High-performance liquid chromatographic determination of amino acids in protein hydrolysates and in plasma using automated pre-column derivatization with o-phthaldialdehyde/2-mercaptoethanol.

ABSTRACT

A sensitive and reproducible method for the routine determination of amino acids in plasma and in protein hydrolysates based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. The resolution of all amino acids was found to be good. The total time for analysis, including separation and reconditioning, ranged from 38 min for protein hydrolysates to 62 min for 29 physiological amino acids. The precision of hydrolysate analysis was within a relative standard deviation of 0.8-7.3% depending on the use of internal or external standards. The relative standard deviations of peak areas for physiological amino acids (standard) ranged between 1.8 and 5.6%. The relative standard deviations of retention times were less than 0.5% for all amino acids. This method can be used for routine analysis. One single column with 4-microns end-capped C18 material was found to be sufficient for 400-500 successive runs.