Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquid chromatography.

An easy, rapid and sensitive method for the simultaneous determination of primary and secondary amino acids in different matrices, e.g. protein hydrolysates, pharmaceutical formulations, plant extracts, food samples and physiological fluids, is described. After minimum sample preparation amino acids were derivatized with two different reagents, o-phthalaldehyde-3-mercaptopropionic acid for primary and 9-fluorenylmethylchloroformate for secondary amino acids, by an automated precolumn derivatization technique. With minor adjustments of separation parameters this method can also be used to determine amino acids in different matrices. Analysis time including reaction, separation and reconditioning ranged from 20 min for hydrolysates to 60 min for physiological fluids. The separation was done on a reversed-phase column with a gradient of acetate buffer-acetonitrile as the mobile phase. The precision for peak areas of the individual amino acids was within a relative standard deviation of 2% for the hydrolysate assay and 2-5% for the physiological assay, and for retention times better than 0.7%. The detection limit with the diode array detector (ultraviolet-visible) was ca. 2-5 pmol, measured at 338 nm for primary and 266 nm for secondary amino acids; with the fluorescence detector 20-50 fmol were detectable at excitation and emission wavelengths of 230 and 455 nm for primary and 266 and 310 nm for secondary amino acids, respectively.