Clinical utility of an ultrasensitive urinary free cortisol assay by tandem mass spectrometry.

BACKGROUND: 24 h urinary free cortisol measurement is a clinically important first-line screening test for Cushing's syndrome (CS). Tandem mass spectrometry (LC-MS/MS) assays have superior sensitivity and specificity compared to immunoassays. Our goal was to improve and validate a LC-MS/MS method to measure urinary free cortisol in both adult and pediatric patients and to characterize its clinical diagnostic performance of CS by chart review. METHODS: We improved a LC-MS/MS method previously reported for urinary free cortisol to be able to measure urinary and salivary cortisol in the same batch for increased efficiency. 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 a retrospective patient chart review was conducted to evaluate the assay's clinical performance in diagnosing CS. RESULTS: The LC-MS/MS assay demonstrated enhanced sensitivity and was linear within an analytical measurement range of 10-10,000 ng/dL. Assay accuracy was satisfactory as determined by spike and recovery studies and highly correlated with a reference LC-MS/MS method. The assay's clinical diagnostic sensitivity and specificity in detecting CS was 96% and 91%, respectively, when compared to a urinary cortisol excretion of at least 50 µg/24 h. CONCLUSIONS: The improved LC-MS/MS method is both sensitive and specific with enhanced analytical performance and clinical diagnostic utility to screen for CS. The clinical diagnostic sensitivity and specificity were superior based on retrospective patient chart review.

Rapid measurement of tacrolimus in whole blood by paper spray-tandem mass spectrometry (PS-MS/MS).

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides sensitivity and specificity for monitoring tacrolimus drug level in blood, but it requires an LC system and sample preparation, which is not amenable to random access testing typical of immunoassays. Paper spray (PS) ionization generates gas phase analyte ions directly from dried blood spots without sample preparation and LC. We evaluated a PS-MS/MS method for tacrolimus drug monitoring in a clinical diagnostic laboratory. METHODS: Whole blood sample was mixed with stable isotope labeled internal standard ([(13)C, (2)H2]-FK506) and spotted onto a cartridge containing triangular shaped card paper. After drying, samples were analyzed by PS MS/MS in the selected reaction monitoring (SRM) mode, with a run time of 3 min/sample. RESULTS: Analytical measurement range was 1.5-30 ng/ml. Assay inter-day imprecision was 13%, 8%, and 5% at tacrolimus concentrations of 4.5, 10.5, and 24.5 ng/ml, respectively. Accuracy was determined by pure tacrolimus solution and was confirmed by result correlation to an immunoassay (slope=1.0, intercept=-0.02; r(2)=0.99), and to a conventional LC-MS/MS method (slope=0.90, intercept=0.4; r(2)=0.94). CONCLUSIONS: PS-MS/MS provides accurate results for tacrolimus with rapid turnaround time amenable to random access testing protocols.