Pattern recognition and method development for qualitative and quantitative assessment of small organic acids in human biological fluids using liquid chromatography-mass spectrometry

Abstract

A simultaneous bio-analytical method for qualitative and quantitative assessment in plasms and urine of eight small organic organic acids potentially contributing to acidosis in severe malaria was developed and validated. High-throughput strong anion exchange solid-phase extraction in a 96-well plate format was used for sample preparation. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative mass spectroscopy was utilized for separation, detection and quantification. Eight possible small organic acids; L-lactic (LA), α-hydroxybutyric acid (aHBA), β-hydroxybutyric acid (aHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and α- ketoglutaric acid (aKGA) were analyzed simultaneously using a ZIC-HILIC column with an isocratic elution containing acetonitrile and ammonium acetate buffer. This method was validated according to U.S. Food and Drug Administration guidelines with additional validation procedures for endogenous substances. Accuracy for all eight acids ranged from 93.1% to 104.0%, and the within-day and between-day precisions (i.e. relative standard deviations) were lower than 5.5% at all tested concentrations. Pattern recognition was applied for evaluation of acid concentration profiles in relation to clinical parameters, in plasma as a main contributor and in urine as additional information. This new approach is a useful tool for the assessment of acidosis in severe malaria patients, and other conditions related to acidosis.