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.     

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.     

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.     

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.     

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 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).     

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 Eschscholtzia californica alkaloids californine and protopine in urine using gas chromatography-mass spectrometry

Eschscholtzia californica preparations are in use as phytopharmaceuticals and as herbal drugs. Studies are described on the metabolism and the toxicological analysis of the Eschscholtzia californica alkaloids californine and protopine in rat urine using gas chromatography-mass spectrometry. The identified metabolites indicated that californine is extensively metabolized by N-demethylation and/or single or double demethylenation with consecutive catechol-O-methylation of one of the hydroxy groups. Protopine, however, only undergoes extensive demethylenation of the 2,3-methylenedioxy group followed by catechol-O-methylation. All phenolic hydroxy metabolites were found to be partly conjugated. The authors' systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of californine and protopine in rat urine after a dose which should correspond to that of drug users. Therefore, use of Eschscholtzia californica preparations should also be detectable in human urine by the authors' systematic toxicological analysis procedure.     

Metabolism of the designer drug 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in mice, after acute administration

4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is a psychoactive drug of abuse often sold under the general street name "Ecstasy". Recent reports on the abuse of 2C-B and analogues denote the lack of knowledge on this drug metabolism. In the present study, we investigated the metabolic profile of 2C-B in the mouse and found unchanged 2C-B and several metabolites, which could be identified by GC/MS in the mice urine. The identification of 2C-B metabolites may give important clues for the biological and toxicological effects of this drug of abuse and provides new important data for forensic analysis on samples taken from 2C-B abusers.     

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.     

Proof of a 1-(3-chlorophenyl)piperazine (mCPP) intake: use as adulterant of cocaine resulting in drug-drug interactions?

Since 2005, increasing numbers of seizures of the designer drug of abuse 1-(3-chlorophenyl)piperazine (mCPP) have been reported. This paper describes the unequivocal proof of a mCPP intake. Differentiation from the intake of its precursor drugs trazodone and nefazodone was performed by a systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation. The found mCPP/hydroxy-mCPP ratio indicated altered metabolism of this cytochrome (CYP) 2D6 catalyzed reaction compared to published studies using the same procedure. Although this might be ascribed to a poor metabolizer (PM) phenotype, genotyping revealed no PM genotype but indications for an intermediate metabolizer genotype. However, a PM phenotype could also be caused by drug-drug interactions with CYP2D6 inhibitors or substrates such as the co-consumed cocaine and diltiazem and/or diltiazem metabolites, respectively. In conclusion, the presented data indicate a possible relevance of CYP2D6 polymorphism and/or drug interactions to mCPP toxicokinetics, which is important for risk assessment of this new designer drug of abuse, in particular if it is used as adulterant of CYP2D6 substrates such as cocaine.     

Studies on the metabolism and toxicological detection of the new designer drug 4'-methyl-alpha-pyrrolidinopropiophenone in urine using gas chromatography-mass spectrometry

4'-Methyl-alpha-pyrrolidinopropiophenone (MPPP) is a new designer drug which has appeared on the illicit drug market. The aim of our study was to identify the MPPP metabolites and to develop a toxicological detection procedure in urine using solid-phase extraction, ethylation and GC-MS. In urine samples of rats treated with MPPP, MPPP was found to be completely metabolized by oxidative desamination, hydroxylation of the 4'-methyl group followed by oxidation finally to the corresponding carboxy compound and/or by hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam. The carboxy groups were found to be partly conjugated. Based on these data, MPPP could be detected in urine via its metabolites by GC-MS using mass chromatography for screening and library search for identification.     

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.     

Detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography–mass spectrometry after extractive methylation

A gas chromatography–mass spectrometry (GC–MS) procedure was developed for the detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 291, 294, 295, 309, 313, 322, 324, 336, 343 and 354, the possible presence of 4-hydroxycoumarin anticoagulants and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of phenprocoumon and warfarin in human urine samples. In absence of human urine, acenocoumarol, coumachlor, coumatetrayl, pyranocoumarin (cyclocumarol) could be detected only in rat 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.     

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.     

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.     

Studies on the metabolism and toxicological detection of the amphetamine-like anorectic mefenorex in human urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay

Studies on the metabolism and on the toxicological analysis of mefenorex [R,S-N-(3-chloropropyl)-α-methylphenethylamine, MF] using gas chromatography-mass spectrometry (GC-MS) and fluorescence polarization immunoassay (FPIA) are described. The metabolites were identified in urine samples of volunteers by GC-MS. Besides MF, thirteen metabolites including amphetamine (AM) could be identified and three partially overlapping metabolic pathways could be postulated. For GC-MS detection, the systematic toxicological analysis procedure including acid hydrolysis, extraction at pH 8-9 and acetylation was suitable (detection limits 50 ng/ml for MF and 100 ng/ml for AM). Excretion studies showed, that only AM but neither MF nor its specific metabolites were detectable between 32 and 68 h after ingestion of 80 mg of MF. Therefore, misinterpretation can occur. The Abbott TDx FPIA amphetamine/methamphetamine II gave positive results up to 68 h. All the positive immunoassay results could be confirmed by the described GC-MS procedure.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.     

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.