垂体后叶注射液含量HPLC法和生物效价测定法等效性研究

目的：建立HPLC法测定垂体后叶注射液中赖氨酸升压素及缩宫素的含量，并研究《中华人民共和国药典》2015年版生物效价测定法与本文的HPLC测定法的相关性与等效性。方法：采用C₁₈（250 mm×4.6 mm，5 μm）色谱柱，以磷酸盐缓冲液（pH 6.0）为流动相A，水-乙腈（1：1）为流动相B，梯度洗脱，流速1.0 mL·min^-1，检测波长220 nm，柱温40℃。结果：在上述色谱条件下，赖氨酸升压素峰与缩宫素峰以及相邻杂质峰之间分离度符合要求；赖氨酸升压素与缩宫素浓度分别在0.42~13.33 IU·mL^-1和0.33~21.00 IU·mL^-1范围内线性关系良好，r=0.999 9；平均回收率（n=9）分别为100.5%和99.7%。6批样品中上述2个成分含量范围分别为6.10~6.40 IU·mL^-1和5.63~5.93 IU·mL^-1。用SPSS软件进行相关性分析，结果表明，生物效价法与HPLC法含量测定呈正相关，Pearson系数分别为0.897和0.947。SAS软件等效性分析表明：升压素生物效价法与HPLC法的测定结果具有等效性，而缩宫素的两法测定结果不具有等效性。结论：本法专属性良好，准确度高，重复性好，与生物效价测定法相关性较好，可用于垂体后叶注射液中赖氨酸升压素含量测定。

Objective:To establish an analysis method of HPLC for simultaneous determination of lysine vasopressin and oxytocin in the posterior pituitary injection, and to study the correlation and equivalence between the biological assay and HPLC method. Methods:A C₁₈ column(4.6 mm×250 mm, 5 μm) was adopted. Phosphate buffer solution(pH 6.0) was used as mobile phase A and the water-acetonitrile(1:1) was used as mobile phase B with gradient elution at a flow rate of 1.0 mL·min^-1. The detection wavelength was 220 nm and the column temperature was set at 40℃. Results:Under the chromatographic condition, resolution between the peaks of lysine vasopressin, oxytocin and other related substances met the requirements. This method showed good linearity between peak area and concentration of lysine vasopressin and oxytocin over the range of 0.42-13.33 IU·mL^-1 and 0.33-21.00 IU·mL^-1, respectively with r²=0.999 9. The average recoveries(n=9) of lysine vasopressin and oxytocin were 100.5% and 99.7%, respectively. The contents of lysine vasopressin and oxytocin in the 6 batches of samples were 6.10-6.40 IU·mL^-1 and 5.63-5.93 IU·mL^-1. The biological titer measurement method was positively correlated with the HPLC method, which Pearson coefficients were 0.897 and 0.947, respectively. SAS software equivalent analysis showed that biological titer measurement and HPLC method were equivalent for lysine vasopressin, but for oxytocin, the two methods were not equivalent. Conclusion:The method has excellent specificity, high accuracy and good reproducibility. It has a good correlation with biological assay, which can be used for the determination of lysine vasopressin in posterior pituitary injection.