A novel high-throughput assay for the measurement of salivary progesterone by liquid chromatography tandem mass spectrometry

Background: Liquid chromatography tandem mass spectrometry (LC-MS/MS) enables specific and sensitive quantification of steroids with a high-throughput. Saliva sampling is advantageous for multi-sample profiling over longer periods of time as it is non-invasive, cheap, can be carried out at home and does not require the attendance of clinical personnel. We developed a rapid LC-MS/MS for the measurement of salivary progesterone, frequently assessed as ovulation marker in patients desiring fertility.



Methods: Samples (300 µL) were prepared by supported liquid extraction using dichloromethane and were reconstituted in 40% methanol. Chromatography was performed using a C8 column with a water/methanol gradient containing 0.1% formic acid and 2mmol/L ammonium acetate. Quantification was performed with a Waters TQ-S mass spectrometer.



Results: Total run time was 5.5 min. The lower limit of quantification was 20 pmol/L (1.2 fmol on column). Inter- and intra-assay comparison showed coefficients of variation and bias between measured and nominal concentrations of less than 11%. Mean recovery was 91%. Interference with a large set of natural and synthetic steroids was excluded. The assay was successfully applied to measure progesterone variation during the menstrual cycle (n=9) and diurnal variations during luteal phase (n=7) in regularly cycling women.



Discussion: We present a novel LC-MS/MS assay for the determination of salivary progesterone with high-throughput potential. The applicability of the assay for progesterone profiling during the menstrual cycle is demonstrated.