Background: Dapivirine (DPV) has been formulated in a vaginal ring (VR), which is currently being studied as an HIV prevention tool in open-label studies. Two clinical pharmacokinetics studies reported average plasma concentration of DPV over 30-days of VR use as 231±64 (MTN-013) and 273.5±98.2 pg/mL (MTN-024). Hair concentrations of antiretrovirals have proven useful as biomarkers of adherence and long-term exposure to orally-administered antiretrovirals. Hair is easier to collect and store than plasma or used VRs. We report, for the first time, the development of an assay to measure DPV concentrations in hair using liquid-chromatography tandem mass-spectrometry (LC-MS/MS).
Methods: Hair samples were collected from participants in the Microbicide Trials Network open-label extension (OLE) DPV-VR trial, MTN-025, for method development and optimization. DPV was extracted from hair (2-mg) by incubation in acidified methanol containing ²H₁₁-DPV (internal standard). The extracted solution was evaporated and reconstituted. DPV was extracted via liquid-liquid extraction using methyl tert-butyl ether. The organic layer was evaporated to dryness and reconstituted, and DPV was analyzed by the LC-MS/MS system (Shimadzu UFLC with Sciex API-5000 triple-quadrupole-mass-spectrometer) via multiple-reaction-monitoring electrospray in positive ionization mode. The standard curve was linear over the range of 0.01-10 ng/mg hair. Quantitation of DPV was determined by plotting peak area ratios of DPV to ²H₁₁-DPV versus the nominal concentration of DPV.
Results: Our analytical method exhibited high sensitivity (0.01 ng/mg hair) and a wide linear dynamic range (0.01-10 ng/mg) using 20-30 strands of hair. Precision (defined by the coefficient variation) and accuracy (defined by relative error) were both < 15%. DPV in hair specimens from four MTN-025 participants (as proof-of-concept) demonstrated a range of concentrations (0.0150, 0.0464, 0.0224, 0.0418 ng/mg).
Conclusions: We describe the development of a sensitive, specific, accurate and precise method per analytic parameters to determine adherence and exposure to DPV for women using the vaginal ring in small hair samples (20-30 strands). As proof-of-concept, this method provided a range of values among four MTN-025 participants. Further work in MTN-025 is ongoing to investigate the use of hair levels as a long-term measure of DPV use and to analyze DPV hair levels as predictors of seroconversion.

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