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Quantitative determination of melatonin in human plasma and cerebrospinal fluid with high‐performance liquid chromatography and fluorescence detection
Author(s) -
Sastre Toraño Javier,
RijnBikker Petra van,
Merkus Paul,
Guchelaar HenkJan
Publication year - 2000
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/1099-0801(200008)14:5<306::aid-bmc986>3.0.co;2-7
Subject(s) - chemistry , chromatography , melatonin , cerebrospinal fluid , fluorescence , plasma , human plasma , high performance liquid chromatography , fluorescence spectroscopy , medicine , physics , quantum mechanics
A validated new and precise reversed‐phase high‐performance liquid chromatographic method for the determination of melatonin in human plasma and cerebrospinal 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 dissolved in eluent and chromatographed on a base‐deactivated octadecyl column, using an eluent composed of 650 mL potassium dihydrogenphosphate solution (0.07 mol/L water), adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 350 mL methanol. Fluorescence detection at an excitation wavelength of 224 nm and an emission wavelength of 348 nm was used for quantitation. Melatonin and 5‐fluorotryptamine chromatographed with retention times of 5.3 and 9.3 min, respectively. Mean recoveries of 96% ( n = 10) and 95% ( n = 5) were found for melatonin in plasma and cerebrospinal fluid respectively. 5‐Fluorotryptamine was found to have a mean recovery of 90% ( n = 10) and 82% ( n = 5) in plasma and cerebrospinal fluid, respectively. The repeatability coefficients of variation for both melatonin and 5‐fluorotryptamine 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) and 0.9–4% ( n = 2, r = 5) for melatonin and internal standard, respectively. In cerebrospinal fluid the repeatability coefficient of variation of the extraction 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 cerebrospinal fluid. The limit of detection was determined at 8 pg/mL which enables to measure melatonin concentrations at physiological concentrations reached during daytime. Copyright © 2000 John Wiley & Sons, Ltd.