A validated LC–MS/MS method of total and unbound lenvatinib quantification in human serum for protein binding studies by equilibrium dialysis

• Lenvatinib (Lenvima™) assay in serum and dialysate has been developed using LC–MS/MS.
• The LLOQ was 40 and 80 pg/mL in dialysate and serum, respectively.
• The validation study demonstrated the method is simple, sensitive, and reproducible.
• This method was successfully applied to in vivo protein binding studies.

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.

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