Preparative isolation of (+)-beta-eudesmol fromAmyris balsamifera

Summary Optically pure (+)-beta-eudesmol is a possible starting material for the synthesis of several termite defense compounds. A two step procedure for the isolation of gram quantities of (+)-beta-eudesmol from commercially availableAmyris balsamifera oil (syn. West Indian sandalwood oil), containing 8% beta-eudesmol, was developed. Step one consisted of an efficient vacuum distillation of the total oil. Step two was a medium pressure LC separation with an AgNO₃ impregnated silica gel stationary phase. Several other separation procedures failed due to the presence of many closely related sesquiterpene alcohols (75% of the oil).     

New cathinone‐derived designer drugs 3‐bromomethcathinone and 3‐fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS and their detectability in urine

3‐Bromomethcathinone (3‐BMC) and 3‐Fluoromethcathinone (3‐FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so‐called “bath salts” or “plant feeders.” The aim of the present study was to identify for the first time the 3‐BMC and 3‐FMC Phase I and II metabolites in rat urine and human liver microsomes using GC–MS and LC–high‐resolution MS (HR‐MS) and to test for their detectability by established urine screening approaches using GC–MS or LC–MS. Furthermore, the human cytochrome‐P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug–drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC–MS and by LC–HR‐MS. The main metabolic steps were N‐demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC–MS could be confirmed by LC–HR‐MS. Furthermore, corresponding Phase II metabolites were identified using the LC–HR‐MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC–MS and LC–MSⁿ after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N‐demethylation of 3‐BMC and 3‐FMC after in vitro–in vivo extrapolation. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of LC-HRMS and GC-NICI-MS methods for stereoselective determination of MDMA and its phase I and II metabolites in human urine

3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography-high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography-mass spectrometry with negative-ion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes.     

New cathinone-derived designer drugs 3-bromomethcathinone and 3-fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC-MS and LC-high-resolution MS and their detectability in urine

3-Bromomethcathinone (3-BMC) and 3-Fluoromethcathinone (3-FMC) are two new designer drugs, which were seized in Israel during 2009 and had also appeared on the illicit drug market in Germany. These two compounds were sold via the Internet as so-called "bath salts" or "plant feeders." The aim of the present study was to identify for the first time the 3-BMC and 3-FMC Phase I and II metabolites in rat urine and human liver microsomes using GC-MS and LC-high-resolution MS (HR-MS) and to test for their detectability by established urine screening approaches using GC-MS or LC-MS. Furthermore, the human cytochrome-P450 (CYP) isoenzymes responsible for the main metabolic steps were studied to highlight possible risks of consumption due to drug-drug interaction or genetic variations. For the first aim, rat urine samples were extracted after and without enzymatic cleavage of conjugates. The metabolites were separated and identified by GC-MS and by LC-HR-MS. The main metabolic steps were N-demethylation, reduction of the keto group to the corresponding alcohol, hydroxylation of the aromatic system and combinations of these steps. The elemental composition of the metabolites identified by GC-MS could be confirmed by LC-HR-MS. Furthermore, corresponding Phase II metabolites were identified using the LC-HR-MS approach. For both compounds, detection in rat urine was possible within the authors' systematic toxicological analysis using both GC-MS and LC-MS(n) after a suspected recreational users dose. Following CYP enzyme kinetic studies, CYP2B6 was the most relevant enzyme for both the N-demethylation of 3-BMC and 3-FMC after in vitro-in vivo extrapolation.     

Investigations into beam monitors at the AEg ̄ IS experiment

Detailed diagnostic of antiproton beams at low energies is required for essentially all experiments at the Antiproton Decelerator (AD), but will be particularly important for the future Extra Low ENergy Antiproton ring (ELENA) and its keV beam lines to the different experiments. Many monitors have been successfully developed and operated at the AD, but in particular beam profile monitoring remains a challenge. A dedicated beam instrumentation and detector test stand has recently been setup at the AE \(\bar {g}\) IS experiment (Antimatter Experiment: Gravity, Interferometry, Spectroscopy). Located behind the actual experiment, it allows for parasitic use of the antiproton beam at different energies for testing and calibration. With the aim to explore and validate different candidate technologies for future low energy beam lines, as well as the downstream antihydrogen detector in AE \(\bar {g}\) IS, measurements have been carried out using Silicon strip and pixel detectors, a purpose-built secondary emission monitor and emulsions. Here, results from measurements and characterization of the different detector types with regard to their future use at the AD complex are presented.     

Convenient Gram-Scale Metabolite Synthesis by Engineered Fission Yeast Strains Expressing Functional Human P450 Systems

The growing need for the characterization of cytochrome P450 (P450) metabolites often necessitates their synthesis up to Gram-scale. This task may in principle be achieved by using various techniques including chemical synthesis, the use of laboratory animals, in vitro P450 systems or microbial biotransformation. However, these approaches are in many instances unfavorable due to low yields, laborious purification, costs of cofactors, or the formation of non-physiologic metabolites. The fission yeast Schizosaccharomyces pombe has previously been shown by others and us to be very well suited for the heterologous expression of human P450s. In this study, we demonstrate whole-cell biotransformation reactions carried out with fission yeast strains that coexpress human cytochrome P450 reductase (CPR) and one of the following P450 isoforms: CYP2B6, CYP2C9, CYP2C19, CYP2D6, or CYP3A4, respectively. These strains could successfully convert their respective standard substrates but showed different responses with respect to incubation pH, the presence of glucose, and temperature, respectively. In addition, the preparative of synthesis of 2.8?g of 4'-hydroxydiclofenac was achieved by whole-cell biotransformation of diclofenac using a CPR-CYP2C9 coexpressing fission yeast strain.     

Qualitative studies on the metabolism and the toxicological detection of the fentanyl-derived designer drugs 3-methylfentanyl and isofentanyl in rats using liquid chromatography–linear ion trap–mass spectrometry (LC-MS<Superscript>n</Superscript>)

The opioid 3-methylfentanyl, a designer drug of the fentanyl type, was scheduled by the Controlled Substance Act due to its high potency and abuse potential. To overcome this regulation, isofentanyl, another designer fentanyl, was synthesized in a clandestine laboratory and seized by the German police. The aims of the presented study were to identify the phase I and phase II metabolites of 3-methylfentanyl and isofentanyl in rat urine, to identify the cytochrome P450 (CYP) isoenzymes involved in their initial metabolic steps, and, finally, to test their detectability in urine. Using liquid chromatography (LC)–linear ion trap–mass spectrometry (MSⁿ), nine phase I and five phase II metabolites of 3-methylfentanyl and 11 phase I and four phase II metabolites of isofentanyl could be identified. The following metabolic steps could be postulated for both drugs: N-dealkylation followed by hydroxylation of the alkyl and aryl moiety, hydroxylation of the propanamide side chain followed by oxidation to the corresponding carboxylic acid, and, finally, hydroxylation of the benzyl moiety followed by methylation. In addition, N-oxidation of isofentanyl could also be observed. All hydroxy metabolites were partly excreted as glucuronides. Using recombinant human isoenzymes, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were found to be involved in the initial metabolic steps. Our LC-MSⁿ screening approach allowed the detection of 0.01 mg/L of 3-methylfentanyl and isofentanyl in spiked urine. However, in urine of rats after the administration of suspected recreational doses, the parent drugs could not be detected, but their common nor metabolite, which should therefore be the target for urine screening.