Abstract Study objectives Loss of cerebrospinal fluid hypocretin-1/orexin-A (Hcrt-1) immunoreactivity is a biomarker for narcolepsy type 1 (NT1). The current radioimmunoassay (RIA) does not allow precise quantification, limiting the assessment of disease severity and evolution. This study aimed to reproduce previously proposed workflows to quantify Hcrt-1 and Hcrt-2 by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), identify RIA antibody targets, and to compare two purification methods in their ability to identify patients with NT1. Methods Solid phase extraction (SPE) and LC–MS/MS measurements of Hcrt-1 and -2 levels from 10 NT1 patients and 21 controls were implemented based on existing literature. Immunoprecipitation (IP) with magnetic beads coupled to the RIA antibody (Phoenix) was performed and peptides identified by MS. Finally, Hcrt-1 (full-length) and Hcrt-1 fragment1-16 levels were analyzed in 4 patients and 4 controls prepared from both workflows by nano-LC coupled to high-resolution mass spectrometry (nLC-HRMS). Results Despite linear calibration curves for Hcrt-1 and Hcrt-2 (R2 0.99) could be assessed with LC–MS/MS, full-length Hcrt-1 concentrations were at the low end of the calibration curve with slightly lower levels in NT1 (p = 0.04). Hcrt-2 was undetectable. The discovery experiment with IP most reliably identified Hcrt-1 fragment1-16. SPE revealed much higher concentrations than IP. Hcrt-11-16-levels allowed distinguishing patients and controls after SPE and IP, whereas full-length Hcrt-1-levels after IP did not. Conclusions Hcrt-11-16 appears stable and abundant in CSF of controls, allows identification of patients with NT1 and is a promising surrogate marker. SPE seems more suitable for further method validation in clinical routine.
Wenz et al. (2026) studied this question.