Enantioselective Analysis of Lactate, Hydroxybutyrates and Malate in Human Physiological Fluids Using a Three-Dimensional HPLC System

For the highly-selective analysis of lactic acid (LA), 2-hydroxybutyric acid (2HB), 3-hydroxybutyric acid (3HB) and malic acid (MA) enantiomers in human physiological fluids, a three-dimensional (3D) HPLC method following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole has been designed and developed. The 3D-HPLC system was composed of reversed-phase, mixed-mode and enantioselective columns, and the conditions of all the dimensions were thoroughly investigated. In the first and second dimensions, the target hydroxy acids were separated from most of the interfering substances in the clinical samples. In the third dimension, the enantiomers were separated with resolution values higher than 3.47. The present method was validated by checking the calibration curves, precision and accuracy using standard solutions and human samples, and satisfactory results were acquired. Using the 3D-HPLC system, D-LA, L-LA, D-3HB, L-3HB and L-2HB were determined in the human plasma and urine (maleic acid enantiomers were not determined). The concentrations of the detected enantiomers in the human plasma were 6.2, 660, 34.0, 1.0 and 19.0 µM, and those in the human urine were 13.4, 88.3, 4.5, 3.7 and 4.5 µM, respectively.