Amino Acids Analysis Using in Loop Derivatization with 4-fluoro-7-nitro- 2,1,3-benzoxadiazole

A determination method for amino acids in mouse plasma was developed by HPLC with fluorescence detection. The developed methodology is based on a pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) carried out in the sample injection loop. The optimized derivatization time and temperature were 10 min and 60 °C, respectively. The separation of 21 amino acid derivatives was achieved on a monolithic silica column with gradient elution. The amino acid derivatives were detected with excitation and emission at 470 nm and 530 nm, respectively. The lower limit of detection for the individual amino acids was 1.52 to 47.0 fmol. The calibration curves for all the amino acids were linear over the tested range of 0.03 to 30 pmol. The validation data showed that the assay is sensitive, selective and reproducible for determination of amino acids. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.