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 100 mu l of plasma, and chromatographic separation was achieved in isocratic mode on Kinetex C18 100 A column with mobile phase of acetonitrile, methanol, and 50 mM phosphate buffer (40:20:40 v/v). The analytical method was fully validated in terms of selectivity, linear range (0.2 to 5.5 ng mL(-1)), linearity (r(2) > 0.997), sensitivity (LOD = 0.05 to 0.75 ng mL(-1) and LOQ = 0.36 to 2.52 ng mL(-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. (C) 2016 Elsevier B.V. All rights reserved.