采用UPLC分析L-氨基酸和D-氨基酸

Yuta Mutaguchi; Toshihisa  Ohshima

doi:10.21769/BioProtoc.1231

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Peer-reviewed

Analysis of L- and D-Amino Acids Using UPLC

采用UPLC分析L-氨基酸和D-氨基酸

YM Yuta Mutaguchi

TO Toshihisa Ohshima email

发布: 2014年09月05日第4卷第17期 DOI: 10.21769/BioProtoc.1231 浏览次数: 13518

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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.

Materials and Reagents

N-Acetyl-L-cysteine (NAC) (Wako Pure Chemical Industries, catalog number: 017-05131 )
N-tert-butyloxycarbonyl-L-cysteine (NBC) (Funakoshi, catalog number: 04-621 )
o-Phthaldialdehyde (OPA) (Nacalai Tesque, catalog number: 27824-74 )
Methanol (Wako Pure Chemical Industries, catalog number: 132-06471 )
Acetonitrile (Wako Pure Chemical Industries, catalog number: 015-08633 )
Trichloroacetic acid (Wako Pure Chemical Industries, catalog number: 203-04952 )
Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 192-15985 )
0.4 M sodium borate buffer (see Recipes)
1. Boric acid (Wako Pure Chemical Industries, catalog number: 021-02195 )
2. Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 195-13775 )
50 mM sodium acetate buffer (see Recipes)
1. Acetic acid (Wako Pure Chemical Industries, catalog number: 192-01075 )
2. Sodium acetate (Wako Pure Chemical Industries, catalog number: 192-01075)
50% TCA (see Recipes)
1 M NaOH (see Recipes)

Equipment

ACQUITY UPLC-FLR system (Waters)
1. AccQ-Tag Ultra 2.1 x 100 mm column (Waters, catalog number: 186003837 )
2. ACQUITY binary solvent manager
3. ACQUITY sample manager
4. ACQUITY FLR detector
5. Sample vial: 12 x 32 mm glass screw neck vial, Quick Thread, LectraBond cap, PTFE/silicone septa, Total Recovery (Waters, catalog number: 186000384c )
6. Empower 2 software
Vortex (Taitec, catalog number: 0061271-000 )
Polytetrafluoroethylene membrane filters (4-mm diameter, 0.2-μm pore size) (Merck KGaA, catalog number: SLLGH04NL )
Amicon Ulta 0.5 ml centrifugal filter 3 K device (Merck KGaA, catalog number: UFC500396 )