Rapid resolution liquid chromatography for monitoring the quality of stockpiled atropine preparations for injection.

We describe a rapid resolution liquid chromatography (RRLC) method for analyzing atropine sulfate, its degradation products (tropic acid, apoatropine, atropic acid) and other components (e.g. phenol, methylparaben) in injectable medicines that are used by the German armed forces in emergency situations. Chromatography is performed using an acetonitrile/phosphate buffer gradient (pH = 1.0) and an RP 18 column (50 x 4.6 mm, 1.8 µm) with the detection wavelength set at 220 nm. The concentration of the active ingredient (atropine sulfate) in the tested products ranges from about 1 mg•ml(-1) to 10 mg•ml(-1) . The concentrations of the detected degradation products range from 0.2% to 4.7% (tropic acid) in relation to the active pharmaceutical ingredient (API). Using shorter separation columns and smaller particle sizes of the stationary phase improved analysis time from 40 to 10 min and reduced the consumption of solvents by approximately 75%. Owing to the pressure conditions (< 200 bar), UHPLC (ultra high performance liquid chromatography) systems are not needed. Comparison of the atropine and tropic acid results obtained with the previously used HPLC (high performance liquid chromatography) method of the MAH (marketing authorization holder) show that there is no indication of a significant difference between the two methods.

Fenproporex N-dealkylation to amphetamine--enantioselective in vitro studies in human liver microsomes as well as enantioselective in vivo studies in Wistar and Dark Agouti rats.

Fenproporex (FP) is known to be N-dealkylated to R(-)-amphetamine (AM) and S(+)-amphetamine. Involvement of the polymorphic cytochrome P450 (CYP) isoform CYP2D6 in metabolism of such amphetamine precursors is discussed controversially in literature. In this study, the human hepatic CYPs involved in FP dealkylation were identified using recombinant CYPs and human liver microsomes (HLM). These studies revealed that not only CYP2D6 but also CYP1A2, CYP2B6 and CYP3A4 catalyzed this metabolic reaction for both enantiomers with slight preference for the S(+)-enantiomer. Formation of amphetamine was not significantly changed by quinidine and was not different in poor metabolizer HLM compared to pooled HLM. As in vivo experiments, blood levels of R(-)-amphetamine and S(+)-amphetamine formed after administration of FP were determined in female Dark Agouti rats (fDA), a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats (mDA), an intermediate model, and in male Wistar rats (WI), a model of the human CYP2D6 extensive metabolizer phenotype. Analysis of the plasma samples showed that fDA exhibited significantly higher plasma levels of both amphetamine enantiomers compared to those of WI. Corresponding plasma levels in mDA were between those in fDA and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher amphetamine plasma levels, which did not significantly differ from those in fDA. The in vivo studies suggested that CYP2D6 is not crucial to the N-dealkylation but to another metabolic step, most probably to the ring hydroxylation. Further studies are necessary for elucidating the role of CYP2D6 in FP hydroxylation.

Chemistry, pharmacology, toxicology, and hepatic metabolism of designer drugs of the amphetamine (ecstasy), piperazine, and pyrrolidinophenone types: a synopsis.

Designer drugs of the amphetamine type (eg, MDMA, MDEA, MDA), of the new benzyl or phenyl piperazine type (eg, BZP, MDBP, mCPP, TFMPP, MeOPP), or of the pyrrolidinophenone type (eg, PPP, MOPPP, MDPPP, MPPP, MPHP) have gained popularity and notoriety as rave drugs. These drugs produce feelings of euphoria and energy and a desire to socialize. Although in the corresponding drug scene designer drugs have the reputation of being safe, studies in rats and primates in combination with human epidemiologic investigations indicate potential risks to humans. Thus, a variety of adverse effects have been associated with the use/abuse of this class of drugs in humans, including a life-threatening serotonin syndrome, hepatotoxicity, neurotoxicity, and psychopathology. Metabolites were suspected to contribute to some of the toxic effects. Therefore, knowledge of the metabolism is a prerequisite for toxicologic risk assessment. The metabolic pathways, the involvement of cytochrome P450 isoenzymes in the main pathways, and their roles in hepatic clearance are described for designer drugs of different groups. In summary, polymorphically expressed CYP2D6 was the major enzyme catalyzing the major metabolic steps of the studied piperazine- and pyrrolidinophenone-derived designer drugs. However, it cannot be concluded at the moment whether this genetic polymorphism is of clinical relevance.

