Validation of a LC-MS/MS method for establishing reference intervals and assessing diagnostic performance of urinary...

24-h urinary free cortisol (UFF) is recommended for screening of Cushing's syndrome (CS), a rare disease characterized by apparent cortisol and cortisone excess. We aimed to validate a simple LC-MS/MS method for accurate measurement of UFF and urinary free cortisone (UFE), establishment of reference ranges, and evaluation of performance for CS diagnosis.

Urine samples were processed using solid-phase extraction cartridges, followed by elution with methanol and acetonitrile. Analysis was performed via tandem mass spectrometry, utilizing multiple reaction monitoring and electrospray ionization source in positive ion mode.

The assay displayed excellent linearity (r > 0.99) in the range of 0.05-100 ng/mL for cortisol and 0.25-500 ng/mL for cortisone, with lower limits of quantification (LLOQ) at 0.05 ng/mL for cortisol and 0.25 ng/mL for cortisone. The obtained results for intra-day and inter-day imprecision for both analytes were within the acceptable range of less than 10 %. The trueness values for both compounds were also within the acceptable limit of 15 %. No significant matrix effects or carry over observed in our method. The reference intervals of UFF, UFE and UFF:UFE ratio were 7.01-45.66 µg/24-h, 27.97-139.21 µg/24-h and 0.17-0.56, respectively. UFF > 56.75 µg/24-h showed 100 % specificity and 100 % sensitivity for CS diagnosis, which was superior to UFF:UFE ratio.

We developed and validated a sensitive LC-MS/MS method to detect UFF and UFE. Our data indicate that UFF measured by the current LC-MS/MS assay exhibited high diagnostic performance for CS.