Impact of glucuronide interferences on therapeutic drug monitoring of posaconazole by tandem mass spectrometry

Background: Posaconazole is a novel antifungal drug for oral application intended especially for therapy of invasive mycoses. Due to variable gastrointestinal absorption, adverse side effects, and suspected drug-drug interactions, therapeutic drug monitoring (TDM) of posaconazole is recommended. Method: A fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of posaconazole with a run-time <3 min was developed and compared to a LC-MS/MS method and HPLC method with fluorescence detection. Results: During evaluation of UPLC-MS/MS, two earlier eluting peaks were observed in the MRM trace of posaconazole. This was only seen in patient samples, but not in spiked calibrator samples. Comparison with LC-MS/MS disclosed a significant bias with higher concentrations measured by LC-MS/MS, while UPLC-MS/MS showed excellent agreement with the commercially available HPLC method. In the LC-MS/MS procedure, comparably wide and left side shifted peaks were noticed. This could be ascribed to in-source fragmentation of conjugate metabolites during electrospray ionisation. Precursor and product ion scans confirmed the assumption that the additional compounds are posaconazole glucuronides. Reducing the cone voltage led to disappearance of the glucuronide peaks. Slight modification of the LC-MS/MS method enabled separation of the main interference, leading to significantly reduced deviation. Conclusions: These results highlight the necessity to reliably eliminate interference from labile drug metabolites for correct TDM results, either by sufficient separation or selective MS conditions. The presented UPLC-MS/MS method provides a reliable and fast assay for TDM of posaconazole. Clin Chem Lab Med 2010; 48:1723-31

Metabolic perturbations associated with the consumption of a ketogenic medium-chain TAG diet in dogs with idiopathic epilepsy

Consumption of diets containing medium-chain TAG (MCT) has been shown to confer neuroprotective effects. We aim to identify the global metabolic perturbations associated with consumption of a ketogenic diet (medium-chain TAG diet (MCTD)) in dogs with idiopathic epilepsy. We used ultra-performance liquid chromatography-MS (UPLC-MS) to generate metabolic and lipidomic profiles of fasted canine serum and made comparisons between the MCTD and standardised placebo diet phases. We identified metabolites that differed significantly between diet phases using metabolite fragmentation profiles generated by tandem MS (UPLC–MS/MS). Consumption of the MCTD resulted in significant differences in serum metabolic profiles when compared with the placebo diet, where sixteen altered lipid metabolites were identified. Consumption of the MCTD resulted in reduced abundances of palmitoylcarnitine, octadecenoylcarnitine, stearoylcarnitine and significant changes, both reduced and increased abundances, of phosphatidylcholine (PC) metabolites. There was a significant increase in abundance of the saturated C17 : 0 fatty acyl moieties during the MCTD phase. Lysophosphatidylcholine (17 : 0) (P=0·01) and PC (17:0/20:4) (P=0·03) were both significantly higher in abundance during the MCTD. The data presented in this study highlight global changes in lipid metabolism, and, of particular interest, in the C17 : 0 moieties, as a result of MCT consumption. Elucidating the global metabolic response of MCT consumption will not only improve the administration of current ketogenic diets for neurological disease models but also provides new avenues for research to develop better diet therapies with improved neuroprotective efficacies. Future studies should clarify the involvement and importance of C17 : 0 moieties in endogenous MCT metabolic pathways

Response to Tsikas et al. comments on Boelaert et al. Determination of asymmetric and symmetric dimethylarginine in serum from patients with chronic kidney disease: UPLC-MS/MS versus ELISA. Toxins 2016, 8, 149

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Metabolite changes in blood predict the onset of tuberculosis

Immunogenetics and cellular immunology of bacterial infectious disease

Metabolic labelling of cholesteryl glucosides in Helicobacter pylori reveals how the uptake of human lipids enhances bacterial virulence.

