Analysis of L- and D-Amino Acids Using UPLC

Abstract

With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism including human and have biologically important roles. D-Aspartate regulates reproductive activity in animals and humans. D-Serine serves as a co-agonist of the N-methyl-D-aspartate receptor, which mediates glutamatergic neurotransmission. D-Alanine plays a role like osmolyte in crustaceans and mollusks. In this protocol, we describe a method for analysis of L- and D-amino acids using ultra-performance liquid chromatography (UPLC). To analyze D- and L-amino acids, the enantiomers are initially converted into diastereomers (diastereomers are stereoisomers that are not related as object and mirror image and are not enantiomers) using pre-column derivatization with o-phthaldialdehyde plus N-acylated cysteine (N-acethyl-L-cysteine or N-tert-butyloxycarbonyl-L-cysteine). The resultant derivatives are fluorescent diastereomers. This is followed by separation of the resultant fluorescent isoindol derivatives on an octadecylsilyl stationary phase using UPLC, and the fluorescence is detected by a fluorescence detector included in UPLC system. Using this method, 16 kinds of D-amino acid can be analyzed., 除了甘氨酸，α-氨基酸具有光学活性，并且可以形成每个氨基酸的两种光学异构体（L-和D-）。分析技术的最新发展表明，几种游离D-氨基酸如D-天冬氨酸，D-丝氨酸和D-丙氨酸存在于包括人在内的许多种生物中，并且具有生物学上的重要作用。 D-天冬氨酸调节动物和人的生殖活性。 D-丝氨酸作为Nε-甲基-D-天冬氨酸受体的共激动剂，其介导谷氨酸能神经传递。 D-丙氨酸在甲壳类动物和软体动物中起到类似渗透物的作用。在该协议中，我们描述了使用超高效液相色谱（UPLC）分析L-和D-氨基酸的方法。为了分析D-和L-氨基酸，首先将对映异构体转化为非对映异构体（非对映异构体是与物镜和镜像无关的立体异构体，并且不是对映异构体），使用前柱衍生化，邻苯二甲醛加上Nε - 酰化的半胱氨酸（Nε - 乙酰基-L-半胱氨酸或Nε - 叔丁基氧羰基-L 。所得衍生物是荧光非对映异构体。然后使用UPLC在十八烷基硅烷基固定相上分离所得荧光异吲哚衍生物，并通过包括在UPLC系统中的荧光检测器检测荧光。使用该方法，可以分析16种D-氨基酸。