Abstract
High-Performance Liquid Chromatography-Tandem Mass Spectrometry has emerged triumphant over the years as a reliable and high throughput clinical instrument to assess different metabolic irregularities. One of the latest applications of LC-MS/MS has been in the field of quantification of glucocorticoids, such as cortisol and cortisone from saliva, that can be an indicator of abnormalities such as Congenital Adrenal Hyperplasia (CAH), Cushing's Syndrome, and Addison's disease. We have developed and validated an LC-MS/MS-based assay for simultaneous detection of cortisol and cortisone in human saliva, which requires only 20 µL of sample, to measure cortisol across a 0.5 - 70 ng/mL range, and cortisone across a 1.2- 100 ng/mL range, respectively. The developed method exhibits linearity of R2>0.99, for both analytes, inclusive of both MRMs, and a percent coefficient of variation that is less than or equal to 20%. Using dilute-and-shoot for sample preparation, we have exhibited sample accuracy of 100±20 for the assay calibrators, and integrated Needle Wash Solvent Chemistry for minimal sample carryover, making it adaptable for crucial for potential diagnostic use. We have exhibited a method that is simplistic, specific, and highly automatable on liquid handling platforms, such as the JANUS® G3 Workstation. With our innovation, we have introduced a potential to test 81-unknown samples in singlicate within an on-deck plating time of fewer than four minutes. We performed additional verification studies, including an accelerated stability study and a freeze-thaw study showcasing the potential long-term usability of our proposed prototype kit. Overall, this work presents an optimized LC-MS/MS method with automated sample preparation that is ready to be utilized for cortisol-cortisone detection for clinical diagnostic contexts related to Cushing's Disease, among other adrenal and endocrine disorders.
Highlights
Elevated levels of cortisol trigger metabolic pathways in the liver, pancreas, and adipose tissue that are responsible for maintaining glucose and protein balance throughout the body [20]
To further adapt the cortisol-cortisone quantification kit to clinical applications, the developed sample preparation method was automated and tested for improved high-throughput capacity using the JANUS® G3 Workstation (PerkinElmer Corp., MA, U.S.)
The sample preparation process previously described was adapted onto the JANUS® workstation using JANUS® Application Assistant and WinPREP® for JANUS®, via the deck arrangement illustrated in Supplemental Fig. 1
Summary
Glucocorticoids (GC), an important class of lipophilic, fat-soluble steroid hormones, regulate a wide variety of physiological functions. Dysregulated levels of cortisol and cortisone may arise from abnormalities in glucocorticoid-activating enzymes such as 11 β-hydroxylase and 21-hydroxylase deficiencies The latter examples can take place in Congenital Adrenal Hyperplasia (CAH), as well as other inflammatory, psychiatric, and metabolic disorders [8,9]. Measuring cortisol from plasma may potentially yield misleading results due to its measurement of the total amount of cortisol, including bound cortisol [14] This encourages the utilization of saliva as a means for rapid recognition, diagnosis, and treatment of diseases affecting the adrenal glands by accurately evaluating circulating glucocorticoid levels. The present work focuses on optimizing streamlined sample preparation and LC-MS/MS analysis method that avoids the need for errorbound extraction steps (liquid-liquid extraction, solid-phase extraction, etc.) often found in other protocols This reduces the complexity of the sample preparation process, allowing for a more streamlined and highthroughput automation using conventional robotic liquid handlers, with minimal needed human interaction. The proposed protocol has the potential to be utilized as a high-throughput research-only kit, that can measure the free cortisol-cortisone in human saliva with great precision and accuracy using only a 20 μL patient sample volume, across the range of 0.5 to 70 ng/mL (CF) and 1.2 to 100 ng/mL (CE) distributed across six calibrators and low and high-Quality Controls (QCs)
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