An ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) with isotope dilution was developed for the analysis of progesterone in human serum.The serum samples were extracted by liquid-liquid extraction(LLE) with ethyl acetate and n-hexane

then separated on an Acquity UPLC BEH C18 column(2.1 mm×100 mm

1.7 μm) by gradient elution in 5 min

and finally analyzed by UPLC-MS/MS with electrospray ionization in positive ion mode under multiple reaction monitoring(MRM) mode and quantified by isotope internal standard.Impact of the two step LLE extraction on progesterone was investigated.Higher separation performance was observed when methanol-water containing 0.1% aqueous ammonia was used as mobile phase.The stability of serum extracts was also investigated.Results showed that the calibration curves for progesterone were linear in the concentration range of 10-10 000 pg/mL with correlation coefficients(r2) of 0.999 8．The limit of detection(LOD

S/N=3) and limit of quantitation(LOQ

S/N=10) were 5 pg/mL and 10 pg/mL

respectively.Recoveries for progesterone ranged from 91.5% to 106% with the intra relative standard deviations(RSD) and inter RSD of 1.2%-8.2% and 4.1%-9.1%

respectively.This method was applied in the determination of progesterone in serum samples from thirty individual donors with the progesterone concentrations ranging from 0.050 2 ng/mL to 1.363 5 ng/mL.With the characteristics of high sensitivity

good accuracy and reliability

this method could be used in the detection of progesterone in clinical serum samples.