Enantioselective and simultaneous determination of lactate and 3‐hydroxybutyrate in human plasma and urine using a narrow‐bore online two‐dimensional high‐performance liquid chromatography system

Abstract

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.

Development of a Three-Dimensional HPLC System for the Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mammalian Urine

“…In the past 10-20 years, various researchers have developed 2D-HPLC systems for the chiral analyses of trace levels of intrinsic compounds in order to obtain high sensitivity and high selectivity. Actually, a large number of reports using 2D-HPLC was published for the trace analysis of enantiomers including amino acids [40][41][42][43][44], dipeptides [45] and hydroxy acids [32,34,35,38,39]. Concerning the hydroxy acids, 2D-HPLC methods for the LA and 3HB enantiomers have been reported including heart-cutting 2D-HPLC [32,34,35] and whole-fraction transfer 2D-HPLC [38,39].…”

“…As a simpler 2D-HPLC system in which the LA and 3HB enantiomers were simultaneously separated by a single enantioselective column, a Chiralpak AD-H column (2.0 mm x 250 mm) was used in the second dimension [39]. A Capcell Pak C18 ACR column (1.5 mm x 250 mm) was used in the first dimension.…”

“…Until now, a variety of 2D-HPLC methods had been developed and applied for the determination of the LA and 3HB enantiomers in real world samples including physiological fluids [32][33][34][35][36][37][38][39]. Among them, some methods were applied to mammalian urine [35,[37][38][39]. For the rat urine, the reported concentration of D-LA was much lower than that of the L-form [35] and to the best of our 3D-HPLC separations of NBD-LA and NBD-3HB enantiomers in the mouse urine.…”

“…Until now, the presence of LA and 3HB enantiomers in living beings has been elucidated using various techniques such as capillary electrophoresis [25,26], gas chromatography [27], liquid chromatography-mass spectrometry (LC-MS) [28][29][30][31] and 2D-HPLC [32][33][34][35][36][37][38][39]. Focusing on the applications in mammalian physiological fluids, LC is the most widely used method.…”

“…Concerning 2D-HPLC, heart cutting methods with partial fraction transfer are reported for the detection of LA enantiomers in rat plasma [32], rat urine [35], human serum [33] and human urine [37], and for the detection of 3HB enantiomers in rat serum [34,36]. The online 2D-HPLC methods with whole fraction transfer are developed for the simultaneous determination of LA and 3HB enantiomers in human plasma and human urine [38,39].…”