Development and validation of LC–MS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine

► Bendamustine is an alkylating agent, used for haematological malignancies. ► Quantification of bendamustine is difficult due to chemical degradation. ► We developed quantitative assays for bendamustine, minimizing its degradation. ► The assays quantify bendamustine and metabolites in human plasma and urine. ► The assays were validated and successfully applied to clinical samples. A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) assay is described for the quantification of the anti-cancer agent bendamustine and its phase I metabolites γ-hydroxy-bendamustine (M3) and N-des-methylbendamustine (M4) and for its product of two-fold hydrolysis, dihydroxy-bendamustine (HP2), in human plasma and urine. Like most alkylating nitrogen mustards, bendamustine is prone to chemical hydrolysis in aqueous solution. To minimize degradation of bendamustine, urine samples were stabilized by a 100-fold dilution with human plasma and then processed identically to plasma samples. Sample aliquots of 200μL were mixed with an internal standard solution and acidified before separation of the analytes from the biomatrix with solid phase extraction. Dried and reconstituted extracts were injected on a Synergi Hydro RP column for the analysis of bendamustine, M3 and M4 or a Synergi Polar RP column for the analysis of HP2. Gradient elution was applied using 5mM ammonium formate with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionized using an electrospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer. The quantifiable range for bendamustine, M3 and M4 was 0.5–500ng/mL in plasma and 0.5–50μg/mL in urine, and that for HP2 was 1–500ng/mL in plasma and 0.1–50μg/mL in urine. The assays were accurate and precise, with inter-assay and intra-assay accuracies within ±20% of nominal and CV values below 20% at the lower limit of quantification and within ±15% of nominal and below 15% at the other concentration levels tested. These methods were successfully applied to evaluate the pharmacokinetic profile of bendamustine and its metabolites in cancer patients treated with bendamustine.