Quantitation of Melatonin and NAS by LC-MS/MS

Details of the analytical method used in the study are reported in (Carter et al. 2012 (link)). Briefly, blood was collected over an 8-h period and immediately placed on ice. Plasma was isolated within 45 min of blood collection. Baseline and PK plasma samples were analyzed in the Vanderbilt Bioanalytical Mass Spectrometry Core Facility by nanoflow LC–MS/MS and HPLC–MS/MS repectively (Carter et al. 2012 (link)). Nanoflow LC was necessary to reliably quantitate the low (pg/ml) endogenous levels of melatonin and N-acetylserotonin (NAS). PK parameters [maximal concentration of melatonin (C_max), time to maximal concentration (T_max), AUC, half-life (t_1/2), clearance (CI), and volume of distribution (V_z)] were derived from plasma concentration versus time curves using the program Win-Nonlin v. 5.2 (Pharsight, Palo Alto, CA, USA). The data were analyzed using a noncompartment model as described in Carter et al. 2012 (link). The LC–MS/MS analytical method was validated on a small group of adult volunteers (two male, two female, weight 72–109 kg, age 28–64 years, height 1.65–1.83 m). For the adult population, calculated PK parameters were consistent with literature values (Table SI and S2). The AUC and C_max showed evidence of linear kinetics. The half-life (t_1/2) and T_max were not a function of dose.