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

Abstract 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- 2 H 5 ]cortisol (6β-OHF- 2 H 5 ), in human urine. 6β-Hydroxy-[1,2,4,19- 13 C 4 ,1,1,19,19,19- 2 H 5 ]cortisol (cortisol- 13 C 4 , 2 H 5 ) 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.