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 C-13(3)-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 C-13(3)-cortisol, and accuracy, precision and robustness were all acceptable per current regulatory guidance for bioanalytical method validation of chromatographic assays for cortisol- and C-13(3)-cortisol-based quality control (QC) samples when quantified against a C-13(3)-cortisol calibration curve. The acceptable robustness of cortisol-based QCs when quantified against a C-13(3)-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. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

A fit-for-purpose LC-MS/MS assay for cortisol in whole blood has been developed using surrogate analytes, showing good accuracy, linearity, and stability for use in clinical trials analyzing cortisol levels. Comparability between whole blood and plasma cortisol concentrations was demonstrated, supporting the trend towards sample miniaturization and at-home sample collection.