柱前衍生化法同时测定人凝血因子Ⅷ中甘氨酸和盐酸赖氨酸的含量

Glycine and lysis hydrochloride assay in human coagulation factor Ⅷ by pre-column derivation

目的建立一种柱前衍生-高效液相色谱法同时测定人凝血因子Ⅷ中2种氨基酸含量的方法。方法以2,4-二硝基氟苯(DNFB)为衍生剂,采用C18色谱柱,流动相A为水,B为乙腈,C为含4mmol/L三乙胺的0.05mol/L醋酸钠缓冲液(pH=6.4),进行梯度洗脱,流速1.0 ml/min,检测波长360nm。结果甘氨酸的线性范围为2.98~14.90g/L(r=0.999 9),盐酸赖氨酸线性范围为1.195~5.975g/L(r=0.999 9),甘氨酸和盐酸赖氨酸的平均回收率分别为99.1%和98.2%。结论此方法检测结果准确,可用于人凝血因子Ⅷ中甘氨酸和盐酸赖氨酸的含量测定。

Objective To establish a method of analysis for glycine and lysis hydrochloride in human coagulation factorⅧ by pre-column derivation HPLC.Methods The sample was derivatized with 2,4-dinitrofluorobenzene（DNFB）.HPLC was performed on a C18 column with gradient elution.The mobile phase A was water,B was acetonitrile and C was 0.05 mol/L sodium acetate buffer containing 4 mmol/L trimethylamine.The flow rate was 1.0 ml/min and detection wavelength was 360 nm.Result There was a linear range for glycine between 2.980 g/L-14.90 g/L（r=0.999 9）and lysis hydrochloride between1.195 g/L-5.975 g/L（r=0.999 9）.The average recovery rate of glycine and lysis hydrochloride was 99.1% and 98.2%.Conclusion The established method is accurate and can be used to determine the contents of glycine and lysis hydrochloride in human coagulation factorⅧ.