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

•For the first time, quantitative determination of cinacalcet urinary metabolites is reported.•The analytical assay is based on derivation of cinacalcet metabolites with a fluorescent labeling reagent (PDAM).•The method was fully validated in terms of linearity, precision, specificity, robustness, LOD and LOQ.•The method is considerable of interest, mainly due to its sensitivity, simplicity, speediness, and cost effectiveness.

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 × 4.6 mm, 5 μm) 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 (r2 > 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 × 10−4 to 3 × 10−3 ng mL−1) and quantification (2 × 10−3 to 1 × 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.