全自动血凝仪动力学法测定肝素类药物抗Ⅹa因子和抗Ⅱa因子效价

Kinetic Determination of Anti-FactorⅩa Activity and Anti-FactorⅡa Activity of Heparins by Automatic Coagulation Analyzer

目的《中国药典》2020年版肝素抗Ⅹa因子和抗Ⅱa因子效价测定采用“终点法”测定吸光度值,本研究尝试建立全自动血凝仪“动力学法”测定反应值,检测肝素类药物的抗Ⅹa因子和抗Ⅱa因子效价。方法使用自定义试剂,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,在405 nm波长检测吸光度随反应时间的变化,测定反应曲线斜率,以反应曲线斜率为纵坐标,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“动力学法”测定肝素类产品抗Ⅹa因子和抗Ⅱa因子效价测定方法,并对方法准确性、重复性、适用性等进行验证,结果良好,对4个厂家的4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“动力学法”测定肝素类产品抗Ⅹa因子和抗Ⅱa因子效价结果与“终点法”检测结果一致,实验误差更小,检测时间更短,并且不使用冰醋酸作为终止剂,对实验人员和环境更为友好。该方法自动化程度高,误差小,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“endpoint method”was used to determine the absorbance value in the method of heparin assay by anti-factorⅩa activity and anti-factorⅡa activity in Chinese Pharmacopoeia.This paper attempts to establish a“kinetic method”to determine the reaction value and detect anti-factorⅩa activity and anti-factorⅡa activity of heparin drugs.METHODS 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.To be detected the change of absorbance with reaction time at 405 nm wavelength,measure the slope of reaction curve,and make linear regression with the slope of the reaction curve as the ordinate and the concentration logarithm of the standard or test sample as the 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 The“kinetic method”of automatic coagulation analyzer was established to determine anti-factorⅩa activity and anti-factorⅡa activity of heparin.The accuracy,repeatability and applicability of the method were verified.The results were good.Four batches of dalteparin sodium injection from four manufacturers were determined,and the results were in accordance with the specifications.CONCLUSION The results determined by“kinetic method”of automatic coagulation analyzer are consistent with the results of“end point method”,the experimental error is smaller,the detection time is shorter,and glacial acetic acid is not used as the terminator,which is friendly to the experimental personnel and the environment.The method has the advantages of high automation,small error and good repeatability.It is helpful to improve the quality control level of heparin drugs and product quality in China.