Sensitive LC-MS/MS Method for the Simultaneous Determination of Bendamustine and its Active Metabolite, γ-Hydroxybendamustine in Small Volume Mice and Dog Plasma and its Application to a Pharmacokinetic Study in Mice and Dogs

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxybendamustine (HBM) in small volume (20 µL) mice and dog plasma using phenacetin as an internal standard (IS) as per regulatory guidelines. Both the analytes and IS were extracted from mice and dog plasma using a liquid-liquid extraction method. Chromatography was achieved on Atlantis dC 18 column using an isocratic mobile phase (0.2% formic acid:acetonitrile, 25:75) at a flow rate of 0.40 mL/min. The total chromatographic run time was 3.0 min and the elution of BM, HBM and IS occurred at ~1.2, 1.2 and 2.0 min, respectively. A linear response function was established 0.11–518 ng/mL for both the analytes in mice and dog plasma. The intra- and inter-day accuracy and precisions were in the range of 3.46–12.9 and 3.63–8.23%; 1.15–9.00 and 7.86–9.49% for BM and HBM, respectively in mice plasma and 2.15–6.49 and 1.73–13.1%; 4.35–13.9 and 4.33–10.5% for BM and HBM, respectively in dog plasma. This novel method has been applied to a pharmacokinetic study in mice and dogs.