Surrogate matrix approach to quantify endogenous progesterone in a fasting bioequivalence study of soft gelatin capsules in postmenopausal women

Abstract

In the present study, endogenous progesterone in plasma was quantified using a validated LC–MS/MS method. This bioanalytical technique was successfully applied to a bioequivalence study in postmenopausal female volunteers. The assay achieved a lower limit of quantification (LLOQ) of 20 pg/mL for endogenous progesterone. Electrospray ionization in positive mode was coupled to a triple-quadrupole mass spectrometer, with deuterated progesterone (progesterone-D9) as the internal standard. Chromatographic separation was performed on a Kinetex Biphenyl column (100 × 4.6 mm, 5 μm) using a time- and flow-gradient program to ensure symmetrical peak shapes and complete resolution from potential interferences. Progesterone was extracted from plasma using methyl tert-butyl ether, affording high recovery and negligible matrix effects. Quantitation was carried out in multiple-reaction monitoring (MRM) mode, monitoring the transitions m/z 315.5 → 97.2 for progesterone and m/z 324.3 → 113.1 for progesterone-D9. The calibration curve was linear over the range of 20.0–40 000.0 pg/mL and was fitted by weighted (1/x²) linear regression. The method fully complies with current regulatory guidelines for bioanalytical assay validation.