Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva

A growing body of literature describes the potential effects of circadian disruption on human health. Indeed, psychiatric diseases, metabolic syndrome, and cancers may be linked to disturbance of the circadian rhythm. Currently, the best practice to assess circadian rhythm is the measurement of melatonin levels. Our goal was thus to develop and validate a highly sensitive LC-MS/MS method to follow salivary melatonin levels throughout the day and night. Our method reached a lower limit of the measuring interval (LLMI) of 0.8 pg/mL. To our knowledge, it is the most sensitive method allowing quantitation of melatonin in saliva. Saliva, obtained from passive drooling or salivette, was extracted by an efficient and quick liquid-liquid extraction with no further needed. The method was validated to the European Medicines Agency (EMA) guidelines and provided excellent results regarding accuracy, precision, linearity, selectivity, and specificity. Comparison between radioimmunoassay and our method was performed and showed differences at low levels, most likely due to cross-reactivity with other indols. To assess daytime melatonin levels in humans, salivary melatonin levels of ten volunteers were monitored throughout the day and showed lower daytime levels than reported in previous studies.

Automated summary

A growing body of literature suggests that circadian disruption may have significant effects on human health, potentially contributing to psychiatric diseases, metabolic syndrome, and cancers. In order to assess circadian rhythm, the measurement of melatonin levels is considered the best practice. This study developed and validated a highly sensitive LC-MS/MS method to track salivary melatonin levels throughout the day and night, establishing a lower limit of the measuring interval (LLMI) of 0.8 pg/mL. The method was validated according to EMA guidelines and showed accurate, precise, and specific results.