A simple and sensitive method for determination of melatonin in plasma, bile and intestinal tissues by high performance liquid chromatography with fluorescence detection

This paper describes the development of a simple and sensitive method for routine quantification of melatonin in low sample amounts by using standard equipment of HPLC with fluorescence detection. A double chloroform extraction with an intermediate cleaning step with 0.1N NaOH allowed to concentrate melatonin and to avoid interferences in extracts of the different tissues assayed. The analytical procedure was found to be precise and linear for a wide range of melatonin concentrations. The retention time of melatonin was about 9 min and the recoveries were in the range of 89–94%. The lower limit of quantification estimated on extracted samples was 8 pg/ml. This method was validated in daytime and nighttime samples of plasma, bile and intestinal tissues of trout.