Measurement of urinary free cortisol by tandem mass spectrometry & comparison with results obtained by gas chromatography-mass spectrometry

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

 

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@cardiffandvale.wales.nhs.uk

 

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.