Identification of human cytochrome P450 2D6 as major enzyme involved in the O-demethylation of the designer drug p-methoxymethamphetamine.

p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.

Cytochrome P450 isoenzymes involved in rat liver microsomal metabolism of californine and protopine.

Studies are described on the cytochrome P450 (CYP) isoenzyme dependence of the main metabolic steps of the Eschscholtzia californica alkaloids californine and protopine using rat liver microsomes. Preparations of E. californica are in use as phytopharmaceuticals and as herbal drugs of abuse. CYP isoenzyme dependences were studied using specific chemical inhibitors for CYP1A2, CYP2D1, and CYP3A2 (alpha-naphthoflavone, quinine, and ketoconazole, respectively). CYP2C11 was inhibited by specific antibodies for lack of specific chemical inhibitors. Californine N-demethylation was mainly catalyzed by CYP3A2 and to a minor extent by CYP1A2 and CYP2D1, but not by CYP2C11. CYP2D1 and CYP2C11 were shown to be mainly involved in demethylenation of both, californine and protopine, while CYP1A2 and CYP3A2 showed only minor contribution. Kinetic parameters of the reactions were established. K(m) and V(max) values for the californine N-demethylation were 4.5+/-4.7 microM and 22.9+/-13.7 min/mg protein (high affinity) and 161.3+/-16.7 microM and 311.8+/-39.4 min/mg protein (low affinity), respectively. Californine demethylenation and protopine demethylenation showed substrate inhibition and K(m) and V(max) values were 5.0+/-0.5 and 7.1+/-0.6 microM and 83.3+/-2.6 and 160.7+/-4.0 min/mg protein, respectively.

Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes.

1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.

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.

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.

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.

Identification of cytochrome p450 enzymes involved in the metabolism of 4'-methyl-alpha-pyrrolidinopropiophenone, a novel scheduled designer drug, in human liver microsomes.

4'-Methyl-alpha-pyrrolidinopropiophenone (MPPP) is a new drug of abuse. It is believed to have an abuse potential similar to that of amphetamines. Previous studies with Wistar rats had shown that MPPP was metabolized mainly by hydroxylation in position 4' followed by dehydrogenation to the corresponding carboxylic acid. The aim of the study presented here was to identify the human hepatic cytochrome p450 (p450) enzymes involved in the biotransformation of MPPP to 4'-hydroxymethyl-pyrrolidinopropiophenone. Baculovirus-infected insect cell microsomes and human liver microsomes were used for this purpose. Only CYP2C19 and CYP2D6 catalyzed this hydroxylation. The apparent Km and Vmax values for the latter were 9.8 +/- 2.5 microM and 13.6 +/- 0.7 pmol/min/pmol p450, respectively. CYP2C19 was not saturable over the tested substrate range (2-1000 microM) and interestingly showed a biphasic kinetic profile with apparent Km,1 and Vmax,1 values of 47.2 +/- 12.5 microM and 8.1 +/- 1.4 pmol/min/pmol p450, respectively. Experiments with pooled human liver microsomes also revealed biphasic nonsaturable kinetics with apparent Km,1 and Vmax,1 values of 57.0 +/- 20.9 microM and 199.7 +/- 59.7 pmol/min/mg of protein for the high affinity enzyme, respectively. Incubation of 2 microM MPPP with 3 microM of the CYP2D6-specific inhibitor quinidine resulted in significant (p < 0.01) turnover inhibition (11.8 +/- 1.6% of control). Based on kinetic data corrected for the relative activity factors, CYP2D6 is the enzyme mainly responsible for MPPP hydroxylation, confirmed by CYP2D6 inhibition studies.

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.

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.