高效液相色谱加校正因子主成分对照法测定注射用缩宫素中有关物质的含量

Determination of related substances in oxytocin for injection by HPLC-principal component self-control with correction factor

目的:测定缩宫素原料药中7个杂质的有关物质含量并对其限度进行探讨。方法:采用高效液相色谱加校正因子的主成分自身对照法进行测定。采用Waters Xbridge C18(150 mm×4.6 mm,5μm)色谱柱,以0.1 mol·L^(-1)磷酸盐缓冲液(pH 5.4)-乙腈(90∶10)为流动相A,乙腈为流动相B,梯度洗脱,流速1.5 mL·min^(-1),柱温32℃,检测波长220 nm,进样量100μL。绘制缩宫素和杂质Ac-缩宫素、[Glu4]缩宫素、[+Gly10]缩宫素、缩宫素[-NH2]、[trisulfide]缩宫素、[cis-dimer]缩宫素和[trans-dimer]缩宫素的线性方程,以斜率计算7个杂质相对于缩宫素的校正因子,测定3批缩宫素原料药中各杂质含量,并与杂质对照品外标法测得的结果进行比较。结果:7个缩宫素杂质的定量限在2.75~5.66 ng,检测限在1.38~2.83 ng。各杂质质量浓度在0.03~3.40μg·mL^(-1)范围内,与相应的峰面积均呈良好线性(r>0.999)。相对于缩宫素,Ac-缩宫素、[Glu4]缩宫素和缩宫素[-NH2]的校正因子为1.1,[+Gly10]缩宫素的校正因子为1.2,[trisulfide]缩宫素的校正因子为0.9,[cis-dimer]缩宫素和[trans-dimer]缩宫素的校正因子为1.3。采用加校正因子的主成分自身对照法,测得3批缩宫素原料药中杂质Ac-缩宫素的含量分别为0.96%、0.93%和1.01%;杂质[Glu4]缩宫素的含量分别为0.07%、0.06%和0.08%;杂质[+Gly10]缩宫素的含量分别为0.07%、0.04%和0.04%;杂质缩宫素[-NH2]的含量分别为0.09%、0.05%和0.07%;杂质[trans-dimer]缩宫素的含量分别为0.27%、0.18%和0.22%。其他单个最大杂质含量为0.18%~0.19%,总杂质含量为1.88%~2.06%。比较采用加校正因子的主成分自身对照法和外标法测得的结果,2种方法的测定结果无统计学差异(p>0.05)。结论:本方法操作简单,可重复性高,能准确、有效地测定缩宫素中有关物质的含量。

Objective:To determine seven impurities in oxytocin for injection and investigate the limit values Methods:HPLC and principal component self-control with correction factor were adopted.The determination was performed on a Waters Xbridge C18 column(150 mm×4.6 mm,5μm).The mobile phase consisted of 0.1 mol·L^(-1)dihydrogen phosphate solution(adjusted to pH 5.4)-acetonitrile(90∶10,phase A),and acetonitrile(phase B)with gradient elution at a flow rate of 1.5 mL·min^(-1).The column temperature was maintained at 32℃,and the detection wavelength was set at 220 nm.The injection volume was 100μL.The linear equations of oxytocin,impurities Ac-Oxy,Oxy[Glu^(4)],Oxy[+Gly 10],Oxy[-NH2],Oxy[trisulfide],Oxy[cis-dimer]and Oxy[trans-dimer]were drawn.The correction factors of each impurity related to oxytocin were calculated by slope.The contents of impurities in 3 batches of oxytocin for injection were determined and compared with the results of impurity reference method.Results:The limits of quantification for seven impurities were 2.75-5.66 ng,while the detection limits were 1.38-2.83 ng.The linear ranges of seven impurities were 0.03-3.40μg·mL^(-1)with good linearity(r>0.999).The correction factors of Ac-Oxy,Oxy[Glu^(4)]and Oxy[-NH2]were 1.1,while the correction factors of Oxy[+Gly 10]and Oxy[trisulfide]were 1.2 and 0.9,respectively.The cor⁃rection factors of Oxy[cis-dimer]and Oxy[trans-dimer]were both 1.3.The seven impurities were determined in 3 batches of samples by principal component self-control with correction factor.The contents of impurity Ac-Oxy were 0.96%,0.93%and 1.01%,respectively.The contents of impurity Oxy[Glu^(4)]were 0.07%,0.06%and 0.08%,respectively.The contents of impurity Oxy[+Gly 10]were 0.07%,0.04%and 0.04%,respec⁃tively.The contents of impurity Oxy[-NH2]were 0.09%,0.05%and 0.07%,respectively.The contents of impurity Oxy[trans-dimer]were 0.27%,0.18%and 0.22%,respectively.The maximum single impurity con⁃tents were 0.18%-0.19%,while the total impurity contents were 1.88%-2.06%.Compared the results measured by principal component self-control with correct factor method and the impurity reference method,there was no significant difference between two methods(p>0.05).Conclusion:The method is proved to be simple,repeatable and accurate for the content determination of related substances in oxytocin for injection.