Development and bioanalytical validation of a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the quantification of the CCR5 antagonist maraviroc in human plasma

• Development of a tandem mass spectrometric method for plasma maraviroc quantification
• Validation of LC-MS/MS assay following FDA guidelines
• Validation of a robust method with a measuring range of 0.5–1000 ng/ml

BackgroundMaraviroc is a CCR5 antagonist that has been utilized as a viral entry inhibitor in the management of HIV-1. Current clinical trials are pursuing maraviroc drug efficacy in both oral and topical formulations. Therefore, in order to fully understand drug pharmacokinetics, a sensitive method is required to quantify plasma drug concentrations.MethodsMaraviroc-spiked plasma was combined with acetonitrile containing an isotopically-labeled internal standard, and following protein precipitation, samples were evaporated to dryness and reconstituted for liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Chromatographic separation was achieved on a Waters BEH C8, 50 × 2.1 mm UPLC column, with a 1.7 μm particle size and the eluent was analyzed using an API 4000 mass analyzer in selected reaction monitoring mode. The method was validated as per FDA Bioanalytical Method Validation guidelines.ResultsThe analytical measuring range of the LC-MS/MS method is 0.5–1000 ng/ml. Calibration curves were generated using weighted 1/x2 quadratic regression. Inter-and intra-assay precision was ≤ 5.38% and ≤ 5.98%, respectively; inter-and intra-assay accuracy (%DEV) was ≤ 10.2% and ≤ 8.44%, respectively. Additional studies illustrated similar matrix effects between maraviroc and its internal standard, and that maraviroc is stable under a variety of conditions. Method comparison studies with a reference LC-MS/MS method show a slope of 0.948 with a Spearman coefficient of 0.98.ConclusionsBased on the validation metrics, we have generated a sensitive and automated LC-MS/MS method for maraviroc quantification in human plasma.