Development and validation of a quantitative assay for the determination of cinacalcet and its main metabolites in human plasma using RP-HPLC method

Abstract

A novel and sensitive methodology for the quantitative determination of cinacalcet (CIN), and its main metabolites (M2a-Glu, M2b-Glu, M5, and M6) in plasma sample is suggested. The analytical assay is based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. The method has been fully validated at range covering the plasma concentrations in patients receiving therapeutic dosages of CIN. In this investigation, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (NI-ETFMS) was used as a pre-column derivation agent according to interaction of amine and carboxylate groups of CIN and its metabolites with NI-ETFMS. The sample pretreatment consists of protein precipitation with acetonitrile using only 100μL of plasma, and chromatographic separation was achieved in isocratic mode on Kinetex C18 100Å column with mobile phase of acetonitrile, methanol, and 50mM phosphate buffer (40:20:40v/v). The analytical method was fully validated in terms of selectivity, linear range (0.2 to 5.5ngmL−1), linearity (r2>0.997), sensitivity (LOD=0.05 to 0.75ngmL−1 and LOQ=0.36 to 2.52ngmL−1), intra and inter-days accuracy (−4.6 to 8.1%), precision (<7.4%), and robustness (<3.5%). As results, the proposed method can be useful in the routine analysis for the determination of CIN and its main metabolites in human plasma samples.