Sensitive method for plasma and tumor Ko143 quantification using reversed-phase high-performance liquid chromatography and fluorescence detection

The fumitremorgin C analogue Ko143 is a potent and selective inhibitor of the ATP-binding cassette transporter ABCG2. To support in vivo ABCG2 resistance studies, we developed a sensitive and selective method for Ko143 quantification in plasma and tumor samples, using the parent compound fumitremorgin C as internal standard. Sample pretreatment by liquid–liquid extraction in diethyl ether yielded a recovery of 50% from human and mouse plasma. Pretreated samples were separated by reversed-phase high-performance liquid chromatography with fluorescence detection at 295 nm excitation and 350 nm emission wavelengths. Sharp chromatographic peaks were obtained with a 5 μm GraceSmart C18 column. The mobile phase consisted of methanol:10 mM ammonium acetate pH 5.0 (62:38, v/v), delivered at a flow rate of 0.2 mL/min. Acceptable accuracy and precision of ±15% were achieved within the linear dynamic range of the calibration curve (2–500 ng/mL) for human and mouse plasma samples. Mouse tumor tissue samples required the use of a calibration curve prepared in the same matrix due to the lower recovery of 40% from this matrix. Then, accuracy and precision were within the generally accepted range of ±15% for bioanalytical assays. Ko143 was stable in human plasma for up to 3 repeated freeze–thaw cycles and when stored at room temperature for up to 72 h. However, when kept at room temperature in mouse plasma, Ko143 was rapidly degraded by esterase activity, which could be prevented by collection of blood into sodium fluoride-containing tubes and maintaining samples on ice during pretreatment. A preliminary pharmacokinetics study in mice demonstrated the applicability of this assay for ABCG2 resistance studies in vivo.

► Fluorescence properties allow accurate Ko143 quantification down to 2 ng/mL.
► Selective sample pretreatment using liquid–liquid extraction with diethyl ether.
► Fumitremorgin C is used as the internal standard.
► The assay is validated for human plasma, mouse plasma and mouse tumor samples.
► Esterase mediated degradation of Ko143 in mouse plasma is inhibited by NaF.