Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays

Ann Clin Biochem 2008;45:380-388
© 2008 Association for Clinical Biochemistry

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Original Articles

Lisa Wood1,
David H Ducroq2,
Helen L Fraser2,
Scott Gillingwater3,
Carol Evans1,
Alan J Pickett1,
Derek W Rees1,
Rhys John1 and
Atilla Turkes1

1 Department of Medical Biochemistry and Immunology, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK;
2 WEQAS, Reference Laboratory, The Quadrant Centre, Cardiff Business Park, Llanishen, Cardiff CF14 5WF, UK;
3 Waters Corporation, MS Technologies Centre, Atlas Park, Simonsway, Manchester M22 5PP, UK

Corresponding author: Dr A Turkes. Email: atilla.turkes{at}

Background: Determination of urinary free cortisol (UFC) is an importantadjunct for the assessment of adrenal function. In this study,we have analysed cortisol concentrations in urine samples bygas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandemmass spectrometry (LC-MS/MS) and two immunoassays. The resultswere compared with GC-MS. The interference of cortisol ring-Ametabolites 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 tothe 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 immunoassayscontribute to overestimation of UFC concentrations. The LC-MS/MSprocedure had the sensitivity, specificity, linearity, precisionand accuracy for the determination of UFC concentrations. Themethod is suitable for routine use provided that method-dependantreference values are established.

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