Determination of free and total cortisol in plasma and urine by liquid chromatography-tandem mass spectrometry

Cortisol is an important adrenal steroid hormone involved in the regulation of metabolic homeostasis. A new liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) multiple reactant monitoring (MRM) procedure for the measurement of cortisol concentration in plasma ultrafiltrate, whole plasma, and urine was developed and validated. Plasma, plasma ultrafiltrate, or urine was extracted by ethyl acetate. The extract was subjected to liquid chromatography with an Inertsil ODS-3 column with an aqueous NH4Cl (1 mM, pH 9.0):methanol mobile phase. The presence of NH4Cl in the mobile phase induced the formation of M+Cl] in the first quadrupole at m/z 397 and 409 for cortisol and 6alpha-methylprednisolone (internal standard), respectively. In the collision cell, the complex dissociated to the neutral parent and the chloride ion at m/z 35; the latter ion was used for quantification. The calibration curve was linear from 0.5 to 100 ng/mL. The lower limit of quantification was 0.50 ng/mL and the limit of detection was 0.25 ng/mL. For quality control samples prepared in water, the intrabatch assay precision was 5.6%, 9.6%, and 9.9% at 50, 10, and 1 ng/mL, respectively. The interbatch assay precision was 4.2%, 6.3%, and 7.5% at 50, 10, and 1 ng/mL, respectively. For measurement of endogenous cortisol in plasma and urine samples, the intra-assay and interassay precision was 10.8% and 4.8% for total plasma cortisol, 13.1% and 5.2% for free plasma cortisol, 10.9% and 13.1% for cortisol protein-binding free fraction, and 8.9% and 14.4% for urine cortisol, respectively. A simple procedure of ultrafiltration coupled with the highly sensitive LC-MS/MS quantification offered a rapid and reproducible assay for plasma free cortisol, which may be useful in the assessment of adrenal function in patients, especially critically ill patients with abnormal protein binding. It may also be useful for plasma and urinary cortisol measurements in pharmacodynamic studies of adrenocorticoid response.