Background: We report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of four commonly prescribed steroid drugs (prednisolone, dexamethasone, betamethasone and beclomethasone dipropionate) while simultaneously measuring 24-h urine free cortisol and cortisone in patients.
Methods: Two hundred and fifty microlitre aliquots of urine were spiked with internal standard and extracted with dichloromethane. The MS instrument was operated with positive electrospray and multiple reaction monitoring. Two transitions were monitored for each analyte of interest and the ratio of the intensities of the product ion fragments was established.
Results: The LC-MS/MS method for the measurement of urine free cortisol and cortisone was established. The assay was linear up to 788 nmol/L for cortisol and 777 nmol/L for cortisone, with a limit of quantitation of 5.0 nmol/L for both. Analysis time per sample was seven minutes. Transitions for four synthetic glucocorticoids were included, and they were identified based on the ratio of the intensities of product ion fragments. Analysis of 219 samples collected from 154 patients (55 male and 99 female) revealed the presence of prednisolone in five samples from three patients. Dexamethasone was detected in samples from four patients, and betamethasone was detected in one sample.
Conclusion: This is the first LC-MS/MS method in routine use to combine quantification of urinary cortisol and cortisone and detection of synthetic glucocorticoids in patients being investigated for Cushing's syndrome. Since the most common quoted cause of Cushing's syndrome is steroid treatment, this is a valuable diagnostic tool.