Sleep-wakefulness is disrupted in most neurological and psychiatric disorders. Although clinical data implicate orexin (hypocretin), a crucial sleep/wake regulatory neuropeptide, in such disorders, limited sample volumes effectively prevent quantification of cerebrospinal fluid (CSF) levels of orexin A in mouse models of brain disorders. Current enzyme- and radio-immunoassays for orexin A generally require 50–100 µL CSF, whereas typical CSF sample volumes from mice are ~5–10 µL/mouse. We therefore aimed to develop and validate a liquid chromatography (LC) targeted mass spectrometry (MS) method for the absolute quantification of orexin A in the CSF of individual mice. LC coupled to tandem MS (LC-MS/MS) and a triple quadrupole (QQQ) mass spectrometer were used to develop a LC electrospray ionization multiple-reaction monitoring (LC-ESI-MRM) method. CSF orexin A levels of C57BL/6JARC mice were quantified using this method at the predicted peak and trough of diurnal orexin A release and following sleep deprivation. The LC-ESI-MRM assay was robust and sensitive, with an intra-assay variation
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