Liquid chromatographic-mass spectrometric method for simultaneous determination of small organic acids potentially contributing to acidosis in severe malaria

Acidosis is an important cause of mortality in severe falciparum malaria. Lactic acid is a major contributor to metabolic acidosis, but accounts for only one-quarter of the strong anion gap. Other unidentified organic acids have an independent strong prognostic significance for a fatal outcome. In this study, a simultaneous bio-analytical method for qualitative and quantitative assessment in plasma and urine of eight small 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 and detection. Eight possible small organic acids; L-lactic acid (LA), alpha-hydroxybutyric acid (aHBA), beta-hydroxybutyric acid (bHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and alpha-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. The calibration ranges were: 2.5-2500 mu g/mL for LA, 0.125-125 mu g/mL for aHBA, 7.5-375 mu g/mL for bHBA, 0.1-100 mu g/mL for pHPLA, 1-1000 mu g/mL for MA, 0.25-250 mu g/mL for MMA, 0.25-100 mu g/mL for EMA, and 30-1500 mu g/mL for aKGA. Clinical applicability was demonstrated by analyzing plasma and urine samples from five patients with severe falciparum malaria; five acids had increased concentrations in plasma (range LA=177-1169 mu g/mL, aHBA = 4.70-38.4 mu g/mL, bHBA = 7.70-38.0 mu g/mL, pHPLA = 0.900-4.30 mu g/mL and aKGA = 30.2-32.0) and seven in urine samples (range LA = 11.2-513 mu g/mL, aHBA = 1.50-69.5 mu g/mL, bHBA=8.10-111 mu g/mL, pHPLA = 4.30-27.7 mu g/mL, MMA = 0.300-13.3 mu g/mL, EMA =0.300-48.1 mu g/mL and aKGA = 30.4-107 mu g/mL). In conclusion, a novel bioanalytical method was developed and validated which allows for simultaneous quantification of eight small organic acids in plasma and urine. This new method may be a useful tool for the assessment of acidosis in patients with severe malaria, and other conditions complicated by acidosis. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved.