Abstract
Despite growing evidence of the importance of melatonin and serotonin in the plant life, there is still much debate over the stability of melatonin, with extraction and analysis methods varying greatly from lab to lab with respect to time, temperature, light levels, extraction solvents, and mechanical disruption. The variability in methodology has created conflicting results that confound the comparison of studies to determine the role of melatonin in plant physiology. We here describe a fully validated method for the quantification of melatonin, serotonin and their biosynthetic precursors: tryptophan, tryptamine and N-acetylserotonin by liquid chromatography single quadrupole mass spectrometry (LC-MS) in diverse plant species and tissues. This method can be performed on a simple and inexpensive platform, and is both rapid and simple to implement. The method has excellent reproducibility and acceptable sensitivity with percent relative standard deviation (%RSD) in all matrices between 1 and 10% and recovery values of 82-113% for all analytes. Instrument detection limits were 24.4 ng/mL, 6.10 ng/mL, 1.52 ng/mL, 6.10 ng/mL, and 95.3 pg/mL, for serotonin, tryptophan, tryptamine, N-acetylserotonin and melatonin respectively. Method detection limits were 1.62 μg/g, 0.407 μg/g, 0.101 μg/g, 0.407 μg/g, and 6.17 ng/g respectively. The optimized method was then utilized to examine the issue of variable stability of melatonin in plant tissue culture systems. Media composition (Murashige and Skoog, Driver and Kuniyuki walnut or Lloyd and McCown's woody plant medium) and light (16 h photoperiod or dark) were found to have no effect on melatonin or serotonin content. A Youden trial suggested temperature as a major factor leading to degradation of melatonin. Both melatonin and serotonin appeared to be stable across the first 10 days in media, melatonin losses reached a mean minimum degradation at 28 days of approximately 90%; serotonin reached a mean minimum value of approximately 60% at 28 days. These results suggest that melatonin and serotonin show considerable stability in plant systems and these indoleamines and related compounds can be used for investigations that span over 3 weeks.