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.