Development of a LC-ESI-MRM method for the absolute quantification of orexin A in the CSF of individual mice

•We developed a mass spectrometry method that allows robust quantification of orexin A in mouse CSF samples as small as 1 µL.•Analysis of individual mouse CSF samples confirmed a diurnal rhythm in orexin A levels.•Orexin A levels in mouse CSF were increased during acute sleep deprivation and reduced in the following dark phase.•Application of this LC-ESI-MRM method presents an opportunity to increase understanding of the role of orexin A in neurological and neuropsychiatric disease using mouse models.