Melatonin Determination in Human Urine by Liquid Chromatography Using Flurometric Detection

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 ones^5,8 by using a very sensitive detector and a good internal standard. The method has good accuracy (96%) and precision (C.V. ∼5%).