Validation parameters, including accuracy (expressed

as bias), precision (percentage coefficient of variation), recovery, specificity, dilution, and stability were evaluated and amply met the acceptance criteria outlined in the FDA guidance [15]. The method for the determination of prucalopride in human heparin plasma was linear in the range of 0.200–100 ng/mL, with a lower limit of quantification (LLQ) of 0.200 ng/mL. Briefly, prucalopride was extracted from 50 μL plasma by liquid–liquid extraction with tertiary butyl methyl ether under alkaline conditions, using an analog (SSP-002392) as the internal standard. High-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) analysis was carried out with an API-4000 mass spectrometer Selleckchem ACY-738 (AB Sciex, Toronto, ON, Canada) coupled with an MK-8931 clinical trial Agilent 1100 HPLC system (Agilent, Santa Clara, CA, USA). The mass spectrometer was operating in positive electrospray ionization (ESI) mode, and the chromatographic separation was achieved on a Zorbax Extend-C18 3.5 μm HPLC column, 4.6 × 75 mm, with a mobile-phase gradient. For ethinylestradiol, the method was linear in the range of 3.00–600 pg/mL,

with an LLQ of 3.00 pg/mL, using 500 μL of plasma. Ethinylestradiol and its deuterated internal standard (ethinylestradiol-d4) were extracted from plasma by solid-phase extraction on Isolute C18 (EC) cartridges (Biotage, Uppsala, Sweden). Subsequently, ethinylestradiol was derivatized with dansyl chloride and the derivate was extracted using liquid–liquid extraction with a mixture of tertiary butyl methyl buy 4SC-202 ether and pentane. BCKDHA HPLC–MS/MS analysis was performed using the API-4000 mass spectrometer coupled with the Agilent 1100 HPLC system. The mass spectrometer was operating in positive atmospheric

pressure chemical ionization (APCI) mode, and the chromatographic separation was achieved on a Hypersil C8 BDS HPLC column (3.0 μm, 4.6 × 150 mm), with a mobile-phase gradient. For norethisterone, the method was linear in the range of 0.0500–20.0 ng/mL, with an LLQ of 0.0500 ng/mL, using 500 μL of plasma. In summary, norethisterone and its stable isotope-labeled internal standard (13C2-norethisterone) were extracted from plasma by online solid-phase extraction on HySphere C8 EC-SE cartridges, using a Symbiosis Pharma system (Spark Holland BV, Emmen, The Netherlands), which was preceded by liquid–liquid extraction with a mixture of chloroform and pentane. Chromatographic separation was achieved on a Zorbax XDB-C8 HPLC column (3.5 μm, 75 × 4.6 mm), with a mobile-phase gradient. The API-4000 mass spectrometer was operating in positive APCI mode. In the current study, each analytical run consisted of a freshly prepared calibration curve, using blank human heparin plasma for all three analytes. Quality control (QC) samples were prepared at three different concentrations (prucalopride: 0.600, 6.00, and 80.0 ng/mL; ethinylestradiol: 9.00, 50.