Abstract
A method for assay of urinary 6-hydroxymelatonin, the major metabolite of the pineal hormone melatonin is described. After addition of an internal standard of deuterated 6-hydroxymelatonin sulfate, human urine was hydrolyzed enzymatically and free 6-hydroxymelatonin extracted, reacted to form a stable t-butyldimethylsilylpentafluoropropionyl derivative which was separated on silica gel column chromatography, and quantified using electron capture negative-ion chemical ionization mass spectrometry. Intrassay variability over an 18-h period was 5.4% [53.8 ng/3 ml urine ± 2.94 (SD)] and interassay variability over a 2-week period was 2.1% [51.8 ng/3 ml urine ± 1.08 (SD)].
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