Member of the 1000 Post Club cat lady Posted June 27, 2008 Member of the 1000 Post Club Report Share Posted June 27, 2008 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: email@example.com 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. Link to comment Share on other sites More sharing options...
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