Quantification of Melatonin with Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry

Melatonin, a key hormone in regulating circadian rhythms and sleep-wake cycles, presents challenges for quantification in C57BL/6 mice. C57BL/6 mice are the most used mouse strain for laboratory studies due to their inherent melatonin deficiency. This strain exhibits very low or undetectable melatonin levels in the pineal gland and blood, complicating standardization efforts in melatonin research. Here we present two primary techniques for melatonin detection in C57BL/6 mouse blood: radioimmunoassay (RIA), using the Novolytix (formerly Buhlmann) kit, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The Novolytix RIA kit offers high sensitivity with a detection limit below 0.3 pg/mL for a 1 mL sample, similar to mass spectrometric detection capability. LC-MS/MS, considered the gold standard, provides even greater sensitivity and specificity allowing for the detection of melatonin at levels as low as 1 pg/mL in mouse plasma. Both methods require careful sample preparation, including extraction steps to reduce matrix impurities and concentrate the melatonin in the sample. This protocol outlines detailed procedures for blood collection, sample preparation, and melatonin quantification using both RIA and LC-MS/MS methods; addressing the unique challenges posed by the low melatonin levels in C57BL/6 mice.