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

Abstract

Background: Topical 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.

Methods: Cervicovaginal 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, 50mm×2.1mm, 1.7μm 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.

Results: Due 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 0.05-25ng/tear strip, and 0.025-25ng/tear strip, respectively. As for the polyester-based swab, the analytical measuring ranges were 0.25-125ng/swab for dapivirine and 0.125-125ng/swab for maraviroc. Dilutional studies were performed for both analytes to extended ranges of 25,000ng/tear strip and 11,250ng/swab. Standard curves were generated via weighted (1/x(2)) 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.

Conclusions: A 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.

Keywords: Antiretroviral; Cervicovaginal fluid; Dapivirine; Maraviroc; Validation.

Figure 1

Structures of (A) the non-nucleoside reverse transcriptase inhibitor dapivirine (C₂₀H₁₉N₅) and B) the CCR-5 antagonist maraviroc (C₂₉H₄₁F₂N₅O). * denotes position of deuterium atoms for the isotopically-labeled internal standards used for analyte quantification.

Figure 2

Representative chromatograms of (A) dapivirine and (C) ²H₄-dapivirine, with retention time demarcated at 1.14 min; and (B) maraviroc and (D) ²H₆-maraviroc, with retention time noted at 0.60 min, from cervicovaginal fluid extracted from polyester-based swabs.

Figure 3

Calibration curves of dapivirine(DPV) from cervicovaginal fluid applied to (A) an ophthalmic tear strip or (C) a polyester-based swab. The analytical measuring range for (A) is 0.05 to 25 ng/tear strip and (C) is 0.25 to 125 ng/swab. Panels (B) and (D) illustrate calibration curves of maraviroc (MVC) from cervicovaginal fluid applied to (A) an ophthalmic tear strip or (D) a polyester-based swab. The analytical measuring range for (B) is 0.025 to 25 ng/tear strip and (D) 0.125 to 125 ng/swab. The curves are fit to the calibrators with either a (A) quadratic or (B-D) linear regression using 1/x² weighting. Representative slopes of the line are illustrated within the figure

Figure 4

Selectivity of dapivirine and maraviroc transitions. (A–B) Chromatograms illustrating the extracted ion current of thedapivirine transition from drug-free cervicovaginal fluid applied to (A) an ophthalmic tear strip or (B) a polyester-based swab. Chromatograms are overlaid with a sample spiked with the lower limit of quantification, (A) 0.05 ng/tear strip and (B) 0.25 ng/swab; the LLOQ is depicted green. (C–D) Chromatograms illustrating the extracted ion current of the maraviroc transition from drug-free cervicovaginal fluid applied to (C) an ophthalmic tear strip or (C) a polyester-based swab. Chromatograms are overlaid with a sample spiked with the lower limit of quantification, (C) 0.025 ng/tear strip and (D) 0.125 ng/swab; the LLOQ is depicted in purple.