Development of a LC-ESI-MRM method for the absolute quantification of orexin A in the CSF of individual mice
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 orexi...
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| Main Authors: | , , , , , |
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| Format: | Book |
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Elsevier,
2021-09-01T00:00:00Z.
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| Summary: | 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 <9% CV, inter-assay variation of 10% CV and limit of quantitation of 1.65 fmoles. CSF orexin A concentrations in C57/Bl6JARC mice were higher in the late active period (2.5 ± 0.5 fmoles/µL) versus the late inactive period (1.2 ± 0.5 fmoles/µL, p < 0.001). Sleep deprivation significantly dysregulated diurnal rhythm, up-regulating orexin A acutely, followed by down-regulation 16 hours after sleep deprivation. We anticipate this validated LC-ESI-MRM assay for the absolute quantification of orexin A in the CSF of individual mice will enhance research using relevant rodent models of sleep or arousal-related brain disorders. |
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| Item Description: | 2590-0986 10.1016/j.medidd.2021.100102 |