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A rapid and simple LC–MS/MS assay for carfilzomib was validated.Sample preparation involved a simple deproteinization with acetonitrile.The total chromatographic run time was 2.5 min.The LLOQ was 0.075 ng/mL in 5 mL of plasma.The assay may allow serial blood sampling from one mouse in pharmacokinetic studies.A highly sensitive and rapid LC–MS/MS method was developed and validated to determine the levels of carfilzomib in mice plasma by using chlorpropamide as an internal standard. Carfilzomib and chlorpropamide were extracted from 5 μL of plasma after protein precipitation with acetonitrile. Chromatographic separation was performed on Phenomenex Luna C18 column (50 × 2.0 mm id, 3 μm). The mobile phase consisted of 0.1% formic acid in acetonitrile −0.1% formic acid in water (1:1 v/v) and the flow rate was 0.3 mL/min. The total chromatographic run time was 2.5 min. Detection was performed on a triple quadrupole mass spectrometer equipped with positive-ion electrospray ionization by selected reaction monitoring of the transitions at m/z 720.20 > 100.15 (for carfilzomib) and m/z 277.05 > 111.05 (for the internal standard). The lower limit of quantification was 0.075 ng/mL and the linear range was 0.075–1250 ng/mL (r ≥ 0.9974). All validation data, including selectivity, precision, accuracy, matrix effect, recovery, dilution integrity, stability, and incurred sample reanalysis, were well within acceptance limits. This newly developed bioanalytical method was simple, highly sensitive, required only a small volume of plasma, and was suitable for application in pharmacokinetic studies in mice that used serial blood sampling.