反相高效液相色谱法同时测定复方盐酸四环素胶囊中盐酸四环素和甲硝唑的含量及有关物质

Simultaneous determination of tetracycline hydrochloride,metronidazole and their related substances in compound capsules by RP-HPLC

目的建立反相高效液相色谱法同时测定复方盐酸四环素胶囊中盐酸四环素和甲硝唑的含量及有关物质的方法。方法高效液相色谱法，Liehrospher C18色谱柱（250mm×4．6mm ID，5μm）；0．1mol·L^-1草酸铵溶液，DMF-0．2mol·L^-1的磷酸氢二铵（68：27：5）为流动相（用氨试液或磷酸调节pH到7．6—7．7）；流速为1mL·min^-1；柱温为35℃；检测波长为280nm。理论塔板数按甲硝唑色谱峰计算，应不低于2000，差向脱水四环素与四环素色谱峰的分离度应不低于1．2。结果用0．01mol·L^-1盐酸制得的供试液置冰水浴中避光保存条件下5h内基本稳定。盐酸四环素和甲硝唑浓度在0．005—1．50mg．mL。范围内峰面积呈现良好的线性关系，r=0．9998。在供试液中两组分浓度均为0．5mg·mL^-1条件下，测得盐酸四环素和甲硝唑平均回收率分别为99．6％和100．9％，精密度良好（RSD％〈1．0）。结论本法分离度高重现性好，可同时测定复方盐酸四环素胶囊中盐酸四环素和甲硝唑的含量及有关物质。

OBJECTIVE To establish a RP-HPLC method for the simultaneous determination of tetracycline hydrochloride,metronidazole and their related substances in tetracycline hydrochloride compound capsules（ each containing 125mg of tetracycline hydrochloride, 125mg of metronidazole and bismuth citrate）. METHODS The HPLC was carried out using a Lichrospher-C18 （250mm × 4.6mm ID, 5μm） analytical column at 35℃ ,a mixture of 680 volumes of 0.1 mol · L^-1 ammonium oxalate solution and 270 volumes of dimethylformamide and 50 volumes of 0.2 mol · L^-1 dibasic ammonium phosphate solution, adjusted with 3 mol · L^-1 ammonium hydroxide or 3 mol · L^-1 phosphoric acid to pH 7.6 to 7.7 as mobile phase at a flow rate of 1 mL · min^-1 ,with UV detection set at 280 nm. The separation was not valid unless the theoretical plates of metronidazole peak was greater than 2000, and the degree of resolution between the 4-epianhydrotetracycline and tetracycline peaks was not less than 1.2. RESULTS The assay solution was prepared with ice-cooled 0.01 mol · L^-1 hydrochloride and was stable for at least 5 hours under the shadowed ice-cooled condition. The linear responses range were from 0.005 to 1.50 mg· mL^-1 for both tetracycline hydrochloride and metronidazole（ r 〉0.999）. The average recoveries of tetracycline hydrochloride and metronidazole were 99.6% （ RSD1.1% ） and 100.9% （ RSD 1.1% ） with the assay concentrations at 0.5 mg mL^-1 for both components,respectively. CONCLUSION This method is accurate, and suitable for the simultaneous determination of tetracycline hydrochloride, metronidazole and its related substances in tetracycline hydrochloride compound capsules.