柱前衍生HPLC法检查壳聚糖中蛋白质残留

Test of protein residue in chitosan with HPLC precolumn derivatization

目的:建立氨基酸的柱前衍生化HPLC测定法用于壳聚糖中残留蛋白质的检查。方法:壳聚糖样品经酸水解并以9-芴甲氧羰酰氯柱前衍生,用5 mmol.L-1醋酸铵缓冲液(pH 6.7)-乙腈(80∶20)和5 mmol.L-1醋酸铵缓冲液(pH 6.7)-乙腈(20∶80)为流动相梯度洗脱,C18柱分离后,以荧光检测器在266 nm激发波长、313 nm发射波长处检测,外标法计算各氨基酸含量,并以总氨基酸含量作为壳聚糖中蛋白质残留量。结果:各氨基酸分离良好,过量衍生试剂和氨基葡萄糖衍生产物仅对Leu、Ile、Phe产生干扰。各氨基酸浓度在0.01～5.0μg.mL-1范围内线性关系良好(r>0.99),精密度(RSD)为1.6%～8.5%,定量限为1.9～10.9 ng.mL-1,回收率为83.4%～99.3%。结论:本方法可准确测定壳聚糖中13个氨基酸并用于壳聚糖中微量蛋白质残留的检查,为评价壳聚糖质量,保证壳聚糖在医药和生物材料使用的安全性提供依据。

Objective： To establish a high performance liquid chromatography method using pre-column derivation for the test of protein residue in chitosan.Methods：Chitosan samples were hydrolyzed by hydrochloric acid and derived using 9-fluorenylmethylchloroformate（FMOC-Cl）.The analysis was carried out on a C18 column using 5 mmol·L^-1 ammonium acetate buffer（pH 6.7）-acetonitrile（80∶ 20）and 5 mmol·L^-1 ammonium acetate buffer（pH 6.7）– acetonitrile（20∶ 80）as mobile phases with linear gradient elution and a fluorescence detection（excitation 266 nm,emission 313 nm）.Amino acids were calculated using external standard method and the total amino acid was counted as the protein residue in chitosan.Results：Good retention and separations were achieved for derivatives of 16 amino acids.Only Leu,Ile and Phe were interfered by the excess derivation reagents and derivation products of glucosamine.The calibrations of amino acids were linear（r0.99）within 0.01-5.0 μg·mL^-1,and the precision（RSD）,recovery and limit of quantification（LOQ）of the established method were 1.6%-8.5%,84.4%-98.6%,and 1.9-10.9 ng·mL^-1,respectively.Conclusion：The established method can accurately quantify 13 amino acids in chitosan and thus can be used for the test of protein residue in chitosan for quality evaluation of chitosan.The method could provide reference for guarantee of the safety for the use of chitosan in medical and biological materials.