Analysis of carbohydrates by anion exchange chromatography and mass spectrometry

A versatile liquid chromatographic platform has been developed for analysing underivatized carbohydrates using high performance anion exchange chromatography (HPAEC) followed by an inert PEEK splitter that splits the effluent to the integrated pulsed amperometric detector (IPAD) and to an on-line single quadrupole mass spectrometer (MS). Common eluents for HPAEC such as sodium hydroxide and sodium acetate are beneficial for the amperometric detection but not compatible with electrospray ionisation (ESI). Therefore a membrane-desalting device was installed after the splitter and prior to the ESI interface converting sodium hydroxide into water and sodium acetate into acetic acid. To enhance the sensitivity for the MS detection, 0.5 mmol/l lithium chloride was added after the membrane desalter to form lithium adducts of the carbohydrates. To compare sensitivity of IPAD and MS detection glucose, fructose, and sucrose were used as analytes. A calibration with external standards from 2.5 to 1000 pmole was performed showing a linear range over three orders of magnitude. Minimum detection limits (MDL) with IPAD were determined at 5 pmole levels for glucose to be 0.12 pmole, fructose 0.22 pmole and sucrose 0.11 pmole. With MS detection in the selected ion mode (SIM) the lithium adducts of the carbohydrates were detected obtaining MDL's for glucose of 1.49 pmole, fructose 1.19 pmole, and sucrose 0.36 pmole showing that under these conditions IPAD is 3-10 times more sensitive for those carbohydrates. The applicability of the method was demonstrated analysing carbohydrates in real world samples such as chicory inulin where polyfructans up to a molecular mass of 7000 g/mol were detected as quadrupoly charged lithium adducts. Furthermore mono-, di-, tri-, and oligosaccharides were detected in chicory coffee, honey and beer samples.     

New designer drug 1-(3-trifluoromethylphenyl) piperazine (TFMPP): gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry studies on its phase I and II metabolism and on its toxicological detection in rat urine

Studies are described on the phase I and II metabolism and the toxicological analysis of the piperazine-derived designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP) in rat urine using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). The identified metabolites indicated that TFMPP was extensively metabolized, mainly by hydroxylation of the aromatic ring and by degradation of the piperazine moiety to N-(3-trifluoromethylphenyl)ethylenediamine, N-(hydroxy-3-trifluoromethylphenyl)ethylenediamine, 3-trifluoromethylaniline, and hydroxy-3-trifluoromethylaniline. Phase II reactions included glucuronidation, sulfatation and acetylation of phase I metabolites. 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 TFMPP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of TFMPP in human urine.     

Volatilization of Pesticides from Plant and Soil Surfaces—Field Versus Laboratory Experiments

Abstract

Comparative volatilization experiments were carried out using isoproturon and parathion-methyl sprayed on French beans in field experiments and on plant stands (0.5 m²) in the volatilization chamber developed by the SLFA Neustadt using both compounds ¹⁴C-labelled. The experimental conditions in the field experiments concerning wind speed, temperature and humidity fluctuations were simulated in the volatilization chamber. The laboratory experiment reflected the actual outdoor situation, showing only a negligible amount of volatile isoproturon directly measured in air samples, and providing no reduction of the A.I. residues in plants compared with the initial value in the corresponding field experiment. 77.2% of the parathion-methyl applied to the plants were volatilized and measured directly in air samples in the volatilization chamber while a reduction by 74.7% was found for the corresponding field experiment by residue analysis of the plants after 24 h. No details could be given concerning the nature of the evaporated portions in the field experiment.     

Differential Response of Pentanal and Hexanal Exhalation to Supplemental Oxygen and Mechanical Ventilation in Rats

High inspired oxygen during mechanical ventilation may influence the exhalation of the previously proposed breath biomarkers pentanal and hexanal, and additionally induce systemic inflammation. We therefore investigated the effect of various concentrations of inspired oxygen on pentanal and hexanal exhalation and serum interleukin concentrations in 30 Sprague Dawley rats mechanically ventilated with 30, 60, or 93% inspired oxygen for 12 h. Pentanal exhalation did not differ as a function of inspired oxygen but increased by an average of 0.4 (95%CI: 0.3; 0.5) ppb per hour, with concentrations doubling from 3.8 (IQR: 2.8; 5.1) ppb at baseline to 7.3 (IQR: 5.0; 10.8) ppb after 12 h. Hexanal exhalation was slightly higher at 93% of inspired oxygen with an average difference of 0.09 (95%CI: 0.002; 0.172) ppb compared to 30%. Serum IL-6 did not differ by inspired oxygen, whereas IL-10 at 60% and 93% of inspired oxygen was greater than with 30%. Both interleukins increased over 12 h of mechanical ventilation at all oxygen concentrations. Mechanical ventilation at high inspired oxygen promotes pulmonary lipid peroxidation and systemic inflammation. However, the response of pentanal and hexanal exhalation varies, with pentanal increasing by mechanical ventilation, whereas hexanal increases by high inspired oxygen concentrations.     

