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

Abstract

For the highly chemoselective separations of lactate (LA) and 3-hydroxybutyrate (3HB) enantiomers in mammalian urine, an online three-dimensional HPLC system (3D-HPLC) composed of reversed-phase (KSAARP, 1.0 mm i.d. x 250 mm), mixed-mode (KSAAMX, 1.5 mm i.d. x 250 mm) and enantioselective (Chiralpak AD-H, 2.0 mm i.d. x 250 mm) columns has been developed. Following the pre-column derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), NBD-LA and 3HB were separated in the first and second dimensions as their scalmic D plus L mixtures and the enantiomer separations were performed in the third dimension with high separation factors (α = 1.70 for LA; α = 1.43 for 3HB). The present system was applied to the urine of mice and humans. For the mice urine, the D and L-forms of NBD-LA and 3HB were well separated without visible interfering compounds, and trace levels of D-LA (less than 5% of total LA) and non-negligible amounts of L-3HB (34.4% of the total 3HB) were clearly observed. Trace levels of the LA and 3HB enantiomers were detected for the human urine samples, although the existence of a few interfering peaks were observed.

Multi-Dimensional High-Performance Liquid Chromatographic Determination of Chiral Amino Acids and Related Compounds in Real World Samples

“…Although the 2D-HPLC system has been considered as one of the suitable methods for the trace analysis of LA and 3HB enantiomers, it still faces some problems of insufficient selectivity, especially for the human urine [81]. In order to increase the selectivity, a 3D-HPLC composed of a reversed-phase separation, a mixed-mode separation and an enantioselective separation has been developed and applied to mammalian urine samples [82]. In the first dimension, a KSAARP column (1.0 mm ID x 250 mm) is used at 40°C.…”

Two-Dimensional Chiral HPLC Analysis of Lactate, Hydroxybutyrates and Malate in Human Plasma

“…Until now, to the best of our knowledge, no method has been developed to simultaneously analyze these hydroxy acid enantiomers in real biological samples by only one injection. However, the methods using an enzymatic reaction [19,20], capillary electrophoresis [21,22], gas chromatography [23,24], high-performance liquid chromatography (HPLC) with various detectors [25][26][27][28][29][30][31][32][33][34] and multi-dimensional HPLC methods [5,14,[35][36][37][38][39][40][41][42] have been reported to separately analyze these chiral hydroxy acids. Among them, HPLC methods (including column-switching/ multi-dimensional systems) are widely used for the chiral analysis of the hydroxy acids.…”

Chiral Analysis of Lactate in Various Food Samples Including Japanese Traditional Amber Rice Vinegar and the Developmental Changes During Fermentation Processes

“…Although these methods are useful, numerous peaks still appeared around the peak region of typical target compounds in the chromatogram and sometimes affected the trace analysis of the minor form of the enantiomers, thus a method with a higher selectivity is required. As a highly selective method, the three-dimensional HPLC (3D-HPLC) equipped with the reversed-phase, mixed-mode and enantioselective separations is one of the most useful methods, and applicable to various complicated matrices including biological and clinical samples [29,30]. In the present study, a 3D-HPLC system was designed/developed for the determination of LA enantiomers in various fermentation food/beverage samples including vinegars.…”