For the enantioselective and simultaneous analysis of lactate and 3-hydroxybutyrate, a validated online two-dimensional high-performance liquid chromatography system using 4-nitro-7-piperazino-2,1,3-benzoxadiazole as a fluorescent derivatization reagent has been developed. For the reversed-phase separation in the first dimension, a Capcell Pak C18 ACR column (1.5 × 250 mm, particle size 3 μm) was used, and the target fractions were isolated by their hydrophobicity. In the second dimension, a polysaccharide-coated enantioselective column, Chiralpak AD-H (2.0 × 250 mm, 5 μm), was used. The system was validated by the calibration curve, intraday precision, interday precision, and accuracy using standards and real human samples, and satisfactory results were obtained. The present method was applied to human plasma and urine, and in the plasma, trace amounts of d-lactate (8.4 μM) and l-3-hydroxybutyrate (1.0 μM), besides high levels of l-lactate (860.9 μM) and d-3-hydroxybutyrate (59.4 μM), were successfully determined. In urine, trace levels of d-lactate (3.7 μM), d-3-hydroxybutyrate (2.3 μM), and l-3-hydroxybutyrate (3.3 μM) in addition to a relatively large amount of l-lactate (15.4 μM) were observed. The present online two-dimensional high-performance liquid chromatography system is useful for the simultaneous determination of all the lactate and 3-hydroxybutyrate enantiomers in human physiological fluids, and further clinical applications are ongoing.
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