screening procedure for pyrrolidinophenones involving enzymatic hydrolysis and mixed-mode solid-phase extraction of urine samples allowed detection of MPBP metabolites. Assuming similar metabolism and dosages in humans, an intake of MPBP should be detectable via its metabolites in urine.     

Metabolism and toxicological detection of the new designer drug 3',4'-methylenedioxy-alpha-pyrrolidinopropiophenone studied in urine using gas chromatography-mass spectrometry

R,S-3',4'-Methylenedioxy-alpha-pyrrolidinopropiophenone (MDPPP) is a new designer drug with assumed amphetamine-like effects, which has appeared on the illicit drug market. The aim of this study was to identify the MDPPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and GC-MS. Analysis of urine samples of rats treated with MDPPP revealed that MDPPP was completely metabolized by demethylenation of the methylenedioxy group followed by partial 3'-methylation of the resulting catechol, oxidative desamination to the corresponding diketo compounds and/or hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam. The hydroxy groups were found to be partly conjugated. Based on these data, MDPPP could be detected in urine via its metabolites by full-scan GC-MS using mass chromatography for screening and library search for identification by comparison of the spectra with reference spectra.     

Studies on the metabolism and toxicological detection of the designer drug 4-ethyl-2,5-dimethoxy-beta-phenethylamine (2C-E) in rat urine using gas chromatographic-mass spectrometric techniques

The phenethylamine-derived designer drug 4-ethyl-2,5-dimethoxy-beta-phenethylamine (2C-E) was found to be mainly metabolized in rats by O-demethylation, N-acetylation, hydroxylation of the ethyl side chain at C2' or at C1' followed by oxidation at C1' to the corresponding ketone, by deamination followed by reduction to the corresponding alcohols or by oxidation to the corresponding acids, and finally combinations of these steps. Most of the metabolites were excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS allowed the detection of an intake of a dose of 2C-E in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-E in human urine.     

Metabolism and toxicological detection of the new designer drug 4'-methoxy-alpha-pyrrolidinopropiophenone studied in rat urine using gas chromatography-mass spectrometry

R,S-4'-Methoxy-alpha-pyrrolidinopropiophenone (MOPPP) is a new designer drug with assumed amphetamine-like effects, which has appeared on the illicit drug market. The aim of this study was to identify the MOPPP metabolites using solid-phase extraction, ethylation or acetylation as well as to develop a toxicological detection procedure in urine using solid-phase extraction, trimethylsilylation and GC-MS. Analysis of urine samples of rats treated with MOPPP revealed that MOPPP [limit of detection (S/N 3) was 100 ng/ml] was completely metabolized by demethylation of the methoxy group, hydroxylation of the pyrrolidine ring with subsequent dehydrogenation to the corresponding lactam and/or oxidative desamination to the corresponding diketo compounds. To some extent, the demethylated MOPPP metabolites were hydroxylated with partial subsequent methylation in position 3'. The hydroxy groups were found to be partly conjugated. Based on these data, MOPPP could be detected in urine via its metabolites by full-scan GC-MS using MS for screening and library search for identification by comparison of the spectra with reference spectra.     

Toxicological detection of the new designer drug 1-(4-methoxyphenyl)piperazine and its metabolites in urine and differentiation from an intake of structurally related medicaments using gas chromatography-mass spectrometry

Studies are described on the toxicological analysis of the piperazine-derived designer drug 1-(4-methoxyphenyl)piperazine (MeOPP) in rat urine using gas chromatography-mass spectrometry (GC-MS). The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of MeOPP and its metabolites 1-(4-hydroxy phenyl)piperazine and 4-hydroxyaniline in rat urine after administration of a single dose corresponding to doses commonly taken by drug users. Therefore, this procedure should also be suitable for detection of a MeOPP intake in human urine. However, the metabolites of MeOPP are not unique and can be produced from other drugs. Therefore, differentiation of use of this designer drug from use of the medicaments dropropizine, oxypertine or others, which are metabolized to the MeOPP isomer 1-(2-methoxyphenyl)piperazine, is discussed.     

