Determination of Steroid Hormones in Oral Contraceptives by High-Performance Liquid Chromatography

ABSTRACT

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 μm, 125×4 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°C).

Calibration curves for ETE–LEVO were obtained using solutions with concentration ranges from 2.40 to 60.0 μg/mL (ETE), and from 12.0 to 300.0 μg/mL (LEVO). Calibration curves for ETE–GEST were obtained using solutions with concentration ranges from 2.40 to 60.0 μg/mL (ETE), and from 9.0 to 160.0 μg/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.

• Steroid hormones
• Oral contraceptives
• High-performance liquid chromatography
• Pharmaceutical preparations