Rapid and sensitive analysis of melatonin by LC-MS/MS and its application to pharmacokinetic study in dogs

A rapid and sensitive liquid chromatography tandem mass spectrometry method was established and validated for determination of melatonin in dog plasma using desvenlafaxine as an internal standard (IS). Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out on a C18 column at a flow rate of 0.2 ml/min by an isocratic mobile phase of methanol : 5 mM ammonium acetate : formic acid (40:60:0.1, v/v/v). Positive ion mode detection was performed using multiple reaction monitoring (MRM) at m/z 233.2→174.2 for melatonin and m/z 264.2→58.2 for desvenlafaxine. The method was linear in the concentration range of 0.020–10 ng/ml with a correlation coefficient ≥0.996. The intra- and inter-assay precision (%RSD) values were within 12.6% (LLOQ 15.2%), and accuracy (%RE) ranged from −1.8% to 5.0% (LLOQ ±16.5%). The total analysis time was 3.0 min. The method was fully validated and successfully applied to a pharmacokinetic study of melatonin prolonged-release tablet in Beagle dogs. The values of half-life and Tmax were similar to the corresponding data reported before.