Studies on the toxicological detection of the designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) in rat urine using gas chromatography-mass spectrometry

The phenethylamine-derived designer drug 4-bromo-2,5-dimethoxy-beta-phenethylamine (2C-B) is known to be extensively metabolized in various species including humans. In rat urine, 2C-B was found to be excreted mainly via its metabolites. In the current study, the toxicological detection of these metabolites in the authors' systematic toxicological analysis (STA) procedure was examined. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug abusers' dose of 2C-B by detection of the O-demethyl deaminohydroxy and two isomers of the O-demethyl metabolites in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-B in human urine.     

Metabolism and toxicological detection of the designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat urine using gas chromatography-mass spectrometry

The amphetamine-derived designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) is an upcoming substance on the illicit drug market. In the current study, the identification of its metabolites in rat urine and their toxicological detection in the authors' systematic toxicological analysis (STA) procedure were examined. DOI is extensively metabolized by O-demethylation and beside small amounts of parent compound it was found to be excreted mainly in form of metabolites. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug users' dose of DOI by detection of the two O-demethyl metabolite isomers in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOI in human urine.     

Studies on the metabolism and toxicological detection of the designer drug 4-methylthioamphetamine (4-MTA) in human urine using gas chromatography-mass spectrometry

4-Methylthioamphetamine (4-MTA) is a scheduled designer drug that has appeared on the illicit drug market and led to several non-fatal or even fatal poisonings. Only few data are available on its metabolism. The first aim of this study was to identify the 4-MTA metabolites in human urine and then to study whether the authors' STA procedure is suitable for screening for and identification of 4-MTA and/or its metabolites in urine. After enzymatic cleavage of conjugates, solid-phase extraction (SPE) and acetylation the following metabolites could be identified by full-scan gas chromatography-mass spectrometry (GC-MS): deamino-oxo 4-MTA, deamino-hydroxy 4-MTA, ring hydroxy and beta-hydroxy 4-MTA. 4-MTA sulfoxide could be identified as possible artifact. In urine samples after enzymatic hydrolysis, acidic extraction, and methylation, 4-methylthiobenzoic acid could be identified. The authors' systematical toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction (LLE) and acetylation allowed detection of 4-MTA as target analyte plus all the above-mentioned metabolites with the exception of 4-methylthiobenzoic acid. The extraction efficiency of 4-MTA was approximately 70% and the limit of detection (LOD) was 30 ng/ml (S/N 3).     

Comparative in vitro metabolism of the ‘designer’ steroid estra-4,9-diene-3,17-dione between the equine, canine and human: Identification of target metabolites for use in sports doping control

Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. ‘Designer’ steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile.In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely the equine, canine and human. In order to allow the retrospective analysis of sample testing data, the use of a high-resolution (HR) accurate-mass Thermo LTQ-Orbitrap LC-MS instrument was employed for metabolite identification of underivatised sample extracts. The full scan HR-LC-MS Orbitrap data was complimented by several further experiments targeted at elucidating more detailed structural information for the most abundant metabolites. These included; HR-LC-MS/MS of the underivatised metabolites, functional group selective chemical derivatisation followed by full scan HR-LC-MS, enzyme inhibition experiments and full scan electron ionization GC-MS analysis of methoxyamine-trimethylsilyl derivatives.The major metabolite detected in all species, and therefore the most suitable candidate for screening of estra-4,9-diene-3,17-dione abuse, was proposed to be an isomer of 17-hydroxy-estra-4,9-dien-3-one. Less significant metabolic pathways in all species included hydroxylation and reduction followed by hydroxylation. Reductive metabolism in the canine was less significant than in the other two species, while the equine was unique in producing a di-reduced metabolite (proposed to be an isomer of estra-4,9-diene-3,17-diol) and also relatively large quantities of d-ring hydroxy and hydroxy-reduced metabolites.     

Study on metabolites of 2,5-dimethoxy-4-bromamphetamine (DOB) in human urine using gas chromatography-mass spectrometry

The study of the biotransformation of a new synthetic drug 2,5-dimethoxy-4-bromamphetamine (DOB) and identification of its metabolites in urine of a poisoned person is described using gas chromatography mass spectrometry (GC-MS) with various ways of derivatization. It has been confirmed that one of its metabolic pathways leads to the corresponding 2-O-desmethyl and 5-O-desmethyl metabolites when the latter is prevailing. It is important to know the metabolism of this neurotoxic and hallucinogenic substance as it is a prerequisite for developing reliable toxicological diagnostic procedures and for assessment of toxicological risks.     

