Development and validation of sensitive liquid chromatography/tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue

A liquid chromatography–tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue was developed and fully validated. Methanol was used to precipitate proteins in brain tissue. Bendamustine and internal standard (chlorambucil) were separated with reverse-phase chromatography on a C-18 column with a gradient of water and 95% methanol in 0.1% formic acid. Positive mode electrospray ionization was applied with selected reaction monitoring to achieve 5 ng/ml lower limits of quantitation in mouse brain tissue. The calibration curve for bendamustine in mouse brain was linear between 5 and 2000 ng/ml. The within- and between-batch accuracy and precision of the assay were within 15% at 10, 100 and 1000 ng/ml. The recovery and matrix effect of bendamustine in mouse brain tissue ranged from 41.1% to 51.6% and 107.4% to 110.3%, respectively. The validated method was then applied to quantitate bendamustine in an animal study. Results indicate the assay can be applied to evaluate bendamustine disposition in mouse brain tissue. This assay will be applied in the future to detect and quantify bendamustine in human brain tissue samples.

► Bendamustine is FDA approved and is under evaluation for use in multiple cancers, including brain malignancies.
► This is the first report of a fully validated LC–MS/MS assay for bendamustine quantification in mouse brain tissue as a surrogate for human brain tissue.
► This assay is being applied to measure plasma and brain tumor concentrations of bendamustine in an ongoing clinical trial in patients with metastatic brain lesions.