Profiling and quantification of isoflavone-C-glycosides impurities in puerarin injection by liquid chromatography coupled to ESI-ion trap mass spectrometry.

An HPLC/DAD/MS(n) method was established for the qualitative and quantitative analysis of the impurities in puerarin injection (PI), a widely used drug in China. The analytical HPLC was performed on an Agela RP-C18 column using 0.1% aqueous formic acid (v:v) and methanol as mobile phase. A total of nine impurities were detected and eight of them were identified as isoflavone-C-glycosides basing on their UV spectra and MS(n) spectra and comparing with the literature data. An HPLC method for the assay of two common impurities in the commercial PI samples, i.e., neopuerarin A and neopuerarin B, was then established. The validation of the method, including sensitivity, linearity, precision, accuracy, was carried out. The calibration curves showed good linearity of R(2)>0.9999 and LOQ (S/N=10) were less than 3.73 ng. The precision was evaluated by intra- and inter-day assays and R.S.D. values were less than 0.94%. The average recovery rates were 97.0% and 99.5%, respectively, with R.S.D. less than 1.38%. The contents of neopuerarin A and neopuerarin B in various commercial brands of PI samples varied over the range of 0.30-1.16% and 0.42-1.66%, respectively. This is the first report on the impurities in PI.