Determination of protein amino acids as benzylthiocarbamyl derivatives compared with phenylthiocarbamyl derivatives by reversed-phase high-performance liquid chromatography, ultraviolet detection and precolumn derivatization

Abstract

All 22 protein amino acids were successfully derivatized to benzylthiocarbamyl (BZTC) derivatives by precolumn reaction at 50°C for 30 min with benzylisothiocyanate (BZITC) and separated in 35 min on a reversed-phase Nova-Pak C 18 column (30 cm×3.9 mm, 4 μm). However, phenylthiocarbamyl-derivatives were not completely separated on this column; in particular cystine and cysteine were resolved into a single peak. The optimun wavelength for determination of BZTC derivatives was 246 nm although the strong absorption wavelengths were 220 nm and 238 nm. The relative standard deivations (R.S.D.) of the relative molar response to the internal standard (norleucine) were less than 5%, except for cysteine (7.22%); however for the PTC derivatives, glutamine, proline, threonine, alanine, leucine, cystine+cysteine and lysine the R.S.D. values exceeded 5%. Standard calibration curves showed good linearity in the measured range from 0.125 to 5 nmol. The correlation coefficient of cystine was the lowest, 0.952. The stability of BZTC derivatives of standard amino acids was about 120 h except for threonine, alanine, cystine and serine. The detection limit was about 3.9 pmol at 0.05 AUFS in both of BZTC and PTC derivatives. The compositions of amino acids of soybean and bovine serum albumin (BSA) analyzed with both derivatives were similar to the results found in the literature and food composition tables, except for cysteine. The BZTC derivatives of cystine and cysteine in soybean and BSA could be determined separately, but the PTC derivatives could not, nor could data on separate dertermination of cysteine and cystine be found in the literature studied.