Salivary cortisone quantification by paper-based ambient mass spectrometry with integrated sample cleanup and enrichment.

Salivary cortisone correlates more closely with serum free cortisol than salivary cortisol itself, making it the most clinically informative non-invasive marker of adrenal status. Yet its trace endogenous concentration has, until now, largely confined accurate quantification to laboratory-based ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). UHPLC-MS/MS demands substantial sample volumes and labour intensive offline preparation. These barriers disproportionately exclude patients who cannot reliably provide the required volumes, including paediatric, elderly and xerostomic populations. Here we report, to our knowledge, the first ambient mass spectrometry method to deliver UHPLC-MS/MS-equivalent quantification of an endogenous steroid biomarker from healthy human saliva across the clinically relevant range. Paper-arrow mass spectrometry (PA-MS), an ambient ionisation technique within the paper spray family, unifies extraction, analyte enrichment and ionisation on a single disposable paper substrate. Cortisone is quantified from 4 μL of saliva, a 50-fold reduction relative to UHPLC-MS/MS, within 10 min. Validated to ICH M10 bioanalytical criteria, the method achieves an LOQ of 0.5 ng/mL, precision ≤8.4% RSD, accuracy 96 to 105%, linearity over 0.5 to 50 ng/mL, and negligible matrix effects. Cross-validation against UHPLC-MS/MS in 28 specimens showed excellent agreement (mean difference 4.17 ± 6.98%). Cortisone, unlike cortisol, was free of co-eluting isobaric interferences, making it well suited to chromatography-free ambient analysis. PA-MS opens a practical route to decentralised endocrine diagnostics for populations underserved by current laboratory-based methods.