Extraction of Melatonin from Chick Pineal and Its Quantification by High-Performance Liquid Chromatography

Abstract

We studied the extraction of melatonin from chick pineals, the preservation of their extracts and the determination of their relatonin by high-perfermance liquid chromatography. A method was established to determine the pineal melatonin content. A stock solution is prepared at a level of 1.00mg/ml using ethanol-acetate buffer (40mM acetic acid and 154mM sodium acetate, pH4.0, containing 0.033% cysteine and 0.029% disodium EDTA (1:9, v/v)). The working standard is prepared to contain 100ng/ml in 50mM perchloric acid containing 0.013% cysteine and 0.012% EDTA. Chicks were killed by decapitation, and their pineals were rapidly removed, homogenized by hand in a 1-ml glass homogenizer chilled in ice and then homogenized after adding each of 0.1, 0.2 and 0.2ml of 0.05M perchloric acid containing 0.013% cysteine and 0.012% disodium EDTA. These procedures in the dark period were performed under dim red light. The supernatant was separated by centrifugation at 3, 000 rpm for 5min at 4°C, and filtered through a 0.02μm-filter. One hundred μl aliquots of the filtrates were applled to a reverse-phase column and eluted with eluents at a flow rate of 1ml/min. The eluates were monitored with a fluorescence spectromonitor at 285nm for excitation and 345nm for emission or with a electrochemical detector at a cell potential of +0.90V versus a saturated silver-silver chloride electrode. The eluents were the mixtures of 0.05M acetate buffer solution (pH4.7) and methanol (65:35 and 75: 25, v/v). The column was used at 35°C. The retention times for melatonin with the eluents containing 25 and 35% methanol were 21.8 and 9.2min, respectively. The pineal extract was stable at 4°C for at least one month.