Determination of steroid hormones in oral contraceptives by high-performance liquid chromatography

The aim of this research was to standardize a high-performance liquid chromatographic method for quantitative determination of steroid hormones, like ethinylestradiol (ETE), levonorgestrel (LEVO), and gestodene (GEST), in commercially available oral contraceptives (OCs). The combination ETE-LEVO was analyzed using a LiChrospher 100 RP-8 column (5 microns, 125 x 4 mm) in LiChroCART, with a mobile phase constituted of acetonitrile: water (60:40 v/v). Using the same column, ETE-GEST was analyzed with a mobile phase constituted of acetonitrile:water (50:50 v/v) at pH 7.5 adjusted with 0.02 M ammonium hydroxide. For both methods, a flow rate of 0.8 mL/min was utilized and detection was carried out at 215 nm. All analyses were performed at room temperature (24 +/- 2 degrees C). Calibration curves for ETE-LEVO were obtained using solutions with concentration ranges from 2.40 to 60.0 micrograms/mL (ETE), and from 12.0 to 300.0 micrograms/mL (LEVO). Calibration curves for ETE-GEST were obtained using solutions with concentration ranges from 2.40 to 60.0 micrograms/mL (ETE), and from 9.0 to 160.0 micrograms/mL (GEST). Correlation coefficients obtained were from 0.9999 to 0.9990. Coefficients of variation for samples containing ETE-LEVO were 0.47% and 0.38%, respectively. For samples with ETE-GEST they were 0.39% and 0.44%, respectively. The average recovery for samples with ETE-LEVO was 103.46% and 100.78%, respectively. For samples containing ETE-GEST it was 100.89% and 101.03%, respectively.