A new strategy for the selective determination of D-amino acids: enzymatic and chemical modifications for pre-column derivatization.
A new strategy for the selective determination of D-amino acids (DAAs) employing a pre-column derivatization was designed with concepts based on both enzymatic and chemical modifications. Selective determination of DAAs was accomplished by following: DAA was enzymatically modified with D-amino acid oxidase (DAAO: EC 1.4.3.3) to form an alpha-keto acid. Subsequently, resulting alpha-keto acid was detected by high-performance liquid chromatography (HPLC) after chemical modification with o-phenylenediamine (PDA) in the presence of 2-mercaptoethanol (2ME) to give the corresponding quinoxalinol derivative (PDA-alpha-keto acid derivative). After optimizing the pre-column derivatization and HPLC separation, five peaks corresponding to DAAs (D-alanine, D-leucine, D-methionine, D-phenylalanine, D-valine (as the standard mixture of DAAs in this paper) were separately eluted and monitored by means of a conventional HPLC system with a gradient elution on octadecyl silica gel (ODS) column and a fluorescence detector (Ex.: 341 nm, Em.: 413 nm), respectively. It was confirmed that the present method was incapable of detecting L-amino acids (LAA) when a sample solution consisting of both LAAs and DAAs was examined. The linearity of the peak-area responses to their concentration range of DAAs from 10 to 500 microM is 0.994-1.000, and their detection limits were 0.2-1 microM (signal/noise = 3). When this method was applied to a methanolic extract of short-necked clams, Ruditapes philippinarum (in Japanese, Asari), a big peak, corresponding to D-alanine was detected, corresponding to 2.9 mg/g D-alanine. In this paper, we present an example of pre-column derivatization method that was newly configured to take into account both the biological and chemical properties of the substances in question.
Oguri
Shigeyuki
S
Laboratory of Food Hygiene, Department of Home Economics, Aichi-Gakusen University, 28 Kamikawanari, Hegoshi-cho, Okazaki-city 444-8520, Japan. s-oguri@gakusen.ac.jp
Nomura
Michiko
M
Fujita
Youko
Y
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010655:Phenylenediamines / Q000737:chemistry*; D011810:Quinoxalines / Q000302:isolation & purification; D015203:Reproducibility of Results; D013237:Stereoisomerism
D000596:Amino Acids; D010655:Phenylenediamines; D011810:Quinoxalines; C034193:1,2-diaminobenzene; D003605:D-Amino-Acid Oxidase