In this study, we report the development and validation of an LC-tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 μL mouse plasma using a liquid-liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC 18 column using an isocratic mobile phase (5 mM ammonium acetate: methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99-2996 and 4.33-3248 ng/mL for bendamustine and copanlisib, respectively. Inter-and intra-day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra-and inter-day precisions were in the ranges of 2.01%-5.05% and 2.74%-6.13% and 1.98%-7.64 and 8.62%-9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at À80 C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.

Copanlisib is a dual PI3K‐δ inhibitor, used in follicular lymphoma treatment. In this research, we report a validated LC–MS/MS method for quantifying copanlisib from a mouse dried blood spot (DBS). We validated the method in line with the guidelines of the US Food and Drug Administration. The liquid–liquid extraction technique was used to extract copanlisib from the DBS discs. We used an Atlantis dC18 column and isocratic mobile phase for the chromatographic separation of copanlisib and the internal standard (idelalisib). The flow was 0.90 ml/min. Under the optimized chromatographic conditions, the retentions of copanlisib and the internal standard were 0.98 and 0.93 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 481.31 → 128.00 and 416.10 → 176.10 for copanlisib and the internal standard (IS) idelalisib, respectively. We have used a broad calibration range (1.01–4,797 ng/ml) with a determination coefficient (r2) of 0.997. All of the evaluated parameters met the acceptance criteria. Hematocrit did not influence the DBS copanlisib concentrations. We have used the validated method to derive the intravenous pharmacokinetic parameters by quantifying copanlisib in mouse plasma.