Objective: To study the main non-steroidal impurities in premarin conjugated estrogens tablets. Methods: An HPLC method for the analysis of non-steroidal impurities in premarin conjugated estrogens tablets was established. The separation was performed on a Hedera ODS-2 C18 (250 mm×4.6 mm, 5 μm) column with a gradient elution of acetonitrile (A)-0.02 mol · L-1 ammonium formate in water (B) as the mobile phase at a wavelength of 215 nm, column temperature of 30 ℃and flow rate of 1.0 mL · min-1. The preliminary characterization of the main non-steroidal impurity components was further performed by LC-Q TOF MS. On the basis of the HPLC profiles of the non-steroidal components, the corresponding non-steroidal impurities were isolated and prepared from estrogen-binding raw material-pregnant horse urine, and the structures were identified by NMR. Results: Five major non-steroidal impurities were found and identified for the first time in premarin conjugated estrogens tablets. They were hippuric acid, N-(2-phenylacetyl) glycine, hndoxyl sulfate, 2, 6-dihydroxybenzoic acid, p-cresyl sulfate. Conclusion: Premarin conjugated estrogens tablets contain non-steroidal impurities, and the established HPLC method can effectively monitor the non-steroidal impurity components, which can provide a reference for the quality evaluation of natural binding estrogens and their preparations.
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