A validated surrogate analyte LC-MS/MS assay for quantification of endogenous cortisol in human whole blood.

Cortisol is a steroid hormone that is frequently measured as a marker of stress, inflammation, and immune function. While commonly analyzed in saliva, hair, blood plasma and urine, a recent trend towards whole blood-based at-home collection devices has emerged, which necessitates development of more sensitive assays for cortisol in whole blood. To support the implementation of a patient-centric sampling approach in a drug development program, a fit-for-purpose surrogate analyte-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for cortisol in whole blood was developed using 13C3-cortisol as a surrogate analyte and cortisol-d6 as the internal standard. The surrogate analyte approach was chosen due to a lack of available cortisol-free whole blood and the absence of appropriately representative surrogate matrices. Samples were prepared using supported liquid extraction, and the LC-MS/MS analysis consisted of a 4.00 min analytical run. The method demonstrated linearity between 0.500 and 500 ng/mL of 13C3-cortisol, and accuracy, precision and robustness were all acceptable per current regulatory guidance for bioanalytical method validation of chromatographic assays for cortisol- and 13C3-cortisol-based quality control (QC) samples when quantified against a 13C3-cortisol calibration curve. The acceptable robustness of cortisol-based QCs when quantified against a 13C3-cortisol-based calibration curve also suggests parallelism between the analytes. These results indicate a viable surrogate analyte method, that is fit-for-purpose to analyze whole blood cortisol levels using a surrogate analyte LC-MS/MS approach. Evaluation of patient samples showed very promising comparability between whole blood and plasma cortisol concentrations, suggesting that whole blood could be used in place of or in addition to a plasma-based sampling protocol in clinical trials analyzing cortisol. Overall, this method presents a novel tool that is a first step in supporting the trend towards sample miniaturization and at-home sample collection, and may be readily used in clinical and diagnostic settings.