高效液相色谱法检测人凝血因子Ⅷ中氨丁三醇残留量  

Determination of tromethamine residues in human coagulation factor Ⅷ by high performance liquid chromatography method

作　　者：陈秀良 罗二华 梁小明 陈家啡 CHEN Xiuliang;LUO Erhua;LIANG Xiaoming;CHEN Jiafei(Boya Bio-Pharmaceutical Group Co Ltd,Blood Product Research Lab of Jiangxi,Fuzhou 344000,China)

机构地区：[1]博雅生物制药集团股份有限公司江西省血液制品重点实验室,江西抚州344000

出　　处：《中国输血杂志》2022年第10期1020-1023,共4页Chinese Journal of Blood Transfusion

基　　金：江西省重点新产品计划(血浆冷沉淀中凝血因子类产品深度开发及产业化)(20173ABC28011)。

摘　　要：目的 建立氨丁三醇的高效液相色谱检测方法,并对该方法进行验证。方法 以丙氨酸为内标,使用6-氨基喹啉基-N-羟基琥珀酰亚氨基氨基甲酸酯(AQC)进行柱前衍生,Inertsil~? ODS-SP为色谱柱,以乙酸-乙酸钠缓冲液和乙腈进行梯度洗脱,流速为1.0 mL/min,柱温37℃,进样体积25μL,使用紫外检测器,以248 nm进行检测。同时对该方法进行验证。结果 辅料甘氨酸、枸橼酸钠和氯化钙对氨丁三醇残留量的检测均无干扰;加标回收率为90.0%~100.8%;重复性验证的相对标准偏差(RSD)分别为2.5%、0.7%和1.2%,中间精密度RSD为0.8%。氨丁三醇对照品在(0.5~4.0)μg/mL范围浓度和与内标峰面积比值呈良好的线性关系,回归方程为Y=0.012 9X-0.000 782,R=0.999 97;定量限为0.04μg/mL。结论 成功的建立了人凝血因子Ⅷ中氨丁三醇残留量的高效液相色谱检测方法,该方法具有良好的线性、专属性、准确度、精密度。Objective To develop and verify high performance liquid chromatography(HPLC) detection for tromethamine(Tris) residues in human coagulation factor Ⅷ. Methods Alanine was used as internal standard, and AQC for pre-column derivation. InertsilODS-SP was adopted, and acetic acid-sodium acetate buffer and acetonitrile were used for gradient elution at the flow rate of 1 mL/min and column temperature of 37℃. The 25 μL sample was loaded and determined by UV-detector with detection wavelength at 248 nm. This method was then verified. Results Glycine, sodium citrate and calcium chloride showed no interference with the detection of tromethamine(Tris) residues. The recovery rate of spike samples was within 90.0%~100.8%. The RSD in repeatability test were 2.5%, 0.7% and 1.2%, respectively, and in intermediate precision test 0.8%. The tromethamine(Tris) at concentrates of(0.5~4.0)μg/mL showed good linear relationship to the peak area to internal standard, of which the regression equation was Y=0.012 9X-0.000 782, R=0.999 97.The quantitative detection limit was 0.04μg/mL. Conclusion The HPLC for determination of tromethamine(Tris) residues in human coagulation factor Ⅷ was successfully developed, which showed good linearity as well as high specificity, precision and accuracy.

关 键 词：氨丁三醇 高效液相色谱法 方法验证 

分 类 号：R446.1[医药卫生—诊断学]