Determination of related substances in ketoprofen injection by RP-HPLC method.

The paper aims to establish a RP-HPLC method for the simultaneous determination of six related substances in ketoprofen injection. The separation was performed on a VP-ODS C18 column (4.6mm×250mm, 5μm) with the mobile phase of 6.8% phosphate buffer solution (adjusted to pH3.5 with 85% phosphoric acid)-acetonitrile-water (2:43:55,v/v/v) at a flow rate of 1.2mL•min-1. The detection wavelength and the injection volume were set at 233nm and 20μL, respectively. Impurity A and C were calculated by external standard method. Main component self-compare method with calibration factor was used to calculate impurity B, D, E, F and main component self-compare method without calibration factor was used to calculate unspecified impurity. Related substances and degraded substances were completely separated from ketoprofen. For impurity A and C, the linear range of determination were separately 0.06 μg•mL-1 ~ 3.6μg•mL-1 and 0.036μg•mL-1 ~ 2.4μg•mL-1 with the correlation coefficient of 0.9999. The average recoveries (n=9) were 98.13% (RSD=0.35%) and 96.32% (RSD=0.43%). The precision and repeatability for method were good. With reference to ketoprofen (retention time =10.06 min), the relative retention time of impurity B, D, E, F were 0.71, 1.46, 0.59, 2.13, respectively, and the relative correction factors were 0.962, 0.938, 0.957, 0.960, respectively. Finally, determined that the contents of impurity A could not be more than 0.3%, any of the contents of impurity B, C, D, E, F and unspecified impurities could not be more than 0.2%, sum of the contents of impurities other than A and C couldn't be more than 0.5%. The method was proved to be simple, rapid, accurate, sensitive and suitable for the simultaneous determination of six related substances in ketoprofen injection.