Development and full validation of an UPLC-MS/MS method for the determination of an anti-allergic indolinone derivative in rat plasma, and application to a preliminary pharmacokinetic study

The natural product (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified some years ago as a nanomolar inhibitor of FcɛRI-receptor dependent mast cell degranulation. To further explore the potential of the compound, we established an UPLC-MS/MS assay for dosage in rat plasma. The method was fully validated according to FDA Guidance for industry. Results of this validation and long term stability study demonstrate that the method in lithium heparinized rat plasma is specific, accurate, precise and capable of producing reliable results according to recommendations of international guidelines. The method was validated with a LLOQ of 30.0 ng/mL and an ULOQ of 3000 ng/mL. The response versus concentration data were fitted with a first order polynomial with 1/X2 weighting. No matrix effect was observed when using three independent sources of rat plasma. The average extraction recovery was consistent over the investigated range. This validation in rat plasma demonstrated that indolinone was stable for 190 days when stored below −65 °C; for 4 days at 10 °C in the autosampler; for 4 h at RT, and during three successive freeze/thaw cycles at −65 °C. Preliminary pharmacokinetic data were obtained in male Sprague–Dawley rats (2 mg/kg BW i.v.). Blood samples taken from 0 to 12 h after injection were collected, and data analyzed with WinNonlin. A short half-life (4.30 ± 0.14 min) and a relatively high clearance (3.83 ± 1.46 L/h/kg) were found.

► Indolinone is stable in rat plasma stored below −65 °C during 190 days.
► Method in rat plasma for indolinone is specific, selective, precise and accurate.
► Indolinone showed a short half-life and a relatively high clearance in rat plasma.