New Ultrasensitive LC–MS/MS Method Allows for Accurate Melatonin Quantification in Human Saliva

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A new study reports the development a highly sensitive LC–MS/MS method to measure salivary melatonin levels. The method has a lower limit of the measuring interval of 0.8 pg/mL, making it the most sensitive method for quantifying melatonin in saliva.

A team of researchers at the University of Liège in Liège, Belgium has developed and validated a highly sensitive LC–MS/MS method for the quantification of melatonin in human saliva. The study, published in the Journal of the American Society for Mass Spectrometry, describes the method as the most sensitive available for measuring melatonin in saliva, with a lower limit of the measuring interval (LLMI) of 0.8 pg/mL (1).

Melatonin is a hormone produced by the pineal gland that plays a critical role in regulating the sleep-wake cycle. Circadian disruption has been linked to a range of health issues, including psychiatric diseases, metabolic syndrome, and cancers. Therefore, the ability to accurately measure melatonin levels throughout the day and night is essential for understanding the effects of circadian disruption on human health.

Saliva was obtained from ten volunteers through passive drooling or salivette, and the method was validated according to the European Medicines Agency (EMA) guidelines. The study found that the method provided excellent results in terms of accuracy, precision, linearity, selectivity, and specificity.

The researchers also compared their method to radioimmunoassay and found differences at low levels, most likely due to cross-reactivity with other indols. Furthermore, the study demonstrated that salivary melatonin levels of the volunteers monitored throughout the day showed lower daytime levels than reported in previous studies.


Indoles are a class of organic compounds containing a bicyclic structure composed of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. They are widely found in nature, including in plants, fungi, and bacteria. Indoles have various biological activities, including acting as signaling molecules, plant growth regulators, and neurotransmitters, and have been studied for their potential pharmaceutical and therapeutic applications. Melatonin is an indole compound.

The development and validation of this ultrasensitive LC–MS/MS method for melatonin quantification in human saliva is an important contribution to the field of circadian rhythm research. This new method could be a valuable tool for researchers investigating the effects of circadian disruption on human health. It could also provide clinicians with a more accurate and sensitive method for assessing melatonin levels in patients with sleep disorders or other health issues related to circadian rhythm disturbances and ultimately lead to improved treatment and prevention of associated health issues.


(1) Demeuse, J. J.; Calaprice, C.; Huyghebaert, L. C.; Rechchad, M.; Peeters, S.; Cavalier, E.; Le Goff, C. Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva. J. Am. Soc. Mass Spectrom. 2023. DOI: