Enantioselective micro‐2D‐HPLC determination of aspartic acid in the pineal glands of rodents with various melatonin contents

Abstract

Enantioselective determination of aspartic acid (Asp) in the pineal gland of rodents with various melatonin contents was performed using a highly sensitive and selective two-dimensional HPLC system. After derivatization of the amino group with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), NBD-Asp was separated using a capillary monolithic ODS column in the first dimension. The fraction of NBD-Asp was automatically collected and transferred to the second dimension, and the D- and L-Asp were separated and determined using a narrowbore enantioselective column. Large amounts of D-Asp were observed in the pineal gland of the rats and specific strains of mice (C3H and CBA) possessing a high concentration of melatonin in their pineal gland. On the other hand, the amounts of D-Asp were small in the pineal gland of mice possessing a trace or no melatonin in their pineal gland (ddY, ICR, C57BL and BALB/c). In other tissues and physiological fluids, no significant strain-dependent changes of the D-Asp amounts were observed. These results indicate that large amounts of D-Asp are present only in the pineal gland containing large amounts of melatonin, and special care should be taken when selecting mouse strains for the investigation of D-Asp.

Enantioselective Two-Dimensional High-Performance Liquid Chromatographic Determination of Amino Acids; Analysis and Physiological Significance of D-Amino Acids in Mammals

“…Therefore, it is a matter of interest whether the amounts of D-Asp in the pineal gland of these mice are related to the amounts of melatonin. In the pineal gland of 3 strains of rats and 6 strains of mice, both D-Asp and melatonin were determined [52]. For the determination of D-Asp, a 2D-HPLC system was used, while a pre-column oxidation column-switching HPLC system was used for the determination of melatonin [53].…”

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

“…Recently, the chiral separations of amino acids have been widely discussed because the minor D enantiomers were found in mammals, including humans, and clarified to have various functions and diagnostic values [13][14][15][16][17]. For instance, D-Ser mediates the neurotransmission [18,19] and D-Asp regulates the endocrine systems [20][21][22]. These results indicate that chiral discrimination is useful to find out new physiologically active substances and biomarkers.…”

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

“…Under these conditions, NBD-Asp and Glu are separated within 60 min in the first dimension, and their enantiomers are separated within 80 min in the second dimension. By using the present 2D-HPLC system, the distributions of D-Asp and D-Glu in the tissues/physiological fluids of rats [24] and mice [25] are demonstrated.…”

“…The simultaneous on-line chiral 2D-HPLC analysis of acidic amino acids, namely, aspartic acid (Asp) and glutamic acid (Glu), has also been reported [24,25]. For the reversed-phase separation, a microbore-monolithic ODS column (0.53 mm ID x 750 mm) is used at 40ºC with the mobile phase of 8% MeCN 0.05% TFA in water.…”