Enantiomeric purity determination of (l)-amino acids with pre-column derivatization and chiral stationary phase: Development and validation of the method

A simple, efficient and general HPLC method for the determination of enantiomeric purity of a series of (l)-amino acids was developed. In order to improve the detection sensitivity, pre-column derivatization was adopted and 7-chloro-4-nitrobenzoxadiazole (NBD-Cl) was selected as derivatization reagent. NBD-amino acid enantiomers were then enantioseparated on a Pirkle-type chiral stationary phase, Sumichiral OA-2500S (250 mm × 4.6 mm, 5 μm), using a mobile phase composed of acetonitrile–methanol (50:50, v/v) containing 5 mmol L−1 citric acid at the flow rate of 0.5 mL min−1. The detection wavelength was 470 nm. All the eleven pairs of tested amino acid enantiomers were well separated, and trace amounts of (d)-amino acids (0.5%) in the presence of a large excess of corresponding (l)-enantiomers could be quantified. The proposed method was validated in terms of selectivity, precision, linearity range, LOD, LOQ and accuracy, and then successfully applied to the determination of enantiomeric purity in bulk samples of (l)-amino acids.