高效液相色谱法测定HFBA原料药有关物质

Determination of the related substances in HFBA by HPLC

目的建立HFBA中有关物质的高效液相色谱测定法。方法色谱柱为Thermo BDS Hypersil-C18(150 mm×4.6 mm,5μm)柱;流动相的有机相为乙腈,水相为体积分数0.5%的冰醋酸水溶液,进行梯度洗脱,流速和柱温分别为1.0 mL·min-1和30℃,检测波长和进样量分别为257 nm和20μL。采用加校正因子的主成分自身对照法计算并进行方法学验证。结果杂质A、杂质B、杂质C的校正因子分别为1.30、1.33、1.61;杂质A、杂质B、杂质C及HFBA的定量限分别为0.1020、0.0988、0.1003和0.1004 mg·L-1,检测限分别为0.0306、0.0297、0.0301、0.0302 mg·L-1。HFBA及其杂质A、B、C在考察的质量浓度范围内均具有良好的线性关系(r≥0.999),样品低、中、高三个水平的加样回收率为93.2%~100.7%(RSD<2.0%,n=9)。结论该方法可用于HFBA中有关物质的测定。

Objective To develop an HPLC method for the determination of related substances in HFBA.Methods The analysis was performed on a Thermo BDS Hypersil-C18 column(150 mm×4.6 mm,5 μm)with 30 ℃ column temperature and the mobile phase was consisted of acetonitrile-0.5% glacial acetic acid solution with gradient elution.The flow rate was 1.0 mL·min-1.The detection wavelength was 257 nm and the injection volume was 20 μL.The self contrast and correction factor method was established to determine the related substances in HFBA and performed the methods validation.Results The relative correction factors for impurity A,impurity B,impurity C were 1.30,1.33 and 1.61.The limits of quantitation for impurity A,impurity B,impurity C and HFBA were 0.102 0,0.098 8,0.100 3 and 0.100 4 mg·L-1 respectively.And the limits of detection for impurities and HFBA were 0.030 6,0.029 7,0.030 1 and 0.030 2 mg·L-1 respectively.The calibration curves for three impurities and HFBA were good linear in the experimental concentration range(r≥0.999).The recovery ranged from 93.2% to 100.7% with RSD<2.0% and n=9.Conclusion This method is accurate,simple and specific for the determination of related substances presented in HFBA.