Machacek, Dwaine

Abstract 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-d4 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 μL of the reconstituted extract was injected onto a reversed-phase C18 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 μg/dL). Interassay CVs were 7.3–16% for mean concentrations of 6–726 nmol/L (0.2–26.3 μg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 μg/dL). Recovery of cortisol and cortisone added to urine was 97–123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 μg cortisol/24 h (r2 = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x − 12.1 μg cortisol/24 h (r2 = 0.67; n = 99). Conclusion: The sensitivity and specificity of the LC-MS/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.

Abstract We compared results obtained by (a) chemical (spectrophotometric) and competitive protein-binding assays for serum corticosteroids, and (b) competitive proteinbinding and radioimmunoassay, with and without CCl4 extraction, for serum cortisol. The two corticosteroid methods gave identical results. Radioimmunoassay with and without a CCl4 extraction step gave identical results for cortisol. Competitive protein-binding assays for cortisol (with and without CCl4 extraction) and radioimmunoassay for cortisol gave similar results. Specimens with above-normal corticosterone or 11-deoxycortisol concentrations are an exception: only methods that included CCl4 extraction gave correct values for cortisol; methods without such an extraction step overestimated cortisol. The normal serum corticosteroid concentration (mean ± SD) was 175 ± 53 µg/liter for morning specimens, and 103 ± 40 µg/liter for specimens collected in the afternoon. For cortisol, the corresponding values were 162 ± 44 and 82 ± 29 µg/liter.