Simultaneous determination of endogenous and stable isotope-labelled 6β-hydroxycortisols in human urine by stable isotope dilution mass spectrometry

This study describes a capillary GC–MS method for the simultaneous determination of endogenous 6β-hydroxycortisol (6β-OHF) and its stable isotope-labelled analogue, 6β-hydroxy-[1,1,19,19,19-2H5]cortisol (6β-OHF-2H5), in human urine. 6β-Hydroxy-[1,2,4,19-13C4,1,1,19,19,19-2H5]cortisol (cortisol-13C4,2H5) was used as an analytical internal standard. The methoxime trimethylsilyl ether (MO-TMS) derivatization was employed for the GC–MS analysis of 6β-OHF. Quantitation was carried out by selected-ion monitoring (SIM) of the characteristic fragment ion ([M−31]+·) of the MO-TMS derivative of 6β-OHF. The sensitivity limit of the present GC–MS-SIM method was found to be 25 pg per injection for 6β-OHF (S/N ratio=5.6). The within-day reproducibility in the amounts of unlabelled and labelled 6β-OHFs determined were in good agreement with the actual amounts added, the relative errors being less than 5.30%. The inter-assay RSDs were less than 4.95% for unlabelled and labelled 6β-OHFs.