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 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.
Sastre Toraño
J
J
Pharmaceutical and Toxicological Laboratory, Department of Pharmacy, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Rijn-Bikker
P v
Pv
Merkus
P
P
Guchelaar
H J
HJ
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008297:Male; D008550:Melatonin / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014363:Tryptamines / Q000032:analysis / Q000097:blood / Q000134:cerebrospinal fluid
D014363:Tryptamines; C034536:5-fluorotryptamine; D008550:Melatonin