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High-Performance Liquid Chromatographic Analysis of Duloxetine and Its Metabolites in Rat and Characterization of Metabolites in Plasma, Urine, Feces and Bile through Retro-Synthesis Followed By NMR and MS Study
Authors: T. K. Laha1*, G. Mishra2, S. Sen1
Number of views: 349
A simple and sensitive reverse phase high performance liquid chromatographic (RP-HPLC) method for determination of duloxetine and twelve of its metabolites, Sulfate conjugate of 4-hydroxy duloxetine (M1), N-desmethyl duloxetine (M2), Glucuronide conjugate of 4-hydroxy duloxetine (M3), Glucuronide conjugate of 6-hydroxy duloxetine (M4), Glucuronide conjugate of 4,6-dihydroxy duloxetine (M5), Glucuronide conjugate of 5-hydroxy-6-methoxy duloxetine (M6), 4-Hydroxy duloxetine (M7), 5-Hydroxy duloxetine (M8), 6-Hydroxy duloxetine (M9), Thienyl alcohol (M10), Glucuronide conjugate of 5-hydroxy duloxetine (M11), Glucuronide conjugate of dihydrodiol duloxetine (M12) in Wistar rat plasma, urine, feces and bile was developed. Analysis was carried out on a µ-Bondapak C18 column (250mm × 4.6mm, 5µm particle size) using methanol: phosphate buffer (pH 7.8, 50 mM) (7:3 v/v) as the mobile phase at a flow rate of 1ml/min. Detection was carried out at 221 nm with an UV detector. The above metabolites were characterized by retro-synthesis followed by 1H-NMR, 13C-NMR and M.S study for structure confirmation and finally injected separately into the HPLC system. All the twelve retention time matches with the metabolites present in the plasma, urine, feces and bile sample. This method has also been successfully applied in the pharmacokinetics study of duloxetine after orally administrating the duloxetine to Wistar rat.