Helicobacter pylori infects approximately half of the human population and is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl and 6'-phosphatidyl α-glucosides (CAGs and CPGs). Owing to a lack of sensitive analytical methods, it is not known if CAGs and CPGs play distinct physiological roles or how the acyl chain component affects function. Herein we established a metabolite-labelling method for characterising these derivatives qualitatively and quantitatively with a femtomolar detection limit. The development generated an MS/MS database of CGds, allowing for profiling of all the cholesterol-derived metabolites. The subsequent analysis led to the unprecedented information that these bacteria acquire phospholipids from the membrane of epithelial cells for CAG biosynthesis. The resulting increase in longer or/and unsaturated CAG acyl chains helps to promote lipid raft formation and thus delivery of the virulence factor CagA into the host cell, supporting the idea that the host/pathogen interplay enhances bacterial virulence. These findings demonstrate an important connection between the chain length of CAGs and the bacterial pathogenicity

Microwave Assisted Synthesis of Py-Im Polyamides

Microwave synthesis was utilized to rapidly build Py-Im polyamides in high yields and purity using Boc-protection chemistry on Kaiser oxime resin. A representative polyamide targeting the 5′-WGWWCW-3′ (W = A or T) subset of the consensus Androgen and Glucocorticoid Response Elements was synthesized in 56% yield after 20 linear steps and HPLC purification. It was confirmed by Mosher amide derivatization of the polyamide that a chiral α-amino acid does not racemize after several additional coupling steps

Plant-based beverages as good sources of free and glycosidic plant sterols

To address the ever-growing group of health-conscious consumers, more and more nutritional and health claims are being used on food products. Nevertheless, only very few food constituents, including plant sterols, have been appointed an approved health claim (European Commission and Food and Drugs Administration). Plant sterols are part of those limited lists of approved compounds for their cholesterol-lowering properties but have been praised for their anti-inflammatory and anti-carcinogenic properties as well. Despite this indisputable reputation, direct quantitative data is still lacking for naturally present (conjugated) plant sterols in beverages. This study aimed to fill this gap by applying a validated extraction and UPLC-MS/MS detection method to a diverse range of everyday plant-based beverages. B-sitosterol--D-glucoside (BSSG) showed to be by far the most abundant sterol in all beverages studied, with concentrations up to 60–90 mg per 100 mL in plant-based milk alternatives and fresh fruit juices. Ergosterol (provitamin D2) could be found in beers (0.8–6.1 g per 100 mL, from the yeast) and occasionally in juices (17–29 g per 100 mL). Overall, the results demonstrated that the concentrations of water-soluble sterol conjugates have been underestimated significantly and that specific plant-based beverages can be good, low-fat sources of these plant sterols

Metabolomics application in maternal-fetal medicine

Metabolomics in maternal-fetal medicine is still an "embryonic" science. However, there is already an increasing interest in metabolome of normal and complicated pregnancies, and neonatal outcomes. Tissues used for metabolomics interrogations of pregnant women, fetuses and newborns are amniotic fluid, blood, plasma, cord blood, placenta, urine, and vaginal secretions. All published papers highlight the strong correlation between biomarkers found in these tissues and fetal malformations, preterm delivery, premature rupture of membranes, gestational diabetes mellitus, preeclampsia, neonatal asphyxia, and hypoxic-ischemic encephalopathy. The aim of this review is to summarize and comment on original data available in relevant published works in order to emphasize the clinical potential of metabolomics in obstetrics in the immediate future

New Method for Simultaneous Determination of Microcystins and Cylindrospermopsin in Vegetable Matrices by SPE-UPLC-MS/MS

Cyanotoxins are a large group of noxious metabolites with different chemical structure and mechanisms of action, with a worldwide distribution, producing effects in animals, humans, and crop plants. When cyanotoxin-contaminated waters are used for the irrigation of edible vegetables, humans can be in contact with these toxins through the food chain. In this work, a method for the simultaneous detection of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR), and Cylindrospermopsin (CYN) in lettuce has been optimized and validated, using a dual solid phase extraction (SPE) system for toxin extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Results showed linear ranges (5–50 ng g−1 f.w.), low values for limit of detection (LOD) (0.06–0.42 ng g−1 f.w.), and limit of quantification (LOQ) (0.16–0.91 ng g−1 f.w.), acceptable recoveries (41–93%), and %RSDIP values for the four toxins. The method proved to be robust for the three variables tested. Finally, it was successfully applied to detect these cyanotoxins in edible vegetables exposed to cyanobacterial extracts under laboratory conditions, and it could be useful for monitoring these toxins in edible vegetables for better exposure estimation in terms of risk assessment.España MINECO AGL2015-64558-