Center for Biomedical Engineering, School of Engineering, Brown University, 182 Hope Street, Providence, RI- 02912, USA.
PerkinElmer Inc., Waltham, MA, USA.
SLAS Technol. 2022 Aug;27(4):237-246. doi: 10.1016/j.slast.2022.01.006. Epub 2022 Feb 21.
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 R>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.
高效液相色谱-串联质谱法多年来一直是一种可靠的高通量临床仪器，可用于评估各种代谢异常。LC-MS/MS 的最新应用之一是在糖皮质激素（如唾液中的皮质醇和可的松）的定量领域，它可以作为先天性肾上腺增生症（CAH）、库欣综合征和艾迪生病等异常的指标。我们开发并验证了一种基于 LC-MS/MS 的测定法，用于同时检测人唾液中的皮质醇和可的松，该方法仅需 20 µL 样本，即可在 0.5-70ng/mL 范围内检测皮质醇，在 1.2-100ng/mL 范围内检测可的松。开发的方法对于两种分析物，包括两种 MRM，均表现出 R>0.99 的线性，且变异系数小于或等于 20%。使用稀释和进样进行样品制备，我们展示了该测定校准器的样品准确度为 100±20%，并集成了针洗溶剂化学以最大限度地减少样品残留，使其适用于潜在的诊断用途。我们展示了一种方法，该方法在液体处理平台（如 JANUS®G3 工作站）上简单、特异性强且高度自动化。通过我们的创新，我们引入了一种潜力，可以在不到四分钟的进样时间内对 81 个未知样本进行单次检测。我们进行了额外的验证研究，包括加速稳定性研究和冻融研究，展示了我们提出的原型试剂盒的潜在长期可用性。总体而言，这项工作提出了一种优化的 LC-MS/MS 方法，具有自动化的样品制备，可用于与库欣病等肾上腺和内分泌疾病相关的临床诊断背景下的皮质醇-可的松检测。
