Simultaneous quantitation of nine kinds of (d)- and (l)-amino acid enantiomers by HPLC-MS/MS: application to the quality control of amino acid tablets

Abstract

A simple and sensitive HPLC-MS/MS method was developed for the simultaneous determination of nine kinds of (D)- and (L)-amino acid enantiomers in amino acid tablets. 7-Chloro-4-nitrobenzoxadiazole (NBD-Cl) was selected as the derivatization reagent, and the derived amino acid enantiomers were enantioseparated on a Sumichiral OA-2500S (250 mm × 4.6 mm, 5 μm) column, using a mobile phase composed of acetonitrile–methanol (50 : 50, v/v) containing 0.5% formic acid at the flow rate of 1.0 mL min⁻¹ with a split ratio of 1 : 3. Sensitive detection was performed with a 4000 Qtrap MS/MS system with an electrospray-ionization source in negative mode. The calibration curves for the determination of all the nine pairs of amino acid enantiomers showed good linearity (R² > 0.999) over the investigated ranges from 0.15 to 30 μg mL⁻¹ for the (D)-enantiomers and from 3.14 to 620 μg mL⁻¹ for the (L)-enantiomers, respectively. The assay was reproducible with overall intra- and inter-day variations of less than 7.7%. The detection for (D)-amino acid enantiomers was selective and sensitive, and a trace amount of them could be detected and quantified, even in the presence of massive corresponding (L)-enantiomers. The validated method was successfully applied to the simultaneous quantitation of the nine kinds of (D)- and (L)-amino acid enantiomers in commercial tablets.