非衍生化HPLC法测定硫酸阿米卡星及注射液的有关物质和含量

The derivatization HPLC determination of the related substances and content of amikacin sulfate and its injection

目的建立HPLC-UV法测定硫酸阿米卡星及注射液的有关物质和含量。方法采用Dikma Spursil C18(250mm×4.6mm,5μm)柱,有关物质测定采用梯度洗脱法,流动相A(取辛烷磺酸钠1.8g和无水硫酸钠20.0g,加pH3.0的0.2mol/L磷酸盐缓冲液50mL和乙腈50mL溶解,用水稀释至1000mL),流动相B(取辛烷磺酸钠1.8g和无水硫酸钠20.0g,加pH3.0的0.2mol/L磷酸盐缓冲液50mL和乙腈100mL溶解,用水稀释至1000mL),含量测定采用等度法,流动相为取辛烷磺酸钠1.8g和无水硫酸钠20.0g,加pH3.0的0.2mol/L磷酸盐缓冲液50mL和乙腈75mL溶解,用水稀释至1000mL,检测波长为200nm,柱温40℃。结果阿米卡星和各杂质完全分离。阿米卡星、杂质A(K29)、杂质E(K6)、杂质F(1,6'双取代杂质)、杂质H(阿米卡星B)在一定的浓度范围内呈较好的线性关系,硫酸阿米卡星注射液含量测定的回收率为100.0%,(RSD=0.5%,n=9),有关物质杂质E,杂质A,杂质H的回收率分别为97.5%、101.3%和104.0%。阿米卡星、杂质A(K29)、杂质E(K6)、杂质F(1,6'双取代杂质)、杂质H(阿米卡星B)的检测限分别为6.7、5.5、5.2、6.4和5.9μg/mL。结论方法简便、灵敏、重复性好,可用于本品的质量控制。

Objective To establish an HPLC-UV method for determining the related substances and the content of amikacin sulfate and its injection. Methods The column was Dikma Spursil C18 （250mm × 4.6mm, 5μm）. The mobile phase for the determination of related substances consisted of A （octane sulfonic acid sodium salt 1.8g, anhydrous sodium sulfate 20.0g, dissolved with 50mL phosphate buffer of pH3.0 0.2mol/L and 50mL acetonitrile, diluted with water to 1000mL） and B （octane sulfonic acid sodium salt 1.8g, anhydrous sodium sulfate 20.0g, dissolved with 50mL phosphate buffer of pH3.0 0.2mol/L and 100mL acetonitrile, diluted with water to 1000mL）. The mobile phase for the determination of the content of amikcin sulfate and its injection was octane sulfonic acid sodium salt 1.8g, anhydrous sodium sulfate 20.0g, dissolved with 50mL phosphate buffer of pH3.0, 0.2mol/L and 100mL acetonitrile, diluted with water to 1000mL. The detection wavelength was 200nm. The column temperature was 40℃. Results Good separation of amikacin from main intermediates could be achieved. The standard curve of amikacin, impurity A （K29）, impurity E（K6）, impurity F（1, 6＇ double replaced）, impurity H （amikacin B） was rectilinear in the certain range. The average recovery of content of amikacin sulfate injection was 100.0%（RSD=0.5%, n=9）. The limit of detection of amikacin, impurity A （K29）, impurity E （K6）, impurity F （1, 6＇ double replaced）, impurity H （amikacin B） was 6.7, 5.5, 5.2, 6.7 and 5.5g/mL respectively. Conclusion The method is simple, sensitive and reproducible, and can be used for quality control of amikacin sulfate.