2019 Volume 40 Issue 1 Pages 25-32
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.