MSACL 2017 EU Abstract

Interference from Alpha- and Beta-Dihydrocortisone in Salivary Cortisol Assays: Ramifications for Patient Care

James Hawley (Presenter)
University Hospital South Manchester

Authorship: James M Hawley (1), Brian G Keevil (1,2)
(1) University Hospital South Manchester, Manchester, UK (2) Manchester Academic Health Science Centre, Manchester, UK.

Short Abstract

We present a novel LC-MS/MS method for the measurement of salivary cortisol and cortisone using protein precipitation for sample preparation. Recent literature has suggested that two isobaric cortisol metabolites, alpha-dihydrocortisone and beta-dihydrocortisone, are difficult to separate chromatographically. Here, we utilise biphenyl chromatography to separate these metabolites from cortisol prior to LC-MS/MS analysis. We show that failure to separate these metabolites from cortisol may result in unnecessary over-investigation of patients. The method is well validated and with an injection-to-injection run time of only 2.5 minutes integrates well into the workflow of a busy, high-throughput clinical laboratory.

Long Abstract


Salivary cortisol measurement is advocated as a screening test in the differential diagnosis of hypercortisolism. Recent literature has suggested that LC-MS/MS salivary cortisol quantitation may be overestimated if the method fails to separate two isobaric metabolites of cortisol: alpha- and beta-dihydrocortisone. Here, we present a novel and well-validated LC-MS/MS method for the measurement of cortisol and cortisone in saliva that is robust and achieves separation of cortisol from these two endogenous metabolites in only 2.5 minutes.


Standards, QCs and salivary samples (50 µL) were precipitated using 0.1 M zinc sulphate and methanolic internal standard. The prepared samples (10 µL) were injected onto a Waters in-line filter connected in series to a Restek Biphenyl 2.7 µm, 30 x 2.1 mm analytical column. Mobile phases consisted of (A) 0.3 mmol/L ammonium fluoride and (B) acetonitrile. Analysis was performed using a Waters Micro TQS® in the positive ionisation mode. Multiple reaction monitoring was used to detect cortisol, cortisone and their respective deuterated internal standards. Routine samples (n=116) were analysed and the alpha- and beta-dihydrocortisone metabolites quantified and added to the final cortisol result to allow assessment of their potential effect to result interpretation.


For both cortisol and cortisone the lower limit of quantitation was determined to be 0.3 nmol/L with recoveries ranging from 99-106% (cortisol) and 92-106% (cortisone), matrix effects were negligible (<15%) for each compound. Intra- and inter-assay imprecision was <5% for each analyte over concentrations of 5.0, 50.0 and 150.0 nmol/L. Linearity-on-dilution was preserved (R2 >0.99) and inter-injection carryover was <0.1%. Analysis of structural analogues of cortisol and ion suppression studies confirmed no interferences. Applying a salivary cortisol cut-off of <2.6 nmol/L, analysis of 116 routine samples identified 54% (n=63) of samples were above this cut-off, when the alpha- and beta-dihydrocortisone metabolites were also accounted for this increased to 77% (n=89) of samples.

Conclusions & Discussion

We have developed and validated a novel and robust LC-MS/MS assay for the quantification of salivary cortisol and cortisone. The method is free from both endogenous and exogenous interferences. Failure to chromatographically separate the alpha- and beta-dihydrocortisone metabolites may have direct ramifications on patient management as over-estimation of the cortisol result may trigger unnecessary and expensive further investigations. Crucially, our data may suggest that salivary cortisol cut-offs may require re-evaluating.

References & Acknowledgements:

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