Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.

The Human Meconium Metabolome and Its Evolution during the First Days of Life.

Meconium represents the first newborn stools, formed from the second month of gestation and excreted in the first days after birth. As an accumulative and inert matrix, it accumulates most of the molecules transferred through the placenta from the mother to the fetus during the last 6 months of pregnancy, and those resulting from the metabolic activities of the fetus. To date, only few studies dealing with meconium metabolomics have been published. In this study, we aimed to provide a comprehensive view of the meconium metabolic composition using 33 samples collected longitudinally from 11 healthy newborns and to analyze its evolution during the first 3 days of life. First, a robust and efficient methodology for metabolite extraction was implemented. Data acquisition was performed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), using two complementary LC-HRMS conditions. Data preprocessing and treatment were performed using the Workflow4Metabolomics platform and the metabolite annotation was performed using our in-house database by matching accurate masses, retention times, and MS/MS spectra to those of pure standards. We successfully identified up to 229 metabolites at a high confidence level in human meconium, belonging to diverse chemical classes and from different origins. A progressive evolution of the metabolic profile was statistically evidenced, with sugars, amino acids, and some bacteria-derived metabolites being among the most impacted identified compounds. Our implemented analytical workflow allows a unique and comprehensive description of the meconium metabolome, which is related to factors, such as maternal diet and environment.

Nitrite Reduction in Aqueous Solution Mediated by Amavadin Homologues: N2 O Formation and Water Oxidation.

Reactions of two vanadium(IV) complex anions that are homologues of amavadin, [V(HIDPA)2 ]2- and [V(HIDA)2 ]2- (HIDPA=N-oxyiminodipropionate, HIDA=N-oxyiminodiacetate), with the nitrite ion (NO2- ) in aqueous solution were investigated by experimental (absorption spectroscopy in the visible range, through measurements of dioxygen formed in solution from water oxidation and identification of nitrogen oxide species of a gaseous atmosphere from nitrite reduction by using an IR analyser) and theoretical methods. Two reactions, mediated by the vanadium complexes, with environmental and biological significance, were observed in this system, namely, reduction of nitrite to N2 O and oxidation of water to molecular oxygen. The reduction of nitrite, as studied by DFT calculations, occurs through the formation of NO (ΔG≠ =14.3 kcal mol-1 ), which is strongly dependent on pH and slightly endergonic, and is then easily converted into N2 O, with an overall activation barrier of ΔG≠ =11.8 kcal mol-1 . The later process includes dimerisation of NO assisted by one molecule of the V complex, protonation and oxidation of the formed ONNO.- ligand by another amavadin molecule or by nitrite, and NO bond cleavage/proton transfer in the ONNOH- ligand. The results indicate that amavadin exhibits an unusual nitrite reductase type activity that could be involved in nitrogen metabolism of Amanita muscaria and other fungi containing this vanadium complex.