Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by µElution solid‐phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive µElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well µElution SPE plate using 70 µL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 × 50 mm, 3.5 µm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor → product ion transitions were monitored at m/z 365.2 [M+H]+ → 167.0 and at m/z 367.3 [M+H]+ → 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL−1 and the linear calibration range was from 0.1 to 100 ng mL−1 for both analytes. This method demonstrated to be very reproducible and reliable. Copyright © 2005 John Wiley & Sons, Ltd.