Monitoring salivary melatonin concentrations in children with sleep disorders using liquid chromatography-tandem mass spectrometry
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Background; Melatonin is S5Tithesized in the pineal gland and is an important circadian phase marker, especially in the detennination of sleep patterns. Both temporary and pennanent abnormal sleep patterns occur in children; therefore, it is desirable to have methods for monitoring melatonin in biological fluids in the diagnosis and treatment of such disorders. Objective: The objective of the study is to develop a liquid chromatography-tandem mass spectrometry method for the detenni¬ nation of melatonin in saliva and to apply it to monitoring salivary concentrations in children with sleep disorders. Methods: A deuterated internal standard (dy-melatonin) was added to a diluted saliva sample (20 p.L) in an autosampler vial insert, and 50 p-L were injected. Plasticware was strictly avoided, and all glass¬ ware was scrupulously cleaned and then baked at 120oC for at least 48 hours to obtain satisfactory perfonnance. Reverse-phase chroma¬ tography was perfonned on a Cg column using a linear gradient elution profile comprising mobile phases A (0.1% aqueous formic acid) and B (15% methanol in acetonitrile containing 0.1% fonnic acid), pumped at a total flow rate of 0.8 mL/min. The run time was 8 minutes. After atmospheric pressure chemical ionization, mass spectrometric detection was in positive ion mode. Mass detection was by selected reaction monitoring mode with the following mass transitions used for quantification; melatonin, m/z 233.0 —> 173.8 and d7-melatonin, m/z 240.0 —> 178.3.
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