HPLC双波长法测定米力农注射液有关物质

Determination of Related Substances in Milrinone Injection by Dual-wavelength HPLC

目的建立高效液相色谱法(HPLC)测定米力农注射液有关物质,有效控制不同生产企业的产品质量。方法采用Shiseido C18(250 mm×4.6 mm,5μm)色谱柱,柱温30℃,流动相为水-甲醇-硼酸钠缓冲液(725:250:25),流速1.0 ml/min,检测波长为284 nm(5-羟甲基糠醛)和254 nm(米力农杂质I、米力农杂质Ⅱ、其他杂质)。结果米力农与各杂质峰均能有效分离,米力农、5-羟甲基糠醛、米力农杂质I、米力农杂质Ⅱ分别在0.2~5μg/ml(r=0.9999)、2~50μg/ml(r=0.9996)、0.2~5μg/ml(r=1.0000)、0.2~5μg/ml(r=0.9999)浓度范围内线性关系良好,5-羟甲基糠醛、米力农杂质I、米力农杂质Ⅱ的回收率分别为99.9%(RSD=0.49%,n=9)、102.0%(RSD=0.48%,n=9)和95.8%(RSD=1.47%,n=9);定量限分别为5 ng/ml,1ng/ml,0.025μg/ml,0.05μg/ml。结论建立的方法灵敏准确,可有效地对米力农注射液的有关物质进行定量测定。

Objective To establish an HPLC method for the determination of related substances to control the quality of Milrinone Injection from different manufacturers. Methods A Shiseido C18 column(4.6 mm×250 mm, 5 μm) was adopted and the column temperature was set at 30 ℃. The mixture of water-methanol-sodium borate buffer(725:250:25) was used as the mobile phase and the flow rate was 1.0 ml/min. The detection wavelengths were 284 nm for 5-hydroxymethyl furfural and 254 nm for milrinone impurity Ι, milrinone impurity Ⅱ and other impurities. Results Milrinone and its related substances were separated well. The calibration curves were linear within the concentration ranges of 0.2-5 μg/ml(r=0.9999) for milrinone, 2-50 μg/ml(r=0.9996) for 5-hydroxymethyl furfural, 0.2-5 μg/ml(r=1.0000) for milrinone impurity Ι and 0.2-5 μg/ml(r=0.9999) for milrinone impurity Ⅱ, respectively. The average recoveries for 5-hydroxymethyl furfural, milrinone impurity Ι and milrinone impurity Ⅱ were 99.9 %(RSD=0.49 %, n=9), 102.0 %(RSD=0.48 %, n=9) and 95.8 %(RSD=1.47 %, n=9), respectively. The limit of quantification(LOQ) of 5-hydroxymethyl furfural, milrinone, milrinone impurity Ι and milrinone impurity Ⅱ were 5 ng/ml, 1 ng/ml, 0.025 μg/ml and 0.05 μg/ml, respectively. Conclusion The method is sensitive and accurate, and can be used to detect the related substances in Milrinone Injection.