Simultaneous determination of melatonin and 6-hydroxymelatonin in human overnight urine by LC-MS/MS

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1181; p. 122938
• Main Authors: Magliocco, G., Le Bloc'h, F., Thomas, A., Desmeules, J., Daali, Y.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2021
• Subjects: calibration; Chromatography, Liquid - methods; CYP450; excretion; Helix pomatia; Humans; LC-MS/MS; Limit of Detection; Linear Models; liquid chromatography; Melatonin; Melatonin - analogs & derivatives; Melatonin - urine; metabolites; Phenotyping; Reproducibility of Results; Solid Phase Extraction; tandem mass spectrometry; Tandem Mass Spectrometry - methods; urine; LC-MS/MS; Melatonin; CYP450; Phenotyping
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2021.122938

•LC-MS/MS was used to quantify endogenous melatonin and 6-hydroxymelatonin in urine.•This method was fully validated using deuterated analogues as internal standards.•Applicability was demonstrated in the analysis of urine samples from 12 volunteers. For the quantification of the pineal hormone melatonin and its metabolite, 6-hydroxymelatonin, in human overnight urine, a single accurate method by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Urine samples were deconjugated using β-glucuronidase/arylsulfatase from Helix pomatia before solid phase extraction (SPE) purification. Chromatographic separation was performed using a reverse phase C18 column with a 7-minute gradient elution. Water was used as matrix to prepare the calibration standards, and deuterated analogues of melatonin and 6-hydroxymelatonin were used as internal standards. This newly developed method was validated in terms of linearity, accuracy, repeatability, intermediate precision, recovery, matrix effect, and stability according to the guidelines of the European Medicines Agency. The method was successfully applied to the analysis of overnight urine samples from 12 healthy volunteers, showing significant correlations of urinary melatonin and 6-hydroxymelatonin excretion rates with age. The urinary 6-hydroxymelatonin to melatonin ratio was also established and will be assessed in further studies as a potential endogenous metric of CYP1A2 activity.