Measurement of urinary free cortisol by stable isotope dilution mass spectrometry using a new cortisol derivative

The reaction of the bismethylenedioxy derivative of cortisol (cortisol-BMD) with heptafluorobutyric anhydride to give the corresponding 3,5-dienol heptafluorobutyrate (cortisol-BMD-HFB) has been shown to proceed with dehydration. Acid-promoted dehydration of either cortisol-BMD or cortisol-BMD-HFB, or concurrent dehydration of both, is the proposed reaction mechanism leading to a trienol heptafluorobutyrate, whose chromatographic properties and mass spectral data are consistent with the additional double bond in the C9-C11 position. Forming the 3,5-dienol heptafluorobutyrate of cortisol-BMD in benzene rather than acetone gave a compound whose chromatographic properties and mass spectral data were different to that of the 3,5,9(11)-trienol heptafluorobutyrate but consistent with that of a cortisol-BMD-HFB. The mass fragmentometry of this new cortisol derivative was found to be more suited to the technique of isotope dilution mass spectrometry than the 3,5,9(11)-trienol heptafluorobutyrate, and thus was applied to our intended goal of measuring urinary free cortisol by gas chromatography-mass spectrometry. An efficient and convenient solid-phase extraction technique is employed in our assay to isolate cortisol from 5 ml of urine.