Abstract
Orexins are two neuropeptides synthesised mainly in the brain lateral hypothalamic area. The orexinergic system provides arousal-dependent cues for a plethora of brain centres, playing a vital role in feeding behaviour, regulation of the sleep–wake cycle and circadian rhythms. Recently, orexins were found to be produced in the retina of an eye; however, their content in the vitreous body and possible daily pattern of expression have not yet been explored. In this manuscript, we describe the development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method designed for quantitative bioanalysis of orexin in the rat vitreous body. Orexin was extracted from vitreous body samples with a water:acetonitrile:formic acid (80:20:0.1; v/v/v) mixture followed by vortexing and centrifuging. Separation was performed on a reverse-phase HPLC column under gradient conditions. Orexin was analysed via multiple-reaction monitoring (MRM) in the positive electrospray mode. The total analysis time for each sample was less than 5.0 min. Once the method was fully optimised, it was then validated, following the 2018 FDA guidance on bioanalytical method validations. The calibration curves for orexin (1–500 ng/mL) were constructed using a linear regression with a 1/x2 weighting. The lower limit of quantitation for orexin was 1.0 pg/mL for the vitreous body. Intra-day and inter-day estimates of accuracy and precision were within 10% of their nominal values, indicating that the method is reliable for quantitation of orexin in the rat vitreous body. From the physiological perspective, our results are the first to show daily rhythm of orexin synthesis by the retina with possible implications on the circadian regulation of vision.
Highlights
The rotation of Earth around its axis within a period of 24 h evokes pronounced cyclic environmental alterations of day and night
Quantitative analysis of the Orexin A (OXA) was performed for the rat vitreous body samples; the validation of the method was performed for this matrix (CS and quality control (QC) samples prepared in the rat vitreous body)
The ion with m/z 858.6 was selected since it was produced in relatively greater abundance, which was reported in previous studies [25]
Summary
The rotation of Earth around its axis within a period of 24 h evokes pronounced cyclic environmental alterations of day and night. Most life on Earth possesses intrinsic timekeeping mechanisms called circadian clocks. The master clock is localised in the hypothalamic brain structure—the suprachiasmatic nuclei (SCN) [1,2]. Accumulating evidence shows the existence of local clocks in other brain areas and peripheral tissues, such as the liver, kidney, lungs [3,4,5,6], or the retina of an eye [7,8,9]. Due to a modern 24/7 lifestyle, shift work or during the course of jet lag, body clocks desynchronise, leading to severe civilisational health problems, including obesity, cardiovascular diseases and some types of cancer [10]. Understanding circadian rhythmicity requires further thorough exploration, including the involvement of new analytical tools for chronobiological investigation
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