加校正因子的主成分自身对照法同时测定咪喹莫特5个杂质

目的：建立加校正因子的主成分自身对照法同时测定咪喹莫特5个杂质。方法：采用高效液相色谱法。以XBridge C₁₈（4.6 mm×250 mm，5 μm）为色谱柱，以0.02 mol·L^-1磷酸氢二钾缓冲液（用磷酸调节pH至8.0）-乙腈（70：30）为流动相，检测波长238 nm，流速1.0 mL·min^-1，柱温25 ℃，进样量10 μL。通过测定咪喹莫特和5个杂质（杂质A、B、C、D、E）的线性方程，以斜率比计算各杂质的相对校正因子，用相对保留时间确定各杂质位置，并进行了方法学验证。用相对校正因子计算咪喹莫特原料药中杂质A、B、C、D、E的含量，并与杂质对照品外标法测定的结果进行比较，验证校正因子的准确性。结果：杂质A、B、C、D、E的相对保留时间分别为0.92、0.32、1.98、0.62、1.37，相对校正因子分别为0.86、1.60、0.95、0.95、2.32，检出限分别为0.02、0.02、0.05、0.04、0.11 ng，定量限分别为0.08、0.05、0.17、0.13、0.35 ng。采用外标法和加校正因子的主成分自身测定的结果基本一致（偏差在±10%之内），无显著性差异。结论：方法学验证结果表明建立的方法符合杂质定量的要求，本法能准确测定咪喹莫特原料药中杂质A、B、C、D、E的含量。

Objective: To establish an HPLC method with the correction factors for simultaneous quantification of five impurities A, B, C, D, E in imiquimod. Methods: The HPLC determination was performed on Waters XBridge C₁₈(4. 6 mm×250 mm, 5 μm) column with a mobile phase consisted of 0. 02 mol·L^-1 dipotassium hydrogen phosphate(adjusted pH 8. 0 with phosphoric acid)-acetonitrile(70:30). The detection wavelength was 238 nm and the flow was 1. 0 mL·min^-1. The column temperature was 25 ℃ and the injection volume was 10 μL. The linear equation of imiquimod and impurities A, B, C, D, E were determined, slope radio was used to calculate the correction factors of impurities to imiquimod. The relative retention time was used for identification of impurities. The results obtained by the proposed method were compared with those by using external standards of the impurities reference substance to verify the accuracy of correction factor. Results: The relative retention time of impurities A, B, C, D, E were 0. 92, 0. 32, 1. 98, 0. 62 and 1. 37, respectively, and the relative correction factor were 0. 86, 1. 60, 0. 95, 0. 95 and 2. 32, respectively compared with imiquimod. The limits of detection and quantification were found to be 0. 02, 0. 02, 0. 05, 0. 04, 0. 11 ng and 0. 08, 0. 05, 0. 17, 0. 13, 0. 35 ng for impurities A, B, C, D, E, respectively. There was no significant difference (The deviation is within ± 10%)between the results by relative correction factors and by external standards. Conclusion: The validation test showed that the proposed method met the requirements for the intended analytical applications. The method can determine the content of impurities A, B, C, D and E in imiquimod.