Simultaneous analysis of cortisol and cortisone in saliva using XLC–MS/MS for fully automated online solid phase extraction

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2012-01, Vol.881 (15), p.42-48
• Main Authors: Jones, Rachel L., Owen, Laura J., Adaway, Joanne E., Keevil, Brian G.
• Format: Article
• Language: English
• Subjects: accuracy; Analysis; Analytical, structural and metabolic biochemistry; Assaying; Automated; Biological and medical sciences; Chromatography; Chromatography, Liquid - methods; Cortisol; Cortisone; Cortisone - analysis; Cortisone - chemistry; Drug Stability; Extraction; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; Hydrocortisone - analysis; Hydrocortisone - chemistry; immunoassays; liquid chromatography; Medical sciences; On-line systems; Online; Pharmacology. Drug treatments; quality control; Reproducibility of Results; Saliva; Saliva - chemistry; salivary glands; Sensitivity and Specificity; solid phase extraction; Solid Phase Extraction - methods; Spark Holland Symbiosis; steroids; tandem mass spectrometry; Tandem Mass Spectrometry - methods; XLC–MS/MS
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2011.11.036

► An XLC–MS/MS assay for salivary cortisol and cortisone has been validated. ► The Spark Holland Symbiosis™ provides fully automated online solid phase extraction. ► We demonstrated excellent precision, accuracy, linearity, sensitivity and specificity. ► For use in the investigation of disorders of the hypothalamic–pituitary–adrenal axis. Salivary cortisol measurements are increasingly being used in the investigation of disorders of the hypothalamic–pituitary–adrenal axis. In the salivary gland, cortisol is metabolised to cortisone by the action of 11β-hydroxysteroid dehydrogenase type 2, and cortisone is partly responsible for the variable interference observed in current salivary cortisol immunoassays. The aim of this study was to validate an assay for the simultaneous analysis of salivary cortisol and cortisone using the Spark Holland Symbiosis™ in eXtraction liquid chromatography–tandem mass spectrometry (XLC–MS/MS) mode for fully automated online solid phase extraction (SPE). Saliva samples were diluted in water with the addition of internal standard (d4-cortisol and d7-cortisone). Online SPE was performed using the Spark Holland Symbiosis™ with HySphere™ C18 SPE cartridges and compounds were eluted onto a Phenomenex ® C18 guard column attached to a Phenomenex ® Onyx monolithic C18 column for chromatography. Mass spectrometry used the Waters ® Xevo™ TQ MS in electrospray positive mode. Cortisol and cortisone eluted with their internal standards at 1.95 and 2.17 min, respectively, with a total run time of four minutes. No evidence of ion-suppression was observed. The assay was linear up to 3393 nmol/L for cortisol and 3676 nmol/L for cortisone, with lower limits of quantitation of 0.75 nmol/L and 0.50 nmol/L, respectively. Intra- and inter-assay imprecision was