Determination of 18 Amino Acids in Gelatin from Bovine and Yak by High Performance Liquid Chromatography Coupled with Precolumn Derivatization

KeyWord:gelatin from Yak  gelatin from bovine  amino acid  precolumn derivatization  phenyl isothiocyanate(PITC)  high performance liquid chromatography(HPLC)

Abstract:

      A rapid quantitative method of high performance liquid chromatograpy(HPLC) was developed for the analysis of 18 amino acids in gelatin from bovine skin and Yak skin.The steps of sample derivatization with PITC were optimized.The method was no need for drying down and extraction with organic solvent.The yak leather was enzymatically hydrolyzed at 37 ¡æ for 72 h with pepsin£¬and then was freeze dried.The sample was then hydrolyzed with 6.0 mol/L HCl at 110 ¡æ.After the hydrolyzation£¬the solution was derivatized with phenyl isothiocyanate(PITC).Gradient HPLC separation was performed on a Shiseido Capcell Pak C18 column(4.6 mm¡Á250 mm,5 ¦Ìm) with 0.14 mol/L sodium acetate-0.5 mL/L triethylamine solution(pH 6.08£¬adjusted with glacial acetic acid) and 60% acetonitrile as mobile phases at a flow rate of 1 mL/min.The detection was carried out with an ultraviolet detector,and the wavelength was set at 254 nm.The calibration curves were linear in the range of 0.2-80 mg/L with correlation coefficients(r2) not less than 0.999 2.The limits of detection for the method were in the range of 0.01-0.39 mg/kg,and the average recoveries were 78.7%-121.0% with RSDs of 0.05%-12.0%.The derivatization steps were simple and sensitive£¬and the sample pretreatment time was short.The method was suitable for the rapid identification and determination of amino components in gelatin from bovid.