Cortisol quantification in human plasma and urine by liquid chromatography coupled to mass spectrometry: Validation, analysis and application in a reference population and patients with adrenal incidentalomas

Cortisol is a glucocorticoid hormone, which is involved in cardiovascular, metabolic, inflammatory and behavioral processes in the human body. Immunoassays, used for routine analysis of analytes, usually lead to erroneous quantification due to the low specificity of these methods. In this study, we developed LC-MS/MS methods with surrogating matrices for evaluating cortisol in both urine and plasma samples and validated them according to Brazilian Health Regulatory Agency guidelines. Urine samples were prepared with an enzymatic hydrolysis stage, followed by a solid-phase extraction (C18 cartridges) using dichloromethane:methanol 9:1 as elution solvent. Plasma samples were prepared by protein precipitation with acetonitrile, using 50 μL of sample. HPLC was performed using a C8 column under 300 mL min⁻¹ flow gradient conditions with water and methanol, both containing 5 mM ammonium formate and 0.1 % formic acid. Mass spectrometer with electrospray ionization in positive mode and selected reaction monitoring as detection technique were employed. Calibration curves were linear over a concentration range of 1–200 ng mL-1 for urine (r² = 0.9950) and 0.5–300 ng mL-1 for plasma (r² = 0.9970). The methods were selective, showed suitable precision, accuracy, and sensibility (limit of quantification = 0.85 ng mL⁻¹ for urine and 0.15 ng mL⁻¹ for plasma). Validated methods were successful applied to 22 real samples and a cohort of patients [n = 63 urines and n = 79 plasmas (from the Clementino Fraga Filho University Hospital, Rio de Janeiro, Brazil)].