Validation of a simple bioanalytical method for the determination of melatonin concentration in human serum using high-performance liquid chromatography with a fluorescent detector

Abstract

Analysis of melatonin in biological samples is a challenge for the analyst both because of its low concentration and because of the numerous interfering substances. Therefore, chromatography, as a powerful analytical technique used for the separation, detection, and quantification of analytes, can be the method of choice for determining melatonin in biological samples for clinical use and pharmacokinetic research. The method was developed and validated according to the internal documents in accordance with Good Laboratory Practice. The concentrations of melatonin in human serum were determined using a high-performance liquid chromatography method with a fluorescence detector and mirtazapine as an internal standard. Preparation of serum samples was performed by liquid–liquid extraction with ethyl acetate: n-hexane (1:1 v/v). High linearity was exhibited over a concentration range of 25–10,000 pg/mL of melatonin, with a correlation coefficient higher than 0.999 during the course of method validation. Repeatability and intermediate precision show a relative standard deviation lower than 6.6 %, and accuracies were from 90.7 % to 110.3 %. The lower limit of quantitation was 30 pg/mL, and the limit of detection was 7.92 pg/mL. The in-house method is simple, fast, reproducible, and applicable for the determination of melatonin in human serum.