Simultaneous determination of gestodene, etonogestrel and ethinylestradiol in plasma by LC-MS/MS following derivatization

To establish a sensitive and specific method for simultaneous determination of gestodene,  etonogestrel and ethinylestradiol in plasma by LC-MS/MS, plasma samples were extracted and derivatized before injection.  An ESI ion source was used and operated in the positive ion mode with multiple reaction monitoring (MRM).  Norgestrel was chosen as internal standard and performed on a C₁₈ (100 mm × 2.1 mm, 5 μm) column.  The concentrations of gestodene, etonogestrel and ethinylestradiol were measured, using step-gradient mobile phase and step-gradient flow rate.  The method was validated over the concentration range of 0.1−20 ng·mL⁻¹ for gestodene and etonogestrel and 0.01−2 ng·mL⁻¹ for ethinylestradiol, and showed excellent linearity.  The  intra- and inter-assay accuracy and precision were below 10.0% and recovery was 93.6%−110.9% over the three concentration levels evaluated.  The method was applied in pharmacokinetic study of the compound gestodene patch and the compound etonogestrel patch in rabbits.  The LC-MS/MS method was selective, accurate and  sensitive, especially the LOQ were 100 pg·mL⁻¹ for gestodene and etonogestrel and 10 pg·mL⁻¹ for ethinylestradiol.  The method was successfully applied in pharmacokinetic study for contraceptives.