柱前衍生HPLC-UV法测定类风湿性关节炎大鼠尿样中16种氨基酸含量

Determination of 16 amino acids in urine of rheumatoid arthritis rats by precolumn derivatization HPLC-UV

目的建立柱前衍生-高效液相色谱-紫外分光光度(柱前衍生-HPLC-UV)测定类风湿性关节炎大鼠尿样中16种氨基酸含量的方法。方法将大鼠尿样样品与异硫氰酸苯酯(PITC)衍生,衍生样品采用Agilent ZORBAX SB C18色谱柱进行分离,以乙腈-甲醇-水(6∶2∶2)和醋酸钠缓冲液为流动相进行梯度洗脱,柱温35℃,检测波长254nm,流速1.0mL/min,进样量10μL。结果 16种氨基酸在线性范围内线性关系良好,相对回收率均在85%~115%,日内RSD均<15%,检出限在3.1~45.3ng/mL范围内。16种氨基酸在大鼠尿液中的含量在2.164~565.966μg/mL范围内。结论本方法操作简便、灵敏度高,适合于类风湿性关节炎大鼠尿样中氨基酸的含量测定。

Objective To establish a pre-column derivatization HPLC-UV method for the determination of16 amino acids in urine of rats with rheumatoid arthritis.Methods The program of biological sample pretreatment method and the pre-column derivatization time were optimized.The column was Agilent ZORBAX SB C（18）,and acetonitrile,methanol and water mixture（acetonitrile-methanol-water =6∶2∶2）and sodium acetate buffer were selected as mobile phase in gradient elution,column temperature 35℃,detection wavelength 254 nm,flow rate 1.0 mL/min,sample volume 10μL,and methodology investigation was carried out.Results 16 amino acids showed good linear relationship in the linear range,the relative recoveries were between 85-115%,the daily RSD was less than 15%,the detection limit was within the range of 3.1-45.3 ng/mL.The contents of 16 amino acids in rat urine ranged from 2.164 to 565.966μg/mL.Conclusion The method is simple,sensitive and can be applied to the determination of 16 kinds of amino acids in urine of rheumatoid arthritis rats.