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

Abstract

For the simultaneous determination of lactate (LA), 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB) and malate (MA) enantiomers, a new separation technique in a two-dimensional high-performance liquid chromatography (2D-HPLC) system following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole has been developed. The 2D-HPLC was composed of the reversed-phase (1D) and enantioselective (2D) separations. In the first dimension, the target hydroxy acids were separated as their D plus L mixtures on a Singularity RP18 column (1.0 mm i.d. x 250 mm), and the enantiomers were separated in the second dimension using a polysaccharide-based chiral stationary phase, Chiralpak IG (2.0 mm i.d. x 250 mm). By using the Chiralpak IG column, the LA, 2HB, 3HB and MA enantiomers were separated with resolution values of 3.47, 3.63, 6.81 and 3.72, respectively. The developed 2D-HPLC system was applied to human plasma, and trace levels of D-LA and L-3HB in addition to their major antipodes were determined. The acquired %D of LA (the percentage of D-LA over total LA) and %L of 3HB (the percentage of L-3HB over total 3HB) were 1.0 and 2.7, respectively. For 2HB, only the L-form was detected, while the MA enantiomers were not found in the human plasma.

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

“…In the third dimension, the enantiomers of NBD-LA, 2HB, 3HB and MA were separated according to the conditions indicated in our previously published study [19]. Briefly, a Chiralpak IG column was used as the stationary phase, and mixed solutions of MeOH/MeCN were used as the mobile phases.…”

“…The 2D-HPLC methods following the derivatization with NBD-PZ were applied to analyze the hydroxy acid enantiomers (e.g. LA, 3HB, 2HB and MA) in mammalian physiological fluids or food products [19,21,28]. Even though these approaches were useful for the chiral separation of the hydroxy acid enantiomers, the accurate determination of the enantiomers in real world matrices was still difficult, since the specimens are comprised of uncountable unknown components.…”

“…With respect to the methods for the simultaneous and enantioselective analysis of LA, 2HB, 3HB and MA by only one injection, to the best of our knowledge, only one 2D-HPLC method has been developed [19]. The 2D-HPLC system was equipped with a Singularity RP18 column (1.0 mm i.d.…”

“…To overcome these difficulties, several two-dimensional (2D) HPLC methods equipped with a reversed-phase column and an enantioselective column have been developed [19][20][21]. A 2D-HPLC analysis following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was performed to simultaneously analyze the LA, 2HB, 3HB and MA enantiomers in human plasma [19].…”

“…To overcome these difficulties, several two-dimensional (2D) HPLC methods equipped with a reversed-phase column and an enantioselective column have been developed [19][20][21]. A 2D-HPLC analysis following the fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was performed to simultaneously analyze the LA, 2HB, 3HB and MA enantiomers in human plasma [19]. Although the 2D-HPLC system has high sensitivity and selectivity, the hydroxy acid enantiomers in several mammalian fluids, such as human urine, could not be successfully determined due to the complexity of the matrices.…”