Tackling the Orexin Conundrum: An Optimized LC–MS/MS Method Demonstrates Accurate CSF Quantification and Absence in Peripheral Blood

Cerebrospinal fluid (CSF) orexin-A is the gold-standard biomarker for narcolepsy type 1 (NT1), however, conventional radioimmunoassays (RIA) often suffer from cross-reactivity and overestimation, fueling long-standing conundrum regarding orexin detection in peripheral blood. In this study, we developed an ultrasensitive LC–MS/MS method (LLOQ = 0.1 pg/mL) incorporating a streamlined one-step protein precipitation protocol coupled with acid-shielding and cocktail-protection strategies to mitigate severe nonspecific adsorption and enzymatic degradation. Verification in paired CSF and blood samples from narcolepsy patients and controls revealed that orexin-B and peripheral orexins remain consistently below the detection limit (<0.1 pg/mL), proving that previously reported ng/mL levels in blood are analytical artifacts. Our LC–MS/MS approach resolved a 50-fold quantitative overestimation by RIA and significantly improved diagnostic resolution for narcolepsy type 2 (AUC = 0.73, P < 0.05) where RIA failed to achieve statistical significance (P = 0.297). This study establishes a high-specificity analytical framework as a practical reference standard for refined sleep disorder diagnostics and confirms that CSF orexin-A remains the currently the most reliable clinical biomarker.