微波辅助蛋白质水解-反相高效液相色谱法测定罗非鱼皮中的羟脯氨酸

摘要

为测定罗非鱼( Oreochromis niloticus)皮中羟脯氨酸含量,建立了微波辅助蛋白质水解-反相高效液相色谱法。实验采用9-芴基甲氧基羰酰氯( FMOC-Cl)为衍生试剂,应用ZORBAX SB-C18色谱柱,以0．05 mol/L的醋酸钠溶液和甲醇为流动相进行梯度洗脱, DAD检测器在262 nm处进行检测,外标法定量。结果显示,羟脯氨酸线性关系良好,相关系数为0．9999,回收率92．00%~103．25%,相对标准偏差2．53%~3．61%,检出限为0．2 mg/g。%A method was established for the determination of hydroxyproline in the skin of tilapia( Oreochromis niloticus) by reversed-phase high performance liquid chromatography after microwave-assisted acid hydrolysis of protein. After derivat-ization by 9-fluorenylmethyl chloroformate ( FMOC-Cl) , separation and quantification was achieved on a ZORBAX SB-C18 column by gradient elution with methanol and 0. 05 mol/L sodium acetate buffer. The detection was carried out with diode array detector (DAD) by absorbance at 262 nm. The external standard calibration curves were used for quantification. There were good linear correlations for hydroxyproline ( r =0. 999 9 ) . Average spike recovery of hydroxyproline was 92. 00% ~103. 25%, with relative standard devitations 2. 53% ~3. 61%, and the limit of detection for hydroxyproline was 0. 2 mg/g. This method is simple, sensitive, reproducible and suitable for the determination of hydroxyproline in fish skin.