Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone

Background: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce. analytical interference, improve accuracy, and shorten the analysis, time, we developed a liquid chromatography-tandem mass' spectrometry (LC-MS/MS) method for urinary cortisol and, cortisone. Methods: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d(4) to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride,washed; and dried, and 10 muL of the reconstituted. extract was injected onto a reversed-phase C-18 column and analyzed using a tandem mass spectrometer operating in the positive mode. Results: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 mug/dL). Interassay CVs were 7.3-46% for mean concentrations of 6-726 nmol/L (0.2-26.3 mug/dL) for cortisol and cortisone. The, detection limit was 6 nmol/L (0.2 mug/dL). Recovery of cortisol and cortisone added to urine wag 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 mug HPLC cortisol/24 h (r(2)=0.992; n = 99) The regression equation for the (y) and immunoassay W methods for cortisol was: y = 0.66x - 12.1 mug cortisol/24 h (r(2) = 0.67; in 99). Conclusion: The sensitivity and specificity Of the LCMS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.(C) 2002 American Association for Clinical Chemistry.