Separation and quantitative determination of cinacalcet metabolites in urine sample using RP-HPLC after derivation with a fluorescent labeling reagent

Abstract

In this investigation, a novel strategy for separation and quantitative determination of four metabolites of cinacalcet (M2a-Glu, M2b-Glu, M7-Gly, and M8-Gly) in human urine is suggested. The analytical assay is based on a pre-column derivation procedure of cinacalcet metabolites with 1-pyrenyldiazomethane (PDAM) as a fluorescent labeling reagent, and subsequently separation and quantitative determination with reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. Metabolites were separated on a Microsorb-MV 100-5 C18 chromatography column (250 x 4.6 mm, 5 mu m) using acetate buffer (pH 3.5):methanol (30:70 v/v) as mobile phase at a flow rate of 1.0 mL min(-1). The method was fully validated in terms of linearity (r(2) > 0.996; 1-10 ng mL(-1)), precision (both intra-day and inter-day; RSD <6.2%), accuracy (92-110%), specificity, robustness (0.15%<RSD<4.1%), limits of detection (5 x 10(-4) to 3 x 10(-3) ng mL(-1)) and quantification (2 x 10(-3) to 1 x 10(-2) ng mL(-1)). According to the results, the proposed method can be useful in the routine analysis for the determination of cinacalcet metabolites in urine samples. (C) 2016 Elsevier B.V. All rights reserved.