苄星氯唑西林原料中二苄基乙二胺和氯唑西林含量HPLC测定方法的改进研究

Research on Improvement of HPLC Determination of Dibenzylethylenediamine and Cloxacillin in Cloxacillin Benzathine API

关键词:  高效液相色谱法  含量测定  抗生素  苄星氯唑西林

Key words:  high-performance liquid chromatography  determination  antibiotics  cloxacillin benzathine

摘要:

建立了高效液相色谱法同时测定苄星氯唑西林原料药中二苄基乙二胺（苄星）和氯唑西林含量的方法。采用十八烷基键合硅胶为填充剂的色谱柱，以0.1 mol/L磷酸二氢钠溶液（pH 4.6±0.2）-乙腈（75:25）为流动相，流速1.0 mL/min，进样体积20 μL，检测波长220 nm，柱温40℃。通过专属性、线性与范围、定量限、准确度、精密度、溶液稳定性和耐用性试验对分析方法进行了验证。结果显示，该方法专属性良好，二苄基乙二胺和氯唑西林峰线性关系良好、定量限分别为2.8 μg/mL和0.26 μg/mL、检测限分别为0.9 μg/mL和0.08 μg/mL、准确度高、精密度RSD分别为0.7%和0.6%、溶液在室温下48小时内稳定且耐用性良好。用该方法测定四批苄星氯唑西林原料药，并与现行版《中国兽药典》一部中苄星氯唑西林原料药含量测定方法进行对比，结果一致。本方法操作简便，可同时测定二苄基乙二胺和氯唑西林含量，提高了检测效率，为苄星氯唑西林质量标准改进提供了依据。

Abstract:

A high-performance liquid chromatography (HPLC) method was established for the simultaneous determination of dibenzylethylenediamine (benzathine) and cloxacillin in benzathine cloxacillin. A chromatographic column with C18 as the filler was used. The mobile phase was a mixture of 0.1 mol/L sodium dihydrogen phosphate solution (pH 4.6 ± 0.2) - acetonitrile (75:25), with a flow rate of 1.0 mL/min, an injection volume of 20 μL, a detection wavelength of 220 nm, and a column temperature of 40℃. The analytical method was validated through specificity, linearity and range, limit of quantitation, accuracy, precision, solution stability, and robustness tests. The results showed that the method had good specificity. The peaks of dibenzylethylenediamine and cloxacillin had good linear relationships. The limits of quantitation of dibenzylethylenediamine and cloxacillin were 2.8 μg/mL and 0.26 μg/mL, respectively. The limits of detection of dibenzylethylenediamine and cloxacillin were 0.9 μg/mL and 0.08 μg/mL, respectively. The method had high accuracy, and the relative standard deviations (RSDs) of precision were 0.7% and 0.6% respectively. The solution was stable within 48 hours at room temperature and had good robustness. This method was used to determine the contents in four batches of benzathine cloxacillin bulk drugs, and the results were consistent with those obtained by the method for the determination of the content of benzathine cloxacillin bulk drugs in the current edition of the Chinese Veterinary Pharmacopoeia. This method is simple to operate, can simultaneously determine the contents of dibenzylethylenediamine and cloxacillin, improves the detection efficiency, and provides a basis for the improvement of the quality standard of benzathine cloxacillin.