High-performance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometry for the detection of 17alpha-ethinylestradiol in hepatocytes

Atmospheric pressure photoionization (APPI) as an interface for the high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) system was employed for the direct determination of 17alpha-ethinylestradiol (EE2) in the incubation mixtures to support in vitro hepatic clearance studies. For the APPI source, the radical cation of the analyte via charge exchange with the dopant radical cation was used for the detection of EE2 in the positive ion mode. It was demonstrated that the major signals of EE2 in the acetonitrile/water mobile phase were substantially increased by replacing toluene with anisole as the dopant. The effects of several experimental conditions on the photoionization efficiency of EE2 in the dopant-assisted APPI source were explored. Electrospray ionization (ESI) source was also suitable for the analysis of the analyte; however, ESI required a derivatization step prior to analysis. The applicability of the proposed HPLC-APPI-MS/MS approach following a protein precipitation procedure for the determination of EE2 at low nano-mole levels was examined with respect to assay specificity and linearity. The assay results obtained by both HPLC-APPI-MS/MS and HPLC-ESI-MS/MS methods were in good agreement.