Dual quantification of dapivirine and maraviroc in cervicovaginal secretions from ophthalmic tear strips and polyester-based swabs via liquid chromatographic–tandem mass spectrometric (LC–MS/MS) analysis

• Development of an LC–MS/MS method for dapivirine and maraviroc quantification in cervicovaginal fluid.
• Drug quantification in cervicovaginal fluid collected on multiple devices.
• Validation of LC–MS/MS assays following FDA guidelines.

BackgroundTopical microbicidal agents are being actively pursued as a modality to prevent HIV viral transmission during sexual intercourse. Quantification of antiretroviral agents in specimen sources where antiviral activity is elicited is critical, and drug measurements in cervicovaginal fluid can provide key information on local drug concentrations. Two antiretroviral drugs, dapivirine and maraviroc, have gained interest as vaginal microbicidal agents, and rugged methods are required for their quantification in cervicovaginal secretions.MethodsCervicovaginal fluid spiked with dapivirine and maraviroc were applied to ophthalmic tear strips or polyester-based swabs to mimic collection procedures used in clinical studies. Following sample extraction and the addition of isotopically labeled internal standards, samples were subjected to liquid chromatographic–tandem mass spectrometric (LC–MS/MS) analysis using a Waters BEH C8, 50 mm × 2.1 mm, 1.7 μm particle size column, on an API 4000 mass analyzer operated in selective reaction monitoring mode. The method was validated according to FDA Bioanalytical Method Validation guidelines.ResultsDue to the disparate saturation capacity of the tested collection devices, the analytical measuring ranges for dapivirine and maravirocin cervicovaginal fluid on the ophthalmic tear strip were 0.05–25 ng/tear strip, and 0.025–25 ng/tear strip, respectively. As for the polyester-based swab, the analytical measuring ranges were 0.25–125 ng/swab for dapivirine and 0.125–125 ng/swab for maraviroc. Dilutional studies were performed for both analytes to extended ranges of 25,000 ng/tear strip and 11,250 ng/swab. Standard curves were generated via weighted (1/x2) linear or quadratic regression of calibrators. Precision, accuracy, stability and matrix effects studies were all performed and deemed acceptable according to the recommendations of the FDA Bioanalytical Method Validation guidelines.ConclusionsA rugged LC–MS/MS method for the dual quantification of dapivirine and maraviroc in cervicovaginal fluid using two unique collection devices has been developed and validated. The described method meets the criteria to support large research trials.

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