Development of a new method for the quantitation of metabolites in the absence of chemically synthetized authentic standards

Abstract A new method for the quantification of metabolites in the absence of a chemically synthetized authentic standard is described herein. Metabolites to be used as reference standards were obtained biologically from microsomes incubation. The method is a stepwise process in which, only the radiolabeled ( 14 C) and non-radiolabeled parent compound are required. Briefly, the separation and principles of equimolar detection of LC-radioactivity were applied and, a calibration curve of the 14 C-parent compound was used to quantify the formation of its 14 C-metabolite. In turn, serial dilutions of this 14 C-metabolite were the base for the calibration curve that allowed the quantification of the non-radiolabeled metabolite. This method was applied in plasma samples obtained from a dog pharmacokinetic study in which, a PharmaMar compound (lurbinectedin) and its N -desmethylated metabolite were quantified and, the results compared to those obtained by the classical approach (with the chemically synthetized N -desmethylated metabolite). Plasma concentrations obtained with the two methods were very similar, with standard relative errors between −11% to −4%. Similar, main pharmacokinetic parameters were calculated with the concentrations obtained either thru this method or by using a chemically synthetized authentic standard