Studies on the metabolism and detectability of the emerging drug of abuse diphenyl‐2‐pyrrolidinemethanol (D2PM) in rat urine using GC‐MS and LC‐HR‐MS/MS

In the last years, the number of new psychoactive substances, so‐called ‘legal highs’, has enormously increased. They are sold via online shops often with inaccurate and false information about the content. The aim of this work was to study the metabolism and the detectability of the drug of abuse diphenyl‐2‐pyrrolidinemethanol (D2PM) in rat urine using gas chromatography‐mass spectrometry and liquid chromatography‐high resolution‐tandem mass spectrometry. Five phase I and two phase II metabolites were identified suggesting hydroxylation at the pyrrolidine and diphenyl part as the main metabolic steps. Assuming similar kinetics, an intake of D2PM should be detectable in human urine mainly via its metabolites. Copyright © 2013 John Wiley & Sons, Ltd.     

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MS n , and LC–HR-MS n

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)–mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS ⁿ ). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors’ standard urine screening approaches by GC–MS and LC–MS ⁿ . Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Coexpression of CPR from Various Origins Enhances Biotransformation Activity of Human CYPs in S. pombe

Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs (and other xenobiotics) in humans, and the corresponding drug metabolites are needed as reference substances for their structural confirmation and for pharmacological or toxicological characterization. We have previously shown that biotechnological synthesis of such metabolites is feasible by whole-cell biotransformation with human CYPs recombinantly expressed in the fission yeast Schizosaccharomyces pombe. It was the aim of this study to compare the activity of seven human microsomal CYPs (CYP2C9, CYP2D6, CYP3A4, CYP3A5, CYP3A7, CYP17, and CYP21) upon coexpression with NADPH-cytochrome P450 oxidoreductases (CPRs) from various origins, namely, human CPR (hCPR) and its homologues from fission yeast (ccr1) and the bishop’s weed Ammi majus (AmCPR), respectively. For this purpose, 28 recombinant strains were needed, with five of them having been constructed previously and 23 strains being newly constructed. Bioconversion experiments showed that coexpression of a CPR does not only influence the reaction rate but, in some cases, also exerts an influence on the metabolite pattern. For CYP3A enzymes, coexpression of hCPR yielded the best results, while for another two, hCPR was equally helpful as ccr1 (both CYP17 and CYP21) or AmCPR (CYP17 only), respectively. Interestingly, CYP2D6 displayed its highest activity when coexpressed with ccr1 and CYP2C9 with AmCPR. These results corroborate the view of CPR as a well-suited bio-brick in synthetic biology for the construction of artificial enzyme complexes.     

Dimethocaine,a synthetic cocaine analogue: studies on its in-vivo metabolism and its detectability in urine by means of a rat model and liquid chromatography–linear ion-trap (high-resolution) mass spectrometry

Dimethocaine (DMC, larocaine), a synthetic derivative of cocaine, is a widely distributed “legal high” consumed as a “new psychoactive substance” (NPS) without any safety testing, for example studies of metabolism. Therefore, the purpose of this work was to study its in-vivo and in-vitro metabolism by use of liquid chromatography–(high resolution) mass spectrometry (LC–HRMS ⁿ ). DMC was administered to male Wistar rats (20 mg kg^?1) and their urine was extracted either by solid-phase extraction after enzymatic cleavage of conjugates or by use of protein precipitation (PP). The metabolites were separated and identified by LC–HRMS ⁿ . The main phase I reactions were ester hydrolysis, deethylation, hydroxylation of the aromatic system, and a combination of these. The main phase II reaction was N-acetylation of the p-aminobenzoic acid part of the unchanged parent compound and of several phase I metabolites. The metabolites identified were then used for identification of DMC in rat urine after application of a common user’s dose. By use of GC–MS and LC–MS ⁿ standard urine-screening approaches (SUSAs), DMC and its metabolites could be detected in the urine samples.