Simultaneous determination of etonogestrel and ethinyl estradiol in human plasma by UPLC-MS/MS and its...

A selective, sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of etonogestrel (ENG) and ethinyl estradiol (EE) in human plasma. The analytes and their deuterated internal standards, ENG-d7 and EE-d4, were extracted from plasma samples by solid-phase extraction on HyperSep™ Retain PEP cartridges. The chromatographic analysis was performed on an Acquity UPLC HSS Cyano column, 100 Å (50 × 2.1 mm, 1.8 microm), column using gradient mobile phase, acetonitrile and 2.0 mm ammonium trifluoroacetate at 0-1.7 min (65:35, v/v) and 1.8-2.7 min (95:5, v/v) with 0.250 mL/min flow rate. Analytes and IS protonated precursor → product ion transitions (ENG, m/z 325.2 → 257.2; EE, m/z 530.2 → 171.2; ENG-d7, m/z 332.2 → 263.2; EE-d4, m/z 534.2 → 171.2) were monitored on a Triple Quadrupole Mass spectrometer (TQMS), operating in multiple reaction monitoring and positive ionization mode. The calibration curves were established at 10.00-2500 pg/mL for ENG and 1.500-150.0 pg/mL for EE with a correlation coefficient (r² ) ≥0.9996 for both. The validated method was successfully applied to support a bioequivalence study of 0.15 mg ENG and EE 0.03 mg tablet formulation, administered in 24 healthy Indian females. Method reliability was assessed by reanalysis of 94 incurred study samples.