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.     

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.     

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.     

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.     

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 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-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 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-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).     

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.     

Preparation of 6,6,1',1',6',6'-hexadeutero sucrose

The preparation of 6,6,1',1',6',6'-hexadeutero sucrose is reported. The synthesis is based on a triple oxidation of a protected sucrose 6,1',6'-triol to the corresponding 6,1',6'-tricarboxylic acid or ester, followed by reduction with lithium aluminium deuteride. This triple oxidation could be achieved either using cat. TEMPO-NaOCl (to the acid) or PDC-Ac(2)O-t-BuOH (to the t-butyl carboxylic ester).     

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.     

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.     

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.     

Plasma protein carbonyl concentration is not enhanced by chronic intake of high-protein diets in adult rats

We tested the hypothesis of whether high dietary protein intake is linked to oxidative stress as measured by the concentration of reactive carbonyl residues in plasma proteins. Three groups of male Wistar rats ( approximately 230 g, n = 10) were fed either 15% (15C), 30% (30C), or 60% (60C) casein diets over a period of 18 weeks. For comparison, a vitamin E deficient diet (60C-E) based on diet 60C was given to an additional group to provoke oxidative stress. Concentrations of alpha-tocopherol in plasma and of reactive carbonyl residues in total plasma proteins were measured by high performance liquid chromatography using fluorescence and by diode array detection after 2,4-dinitrophenylhydrazine reaction, respectively. After 1 week the concentration of reactive carbonyl residues in plasma proteins was found to be significantly (P < 0.05) higher in the 60C and 60C-E groups ( approximately 2.7 nmol/mg protein) compared with the 15C and 30C groups ( approximately 1.7 nmol/mg protein). After 14 weeks the 15C (3.4 +/- 1.2 nmol/mg protein) and 60C-E groups (3.9 +/- 1.7 nmol/mg protein) showed a significantly increased concentration of reactive carbonyl residues in plasma protein compared with the 30C and 60C groups (2.5 +/- 1.0 nmol/mg protein; 2.6 +/- 0.8 nmol/mg protein). As expected, chronic vitamin E deficiency (60C-E) resulted in significantly decreased alpha-tocopherol concentrations (3.91 +/- 2.42 micromol/mL vs. 31.3 +/- 4.8 micromol/mL) and a higher concentration of reactive carbonyl residues in plasma proteins. These results do not support the hypothesis that a chronic intake of high-protein diets leads to oxidative stress in adult rats. However, in the non-adapted state (1 week) a high protein intake contributes to oxidative modifications of protein-bound amino acid residues.     

Expression of cloned Saccharomyces diastaticus glucoamylase under natural and inducible promoters

Any one of three homologous genes - STA1, STA2 and STA3 - encoding glucoamylase isozymes I, II and III respectively, allows the Saccharomyces species to utilize starch as a sole carbon source. We show in this paper that glucoamylase II production can be increased 4-fold over the level produced by STA2 strains, by using a two-step fermentation and a yeast strain transformed with a high-copy-number plasmid carrying the STA2 gene. The accumulation of anomalous STA2 mRNA species, mainly differing at their 5' ends, and saturation of step(s) in the secretory pathway appear to be among the major factors limiting glucoamylase expression in synthetic media.     

Mitomycin C pharmacokinetics in patients with recurrent or metastatic colorectal carcinoma

The pharmacokinetics of mitomycin C as a single agent have been determined in 25 treatment courses given to 18 patients with recurrent or metastatic colorectal carcinoma using a high performance liquid chromatography (HPLC) assay to analyze plasma and urine samples. The plasma pharmacokinetics conformed to a two-compartment linear model in 21 of 25 courses monitored with a mean t1/2 lambda 1 of 9.8 +/- 1.2 (SEM) min and mean t1/2 lambda z of 64.1 +/- 8.9 (SEM) min. The large variation observed in t1/2 lambda z was not related to dose or treatment, but an interaction of these two factors approached significance (p = 0.057). Renal excretion in the 12 courses in which it was determined averaged only 2.3% of the total administered dose during the first 4 h monitored and no mitomycin C metabolites were detected in plasma or urine by the HPLC technique used. The most common toxicity, thrombocytopenia, did not correlate with t1/2 lambda z or the area under the curve. This may be due to a failure to monitor active metabolites of mitomycin C; other factors besides plasma drug concentrations that mediate toxicity towards marrow elements; or the small number of courses associated with thrombocytopenia (less than 100,000/mm3). Our study indicates that an interaction of drug dose and treatment course may be associated with increasing t1/2 lambda z; the renal clearance contributes a small component of mitomycin C elimination; metabolites of mitomycin C cannot be detected by the present HPLC technique; and routine monitoring of mitomycin C using present methods cannot be recommended for clinical use to predict toxicity.     

The metabolism of benzyl isothiocyanate and its cysteine conjugate.

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.     

The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview

Saccharomyces cerevisiae has been used widely both as a model system for unraveling the biochemical, genetic, and molecular details of gene expression and the secretion process, and as a host for the production of heterologous proteins of biotechnological interest. The potential of starch as a renewable biological resource has stimulated research into amylolytic enzymes and the broadening of the substrate range of S. cerevisiae. The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched glucose polymers (amylopectin), is catalyzed by alpha- and beta-amylases, glucoamylases, and debranching enzymes, e.g., pullulanases. Starch utilization in the yeast S. cerevisiae var. diastaticus depends on the expression of the three unlinked genes, STA1 (chr. IV), STA2 (chr. II), and STA3 (chr. XIV), each encoding one of the extracellular glycosylated glucoamylases isozymes GAI, GAII, or GAIII, respectively. The restriction endonuclease maps of STA1, STA2, and STA3 are identical. These genes are absent in S. cerevisiae, but a related gene, SGA1, encoding an intracellular, sporulation-specific glucoamylase (SGA), is present. SGA1 is homologous to the middle and 3' regions of the STA genes, but lacks a 5' sequence that encodes the domain for secretion of the extracellular glucoamylases. The STA genes are positively regulated by the presence of three GAM genes. In addition to positive regulation, the STA genes are regulated negatively at three levels. Whereas strains of S. diastaticus are capable of expressing the STA genes, most strains of S. cerevisiae contain STA10, whose presence represses the expression of the STA genes in an undefined manner. The STA genes are also repressed in diploid cells, presumably by the MATa/MAT alpha-encoded repressor. STA gene expression is reduced in liquid synthetic media, it is carbon catabolite repressed by glucose, and is inhibited in petite mutants.