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

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF, 6β,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione), 6α-hydroxycortisol (6α-OHF, 6α,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione) and 6β-hydroxycortisone (6β-OHE, 6β,17α,21-trihydroxypregn-4-ene-3,11,20-trione) in human urine is described. Deuterium-labelled compounds, 6β-[1,1,19,19,19-2H5]OHF (6β-OHF-d5), 6α-[1,1,19,19,19-2H5]OHF (6α-OHF-d5) and 6β-[1,1,19,19,19-2H5]OHE (6β-OHE-d5) were used as internal standards. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-31]+) of the methoxime–trimethylsilyl (MO–TMS) derivatives of 6β-OHF, 6α-OHF and 6β-OHE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring 6β-OHF, 6α-OHF and 6β-OHE in human urine.