Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9748992 | Journal of Chromatography A | 2005 | 8 Pages |
Abstract
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 μM is 0.994-1.000, and their detection limits were 0.2-1 μM (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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Shigeyuki Oguri, Michiko Nomura, Youko Fujita,