Designer drug 2,5-dimethoxy-4-methyl-amphetamine (DOM, STP): involvement of the cytochrome P450 isoenzymes in formation of its main metabolite and detection of the latter in rat urine as proof of a drug intake using gas chromatography-mass spectrometry

The designer drug 2,5-dimethoxy-4-methyl-amphetamine (DOM, STP) is known to be extensively metabolized in various species. The current study showed that cytochrome P450 2D6 was the only isoenzyme involved in formation of the main metabolite hydroxy DOM. In addition, the authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS was suitable to prove an intake of a common drug users' dose of DOM by detection of hydroxy DOM in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOM in human urine. However, DOM and/or other metabolites such as deamino-oxo-hydroxy DOM might be the target analyte in urine of CYP2D6 poor metabolizers.     

Metabolism of [14C]methamphetamine in man, the guinea pig and the rat

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.     

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

Studies are presented on the toxicological detection of the designer drug methylenedioxyethylapphetamine [MDE, rac-N-ethyl-(3,4-methylenedioxyphenyl)-propane-2-amine] in urine after a single oral dose of 140 mg of MDE by GC-MS and fluorescence polarization immunoassay (FPIA). After acid hydrolysis, extraction and acetylation MDE and its metabolites could be detected by mass chromatography with the selected ions m/z 72, 86, 114, 150, 162 and 164, followed by identification of the peaks underlying full mass spectra by computer library search. The following metabolites could be detected: unchanged MDE and 3,4-dihydroxyethylamphetamine (DHE) for 33,62 h, 3,4-methylenedioxyamphetamine (MDA) for 32–2036 h, and 4-hydroxy-3-methoxyethylamphetamine (HME) for 7 4-hydroxy-3-methoxyamphetamine (HMA), piperonul aceton, 3,4-Dihydroxyphenyl acetone and 4-hydroxy-3-methoxy-phenyl acetone could only be detected in trace amounts within the first few hours. The Abbott TD_x FPIA assay amphetamine/metamphetamine II gave positive results in urine for 33--62 h. Therefore, positive immunoassay results could be confirmed by the GC-MS procedure which also allowed the differentiation of MDE and its homologues 3,4-methylenedioxymethamphetamine (MDMA) and MDA as well as other amphetamine derivatives interfering with the TD_x assay. Furthermore, this GC-MS procedure allowed the simultaneous detection of most of the toxicologically relevant drugs.     

Gas and liquid chromatography-mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking mixtures

Prohibition of some synthetic cannabimimetics (e.g., JWH-018, JWH-073 and CP 47497) in a number of countries has led to a rise in new compounds in herbal mixtures that create marijuana-like psychotropic effects when smoked. The cannabimimetic JWH-250 (1-pentyl-3-(2-methoxyphenylacetyl)indole) was identified in May 2009 by the German Federal Criminal Police as an new ingredient in herbal smoking mixtures. The absence or low presence of the native compound in urine samples collected from persons who had consumed JWH-250 necessitates a detailed identification of their metabolites, which are excreted with urine and present in blood. Using gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS/MS), we identified a series of metabolites in urine samples and serum sample from humans and urine samples from rats that were products of the following reactions: (a) mono- and dihydroxylation of aromatic and aliphatic residues of the parent compound, (b) trihydroxylation and dehydration of the N-alkyl chain, (c) N-dealkylation and (d) N-dealkylation and monohydroxylation. The prevailing urinary metabolites in humans were the monohydroxylated forms, while N-dealkylated and N-dealkyl monohydroxylated forms were found in rats. The detection of the mono- and dihydroxylated metabolites of JWH-250 in urine and serum samples by GC-MS and LC-MS/MS proved to be effective in determining consumption of this drug.     

Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose

Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.     

Identification of the major urinary metabolite of alprenolol in man, dog and rat

After oral administration of ³H-alprenolol to man, dog and rat, urinary metabolites of the drug have been separated by ion-exchange chromatography on Bio-Rex 70, a carboxylic acrylate resin. The major metabolite has been identified by GC-MS as 4-hydroxyalprenolol. Occurring in the urine largely in a conjugated form, it represents about 40 % of the excreted amount in man and dog and about 30 % in rat. Including alprenolol, which also appears largely as a conjugate, about 80 % of the amount of radioactivity excreted in human urine can be accounted for.     

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A.Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine.In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.