Separation and quantitative determination of cinacalcet metabolites in urine sample using RP-HPLC after derivation with a fluorescent labeling reagent.
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 (250×4.6mm, 5μm) using acetate buffer (pH 3.5):methanol (30:70 v/v) as mobile phase at a flow rate of 1.0mLmin(-1). The method was fully validated in terms of linearity (r(2)>0.996; 1-10ngmL(-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)ngmL(-1)) and quantification (2×10(-3) to 1×10(-2)ngmL(-1)). According to the results, the proposed method can be useful in the routine analysis for the determination of cinacalcet metabolites in urine samples.
Amir
A
Farnoudiyan-Habibi
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, P.O. Box 51656-65811, Tabriz, Islamic Republic of Iran. Electronic address: m_jaymand@yahoo.com.
Mehdi
M
Jaymand
D057966:Calcimimetic Agents / Q000032:analysis / Q000378:metabolism / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D000069449:Cinacalcet / Q000032:analysis / Q000378:metabolism / Q000652:urine*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D011721:Pyrenes / Q000737:chemistry
D057966:Calcimimetic Agents; D005456:Fluorescent Dyes; D011721:Pyrenes; C058827:1-pyrenyldiazomethane; D000069449:Cinacalcet
Cinacalcet; Fluorescent labeling; Metabolites; RP-HPLC; Urine; Validation