@article{745f9c0dacc84ca0aa067147127690d2,
title = "Development of a LC-ESI-MRM method for the absolute quantification of orexin A in the CSF of individual mice",
abstract = "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.",
keywords = "Animal models, Arousal, Mass spectrometry, Mice, Orexins/hypocretins, Sleep",
author = "Katherine Hopkins and Soumya Mukherjee and Dalia Ponce and Jonathan Mangum and Jacobson, {Laura H.} and Daniel Hoyer",
note = "Funding Information: This work was supported by the Alzheimer's Association (2016-NIRG-396905; DH, LHJ) and National Health and Medical Research Council (Ideas Grant 2003370, Project Grant 1105284; DH, JM, LHJ). The Florey Institute of Neuroscience and Mental Health acknowledge support of the Victorian Government and in particular funding from the Operational Infrastructure Support Grant. The authors thank Dr Blaine R. Roberts (Emory University) for his helpful discussion on method development. We are grateful to Prof Colin Masters (University of Melbourne and The Florey Institute of Neuroscience and Mental Health) for his unconditional support. We gratefully thank Dr John Karas (University of Melbourne) for synthesizing the heavy orexin A peptide, and Ms Heather Daykin and Ms Sara Oberrauch (University of Melbourne and The Florey Institute of Neuroscience and Mental Health) for expert technical assistance. We deeply thank Prof Sigrid Veasey (University of Pennsylvania) for her expert advice on the sleep deprivation through novelty protocol. Funding Information: This work was supported by the Alzheimer{\textquoteright}s Association ( 2016-NIRG-396905 ; DH, LHJ) and National Health and Medical Research Council (Ideas Grant 2003370 , Project Grant 1105284 ; DH, JM, LHJ). The Florey Institute of Neuroscience and Mental Health acknowledge support of the Victorian Government and in particular funding from the Operational Infrastructure Support Grant. The authors thank Dr Blaine R. Roberts (Emory University) for his helpful discussion on method development. We are grateful to Prof Colin Masters (University of Melbourne and The Florey Institute of Neuroscience and Mental Health) for his unconditional support. We gratefully thank Dr John Karas (University of Melbourne) for synthesizing the heavy orexin A peptide, and Ms Heather Daykin and Ms Sara Oberrauch (University of Melbourne and The Florey Institute of Neuroscience and Mental Health) for expert technical assistance. We deeply thank Prof Sigrid Veasey (University of Pennsylvania) for her expert advice on the sleep deprivation through novelty protocol. Publisher Copyright: {\textcopyright} 2021 The Authors",
year = "2021",
month = sep,
doi = "10.1016/j.medidd.2021.100102",
language = "English",
volume = "11",
journal = "Medicine in Drug Discovery",
issn = "2590-0986",
}