Targeted Metabolomic Profiling of Plasma

LC-MSmass spectrometry (LC-MS/MS) is increasingly used in clinical settings for quantitative assay of small molecules and peptides such as vitamin D, serum bile acid and parathyroid hormone under Clinical Laboratory Improvement Amendments environments with high sensitivities and specificities³⁴ . In this study, targeted metabolomic analysis of plasma samples was performed using the Biocrates Absolute-IDQ P180 (BIOCRATES, Life Science AG, Innsbruck, Austria). This validated targeted assay allows for simultaneous detection and quantification of metabolites in plasma samples (10 µL) in a high-throughput manner. The methods have been described in detail^{35 ,36} . The plasma samples were processed as per the instructions by the manufacturer and analyzed on a triple-quadrupole mass spectrometer (Xevo TQ-S, Waters Corporation, USA) operating in the MRM mode. The measurements were made in a 96-well format for a total of 148 samples, and seven calibration standards and three quality control samples were integrated in the kit. Briefly, the flow injection analysis tandem mass spectrometry (MS/MS) method was used to quantify a panel of 144 lipids simultaneously by multiple reaction monitoring. The other metabolites are resolved on the UPLC and quantified using scheduled MRMs. The kit facilitates absolute quantitation of 21 amino acids, hexose, carnitine, 39 acylcarnitines, 15 sphingomyelins, 90 phosphatidylcholines and 19 biogenic amines. Data analysis was performed using the MetIQ software (Biocrates), and the statistical analyses included the nonparametric Kruskal-Wallis test with follow-up Mann-Whitney U-tests for pairwise comparisons using the STAT pack module v3 (Biocrates). Significance was adjusted for multiple comparisons using Bonferroni’s method (P < 0.025). The abundance is calculated from area under the curve by normalizing to the respective isotope labeled internal standard. The concentration is expressed as nmol/L. Human EDTA plasma samples spiked with standard metabolites were used as quality control samples to assess reproducibility of the assay. The mean of the coefficient of variation (CV) for the 180 metabolites was 0.08, and 95% of the metabolites had a CV of <0.15.