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Article Title

Determination of melatonin in rat plasma by high performance liquid chromatography with electrochemical detection

Abstract

Melatonin ( 5-methoxy-N-acetyltryptamine ) plays a role in a number of distinct physiological functions. We have developed a convenient high performance liquid chromatography assay with electrochemical detection ( HPLC / ECD ) for the accurate determination of melatonin in biological samples. This assay involves direct addition of rat plasma to acetonitrile for simultaneous protein precipitation and melatonin extraction. The assay was validated by addition of melatonin to rat plasma with a recovery rate between 92 and 99 %. The correlation coefficient of calibration curve was 1.000. The relative standard deviations for intraday and interday analyses were 1.5-2.2 % and 3.5-5.0 %, respectively. Melatonin clearance rate of biological samples were determined by this method. Melatonin ( 0.67 mg / kg ) were given to Sprague Dawley rats by femoral vein injection. The melatonin concentrations were determined by HPLC / ECD at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hrs. The concentrations were reached the peak levels at 0.5-1.0 hrs and cleared after 3.0 hrs. The concentrations of melatonin were 102.8 ± 24.3, 51.5 ± 13.0, 30.2 ± 7.1, 22.2 ± 7.2, 20.4 ± 9.8 and 17.9 ± 8.1 nM ( mean ± S.E.M., n = 8 ) at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hrs, respectively. This HPLC / ECD method can be useful in its application to biomedical studies of melatonin.

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