Melatonin Determination in Human Urine by Liquid Chromatography Using Flurometric Detection

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Abstract
A new, simple, very sensitive, highly reproducible, isocratic, and reverse phase liquid chromatographic method has been developed for determining urine melatonin concentrations using a fluorometric detector. 6-methoxymelatonin is used as an internal standard. A C18 column (150 × 0.46 cm) and a “Waters” fluorometric detector (Model 474 with a 16 uL flow cell are the main components of the liquid chromatographic system. The excitation and emission wavelengths are 285 and 335 nm respectively. Melatonin is extracted from urine at pH 7 using chloroform. The method is sensitive to 0.5 pg. The mean (n=6) inter-assay precision (CV) is 4.3% and intra-assay precision is 5.8%. The mean absolute recovery of the method is about 90%. In summary, when there is a choice between fluorometric and electrochemical detection for a compound, the former is far superior and several orders of magnitude more sensitive than the latter and easier to use. Our fluorometric HPLC assay for melatonin improves upon the earlier ones5,8 by using a very sensitive detector and a good internal standard. The method has good accuracy (96%) and precision (C.V. ∼5%).