Quantitative determination of melatonin in human plasma and cerebrospinal fluid with high-performance liquid chromatography and fluorescence detection

A validated new and precise reversed-phase high-performance liquid chromato graphic method for the determination of melatonin in human plasma and cereb rospinal fluid, with 5-fluorotryptamine as internal standard, is described. Liquid-liquid extraction with dichloromethane was performed under alkaline conditions. After evaporation of the organic solvent, the extract was diss olved in eluent and chromatographed on a base-deactivated octadecyl column, using an eluent composed of 650 mt potassium dihydrogenphosphate solution (0.07 mol/L water), adjusted to a pH of 3.0 with a 43% phosphoric acid solu tion, mixed with 350 mt methanol. Fluorescence detection at an excitation w avelength of 224 nm and an emission wavelength of 348 nm was used for quant itation. Melatonin and 5-fluorotryptamine chromatographed with retention ti mes of 5.3 and 9.3 min, respectively. Mean recoveries of 96% (n = 10) and 9 5% (n = 5) were found for melatonin in plasma and cerebrospinal fluid respe ctively. 5-Fluorotryptamine was found to have a mean recovery of 90% (n = 1 0) and 82% (n = 5) in plasma and cerebrospinal fluid, respectively. The rep eatability coefficients of variation for both melatonin and 5-fluorotryptam ine in plasma were 4-5% [five different samples (r = 5) on two consecutive days (n = 2)], with reproducibility coefficients of 1.6-7% (n = 2, r = 5) a nd 0.9-4% (n = 2, r = 5) for melatonin and internal standard, respectively, In cerebrospinal fluid the repeatability coefficient of variation of the e xtraction procedure was 5% (n = 1, r = 5) for melatonin and 7% (n = 1, r = 5) for 5-fluorotryptamine. The correlation coefficients of the calibration curves were 0.9998 (n = 2) in plasma at a concentration range of 0.108-25.9 ng/mL and 0.9994 (n = 2) at a concentration range of 0.108-25.9 ng/mL in c erebrospinal fluid. The limit of detection was determined at 8 pg/mL which enables to measure melatonin concentrations at physiological concentrations reached during daytime. Copyright (C) 2000 John Wiley & Sons, Ltd.