title: A validated LC-MS/MS method of total and unbound lenvatinib quantification in human serum for protein binding studies by equilibrium dialysis.
abstract: A sensitive method for the determination of total and unbound lenvatinib (Lenvima™), a novel tyrosine kinase inhibitor, in human serum was developed for protein binding studies using an equilibrium dialysis and liquid chromatography with tandem mass spectrometry. Serum samples (0.8 mL) were dialyzed against phosphate buffered saline (PBS) in dialyzer for 18 h at 37 °C to obtain dialysate and serum for unbound and total lenvatinib, respectively. After extraction by organic solvent, separation was achieved on a Symmetry Shield RP8 column with isocratic elution of 2 mM ammonium acetate (pH 4.0)-acetonitrile (3:2, v/v) at the flow rate of 0.2 mL/min. Detection was performed using API4000 with multiple reaction monitoring mode using positive electrospray ionization. The standard curve ranged from 0.0400 to 16.0 ng/mL and 0.0800 to 400 ng/mL as lenvatinib free base in PBS and serum, respectively. Accuracy and precision in the intra- and inter-batch reproducibility study were within the acceptance criteria. Various stability assessments including bench-top, freeze/thaw, processed samples, and frozen stability confirmed that lenvatinib was stable in serum and PBS. Application to in vivo protein binding studies in clinical studies was successfully performed and results showed that lenvatinib was highly protein bound in serum.
authors.affiliation: Drug Metabolism and Pharmacokinetics, Biopharmaceutical Assessment Core Function Unit, Eisai Co., Ltd., 1-3, 5-chome, Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan. Electronic address: y2-mano@hhc.eisai.co.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Mano
authors.affiliation: Drug Metabolism and Pharmacokinetics, Biopharmaceutical Assessment Core Function Unit, Eisai Co., Ltd., 1-3, 5-chome, Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan.
authors.forename: Kazutomi
authors.initials: K
authors.lastname: Kusano
mesh_terms: D000085:Acetates / Q000737:chemistry; D002138:Calibration; D002482:Cellulose / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D057230:Limit of Detection; D010671:Phenylurea Compounds / Q000032:analysis* / Q000097:blood*; D011485:Protein Binding; D011786:Quality Control; D011804:Quinolines / Q000032:analysis* / Q000097:blood*; D015203:Reproducibility of Results; D012997:Solvents / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000085:Acetates; D007477:Ions; D010671:Phenylurea Compounds; D011804:Quinolines; D012997:Solvents; D002482:Cellulose; C531958:lenvatinib; C018824:ammonium acetate
keywords: Human serum; LC–MS/MS; Lenvatinib; Protein binding; Validation