Clinical utility of an ultrasensitive late night salivary cortisol assay by tandem mass spectrometry.

Late night salivary cortisol measurement is a clinically important and convenient screening test for Cushing's syndrome. Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve a LC-MS/MS method to measure salivary cortisol in both adult and pediatric patients and to characterize its analytical performance by method validation and clinical performance by chart review. We improved a LC-MS/MS method originally developed for urine cortisol to measure low level salivary cortisol. The sample preparation was by liquid-liquid extraction using dichloromethane followed by stepwise washing with acidic, basic and neutral solutions. The assay's analytical performance was characterized and retrospective patient chart review was conducted to evaluate the assay's clinical diagnostic performance. The LC-MS/MS assay showed enhanced functional sensitivity of 10 ng/dL for salivary cortisol and was linear within an analytical measurement range of 10-10,000 ng/dL. Assay accuracy was within 84-120% as determined by recovery studies and correlation with a reference method. Data from healthy adult volunteers was compiled to establish the reference interval for late night salivary cortisol. Patient chart review determined subjects with diagnosis of Cushing's syndrome or disease, and assay's clinical diagnostic sensitivity of 100% and specificity of 92% when the cutoff value was 70 ng/dL. The improved LC-MS/MS method is sensitive and specific with enhanced analytical performance and clinical diagnostic utility for screening Cushing's syndrome. The assay may have broad clinical application due to its high sensitivity and wide dynamic range.