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A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of progesterone in human plasma was validated. The analyte was eluted in 1.9 minutes on a reversed phase column (Zorbax SB-C18, 100 mm x 3.0 mm I.D., 3.5 μm) under isocratic conditions using a mobile phase of a 20:80 (v/v) mixture of formic acid 0.1% (v/v) and methanol. The flow rate was 1 ml/min at the column temperature of 45 ºC. The detection of the analyte was in MS/MS mode using an atmospheric pressure chemical ionization source (APCI+, m/z 315.2 → m/z 279.2). The sample preparation was very simple and rapid and consisted in plasma protein precipitation from 0.2 ml plasma using 0.5 ml methanol. Calibration curves were generated over the range of 0.8-80 ng/ml with values for coefficient of determination greater than 0.995 and by using a weighted (1/y²) linear regression. The values of precision (coefficient of variation %) and accuracy (relative error %) were less than 9.4% and 14.2%, respectively, both for within- and between-run analysis. The mean recovery of the analyte was 98.6%. The developed LC/MS/MS method could be applied for determination of progesterone in human plasma for therapeutic drug monitoring in pregnancy and gynecological disorders.

Keywords: progesterone, human plasma, LC/MS/MS, method validation