OBJECTIVE To establish a RP-HPLC method for determination of the contents of eplerenone and its related substances.METHODS A RP-HPLC method was developed by using a C18column (Agilent Poroshell, 4.6 mm × 100 mm, 2.7μm), the mobile phase consisted of H2O-CH3CN-MeOH (64:18:18), the flow rate was 1.0 mL·min-1, and the detection wavelength was set at 240 nm.The diluent was H2O-CH3CN-MeOH (50:25:25).RESULTS Eplerenone was well separated from the impurities.For the determination of related substances, the LODs of eplerenone, its intermediate (III and IV) and related substances (A-E) were 0.03, 0.13, 0.25, 0.05, 0.05, 0.26, 0.07 and 0.12 ng, and the LOQs were 0.12, 0.42, 0.84, 0.18, 0.18, 0.86, 0.22 and 0.41 ng, resp.The calibration curves of eplerenone and related substances (A-E) had good linear relationship within 0.13-1.53, 0.25-3.03, 0.25-2.99, 0.13-1.61, 0.14-1.63, and 0.13-1.54μg·mL-1 (r = 0.999 9, n = 7), and the correction factors of the related substances (A-E) were all in the range of 0.90-1.10.The average recoveries of the related substances (A-E) were all within 95.0%-105.0% (n = 9).For the content determination of eplerenone, the validation test showed good linearity over the range of 12.60-75.63μg·mL-1 (r = 0.999 6, n = 6) with good repeatability (RSD = 0.19%, n = 6).CONCLUSION The developed method proves to be simple, accurate, specific and reliable.It can be applied to the determination of eplerenone and its related substances.