Design of experiments and bioanalytical LC–MS/MS method for analysis of melatonin and tasimelteon in rat brain homogenate with whiteness assessment

Sleep disorders include a range of common problems that affect the quality of sleep at night and, as a result, impact an individual's daily functioning. Treatment protocols vary from over-the-counter products to regulated pharmaceuticals. Melatonin and Tasimelteon are two compounds utilized for severe to moderate sleeping disorders. This study developed and validated a sensitive, simple bioanalytical LC-MS/MS method for the measurement of Melatonin and Tasimelteon in spiked rat brain tissue. Chromatographic analyses were conducted in isocratic mode, with Citalopram selected as an appropriate internal standard. The Supelco Ascentis® Express Phenyl-Hexyl column was used for the stationary phase, and the mobile phase comprised 0.2% formic acid in a mixture of acetonitrile and water (65:35, v/v). A response surface methodology is applied. The Box-Behnken design was used to optimize the influence of three independent factors (acetonitrile%, formic acid%, and flow rate (mL/min)) on the response. The study focused on finding the most significant factors influencing chromatographic separation, namely the resolution between Tasimelteon and Melatonin, as well as the tailing factors of both. Statistical analysis of variance provided the optimal conditions for separating the substances as well as the most influential factors. Validation of the analytical method was conducted in accordance with the International Council for Harmonization guideline M10 related to bioanalytical method validation. The method validated was precise and linear in 55.00-1650 (ng/mL) and 20-600 (ng/mL) for the Melatonin and Tasimelteon, respectively. The validated method's lower limit of quantification values was 55 and 20 ng/mL for Melatonin and Tasimelteon, respectively. For Melatonin, intraday accuracy (recovery, %) ranged from 96.53% to 102.68%, and precision (expressed as relative standard deviation) ranged from 0.26% to 0.96%. And inter-day accuracy ranged from 96.58% to 103.08%, and inter-day precision ranged from 0.33% to 3.55%. Intraday accuracy results for Tasimelteon 99.61%-103. 75% precision results were in the range 0.23%-0.93%; additionally, inter-day accuracy was 99.37-103.87%, and the precision range was 1.04-2.11%. The total run time was 3 min, with retention time for Melatonin and Tasimelteon at 1.9 and 2.5 min, respectively, achieving effective chromatographic separation under optimum conditions. The Red Green Blue 12 score for whiteness was determined to be 79.2%.