目的: 建立高效液相色谱法测定盐酸左旋咪唑片的有关物质及含量。方法: 有关物质检测采用Thermo Hypsil C18(100 mm×4.6 mm,3 μm)色谱柱,以0.75%磷酸二氢铵溶液(用二异丙胺调节pH至7.0)-乙腈为流动相,梯度洗脱;含量测定采用InertSustain C18(150 mm×4.6 mm,5 μm)色谱柱,以0.75%磷酸二氢铵溶液(用二异丙胺调节pH至7.0)-乙腈(70∶30)为流动相。流速均为1.0 mL·min-1,检测波长均为215 nm,进样体积均为10 μL。结果: 建立的有关物质检测方法能较好地分离盐酸左旋咪唑与5个已知杂质;已知杂质A、B、C、D、E质量浓度在10.87~25.37、11.62~27.10、12.38~28.90、30.89~72.07、12.41~28.95 μg·mL-1的范围内与峰面积呈良好的线性关系(r＞0.999),平均校正因子分别为1.6、1.4、2.6、1.2、2.4,平均回收率(n=9)分别为98.1%、99.0%、98.6%、100.1%和99.9%,RSD分别为0.63%、0.79%、0.92%、0.96%、0.33%。建立的含量测定方法能分离盐酸左旋咪唑与相邻杂质C;盐酸左旋咪唑质量浓度在0.101 4~0.304 3 mg·mL-1的范围内与峰面积呈良好的线性关系(r=0.999 9);盐酸左旋咪唑的平均回收率(n=9)为100.2%,RSD=0.52%;盐酸左旋咪唑的定量限与检测限分别为0.076 ng和0.030 ng。采用建立的方法实际测定5家企业生产的10批样品,总杂质量为0.05%~0.62%,含量为99.5%~104.3%。结论: 研究建立的盐酸左旋咪唑片的有关物质和含量测定的方法专属性强,结果准确度高,可用于盐酸左旋咪唑片中杂质的控制研究以及质量标准的提高。
Objective: To develop an HPLC method for the determination of related substances and assay of levamisole hydrochloride tablets. Methods: The chromatographic separation of related substances was performed on a Thermo Hypsil C18 column(100 mm×4.6 mm, 3 μm). A gradient elution was applied with a mobile phase composed of 0.75% monobasic ammonium phosphate solution(adjusted at pH 6.5 with diiopropylamine) and acetonitrile. The assay analytical column was packed with IntertSustain C18 column(150 mm×4.6 mm, 5 μm). The mobile phase was 70 volumes of 0.75% monobasic ammonium phosphate solution(adjusted at pH 7.0 with diiopropylamine) with 30 volumes of acetonitrile. The wavelength detection was set at 215 nm, the injection volumn was 10 μL, and the flow rate was 1.0 mL·min-1. Results: Levamisole and its impurities were separated well by related substances HPLC method above. Impurities A, B, C, D, E showed the good linearities in the concentration ranges of 10.87-25.37 μg·mL-1, 11.62-27.10 μg·mL-1, 12.38-28.90 μg·mL-1, 30.89-72.07 μg·mL-1, 12.41-28.95 μg·mL-1 (r＞0.999). The average correction factors of impurities A, B, C, D, E determined by three columns and liquid chromatographies were 1.6, 1.4, 2.6, 1.2, 2.4, respectively, the recovery rates(n=9) were 98.1%, 99.0%, 98.6%, 100.1%, 99.9% and the RSDs were 0.63%, 0.79%, 0.92%, 0.96%, 0.33%. The separation of levamisole and impurity C was good by assay HPLC method. Levamisole showed the good linearity in the concentration range of 0.10 14-0.304 3 mg·mL-1 (r=0.999 9). The average recovery rate(n=9) of levamisole was 100.2%, RSD=0.52%. Ten batches levamisole hydrochloride tablets from five domestic pharmaceutical enterprises were determined by the above related substances and assay HPLC method, the total impurities mass were 0.05%-0.62%, and the assays were 99.5%-104.3%. Conclusion: The established method is high selection and accurate. It is suitable applied for determination of related substances and assay of levamiole hydrochloride tablets.
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