Quantification of lactate enantiomers in human sweat samples using two-dimensional liquid chromatography

Abstract

Lactate (LA) is primarily produced by the reduction of pyruvate in the human body and is crucial for energy production via anaerobic glycolysis. Although the D-LA concentration is considerably lower than that of L-LA, a significant increase in D-LA concentration alone has been reported in some diseases. Quantifying LA enantiomers in human biofluids has the potential for disease diagnosis. Sweat has recently been recognized as a novel biological alternative to blood because it can be sampled non-invasively. Therefore, in this study, heart-cutting two-dimensional liquid chromatography (2D-LC) using a highly sensitive fluorescence detection method was developed for the analysis of LA enantiomers in small amounts of human sweat. LA was derivatized with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) and separated from endogenous compounds using an octadecyl silica column in the first dimension. Subsequently, the NBD-PZ-LA peak was fractionated and enantiomerically separated in the second dimension on a chiral column. Sufficient linearities (R² > 0.999) were observed in the ranges of 1–100 and 10–1000 µM for NBD-PZ-D-LA and NBD-PZ-L-LA, respectively. The corresponding limits of quantification were 0.97 and 1.12 µM. The precision values were 1.04 %–12.03 %, and the accuracies were 85.6 %–100.4 %. The developed method was successfully applied to ∼5 µL of human sweat collected from five healthy subjects. The concentrations of D-LA and L-LA in sweat were 30.29 ± 20.18 µM and 23.69 ± 12.15 mM, respectively. The developed 2D-LC system should be clinically applicable to LA enantiomer analysis in human sweat as a non-invasive biomarker.