全自动血凝仪测定肝素类药物抗Xa因子和抗Ⅱa因子效价  

Determination of Anti-factor Xa Activity and Anti-factorⅡa Activity of Heparins by Automatic Coagulation Analyzer

作　　者：李京[1] 龚益妮 张佟 范慧红[1] LI Jing;GONG Yi-ni;ZHANG Tong;FAN Hui-hong(National Medical Products Administration Key Laboratory for Quality Research and Evaluution of Chemical Drugs,National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院,国家药品监督管理局化学药品质量研究与评价重点实验室,北京102629

出　　处：《中国药学杂志》2021年第21期1755-1763,共9页Chinese Pharmaceutical Journal

摘　　要：目的肝素类药物效价测定方法较为复杂,手工操作步骤多,实验误差较大,实验效率不高。本研究探索使用全自动血凝仪的微量生色底物法测定肝素类药品抗Xa因子和抗Ⅱa因子效价,提高实验自动化程度。方法摸索剂量-反应关系,选择适合全自动血凝仪的剂量范围、剂间比、试剂使用量、反应时间、标准品及供试品溶液配制方式、测定方式等。不使用血凝仪自带测定程序及配套试剂,使用自定义试剂,按《中国药典》2020年版四部通则1208"肝素生物检定法"中新增的抗Xa因子和抗Ⅱa因子效价测定法编写血凝仪抗Xa因子和抗Ⅱa因子测定程序。以405 nm波长吸光度为纵坐标,标准品或供试品的浓度对数值为横坐标分别作线性回归,按2020年版《中国药典》四部通则1431中的量反应平行线原理4×4法实验设计,通过BS2000或Biostat 1.0软件计算效价和实验误差。结果建立了全自动血凝仪测定肝素类产品抗Xa因子和抗Ⅱa因子效价测定方法,并对方法的线性范围、准确性、重复性等进行验证,结果良好,对4个厂家的40批达肝素钠注射液进行了测定,结果符合规定。结论全自动血凝仪测定肝素类产品抗Xa因子和抗Ⅱa因子效价方法可以用于肝素类药品的质量控制,该方法自动化程度高,提高了实验效率,降低了人工操作的实验误差,重复性好,有助于提高我国肝素类药品质控水平,提高产品质量。OBJECTIVE The assay of heparin is quite complicated. There are many steps for manual operations. The test error is large. The efficiency is not good. To determine the activities of anti-factor Xa and anti-factor Ⅱa by highly automated coagulation analyzer, using micro-chromogenic substrate methods. METHODS Suitable experimental conditions were explored for automatic coagulation analyzer, including dose-response relationship, dose range, dose ratio, the quantity of the reagents, reaction time, preparation of the standard and test solution, and determination methods. Without using the program and reagents of coagulation analyzer, user defined programs and reagents were established according to “bioassay of heparin” in the general rules 1208 of Chinese Pharmacopoeia(2020 Edition). Micro-chromogenic substrate methods were established to determine the anti-FXa and anti-FⅡa activity of heparins. Linear regression was performed with absorbance at 405 nm as ordinate and logarithm of standard or test sample concentration as abscissa. According to the principle of parallel line of dose-response in the general rule 1431 of Chinese Pharmacopoeia(2020 Edition), the potency and experimental error were calculated by BS2000 or Biostat 1.0 software. RESULTS A new method to determine the anti-factor Xa and anti-factor Ⅱa activity of heparins by automatic coagulation analyzer was established. The linear range, accuracy and repeatability of the method were verified. Forty batches of dalteparin sodium injection from four manufacturers were determined and the results were in accordance with the specifications. CONCLUSION The automated method can be used for the quality control of heparin drugs. It can improve the experiment efficiency and reduce the error of manual operation. The accuracy and repeatability of the method are good. It is helpful to improve the quality control level of heparin drugs and improve the quality of the products.

关 键 词：肝素 低分子量肝素 达肝素钠 抗XA因子 抗Ⅱa因子 效价 生色底物 血凝仪 

分 类 号：R917[医药卫生—药物分析学]