Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays.
BACKGROUND
Determination of urinary free cortisol (UFC) is an important adjunct for the assessment of adrenal function. In this study, we have analysed cortisol concentrations in urine samples by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and two immunoassays. The results were compared with GC-MS. The interference of cortisol ring-A metabolites in immunoassays was also assessed.


METHODS
The GC-MS technique involved solvent extraction, LH-20 clean-up and derivatization. Only solid-phase extraction procedure was used for LC-MS/MS. The samples were analysed in positive electro-spray ionization mode, monitoring the transitions for cortisol and deuterated-cortisol at m/z 363.3 > 121.2 and m/z 365.3 > 122.2, respectively. Immunoassays were performed according to the manufacturer's instructions.


RESULTS
When compared with GC-MS results both immunoassays (Coat-A-Count; approximately 1.9-fold, Centaur; approximately 1.6-fold) overestimated UFC concentrations. Cortisol ring-A dihydro- and tetrahydrometabolites contribute significantly to this overestimation. There was no interference by these metabolites in either GC-MS or LC-MS/MS methods. The sensitivity of the LC-MS/MS procedure was 2 nmol/L and the intra- and inter-assay variations were <5% in each quality-control sample. The comparison of the UFC results achieved by assaying the study samples with GC-MS and LC-MS/MS indicated that the agreement between the two methods was excellent (LC-MS/MS = 1.0036GC-MS - 0.0841; r2 = 0.9937).


CONCLUSIONS
The interference of cortisol ring-A metabolites in immunoassays contribute to overestimation of UFC concentrations. The LC-MS/MS procedure had the sensitivity, specificity, linearity, precision and accuracy for the determination of UFC concentrations. The method is suitable for routine use provided that method-dependant reference values are established.
Wood
Lisa
L
Department of Medical Biochemistry and Immunology, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK.
Ducroq
David H
DH
Fraser
Helen L
HL
Gillingwater
Scott
S
Evans
Carol
C
Pickett
Alan J
AJ
Rees
Derek W
DW
John
Rhys
R
Turkes
Atilla
A
D002853:Chromatography, Liquid; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis* / Q000378:metabolism; D007118:Immunoassay; D008297:Male; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D014556:Urine
D006854:Hydrocortisone