Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography–tandem mass spectrometry: Application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory

We describe an ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3 min. The limit of quantitation for cortisol and cortisone in plasma was 3.75 nmol/L and linearity extended to 2000 nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6 nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5 nmol/L and for dexamethasone 1 nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC = 0.79 × IA + 31.12 with R2 = 0.960 (p < 0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC = 1.06 × HPLC + 9.82, R2 = 0.992 (p < 0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.