title: 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.
authors.affiliation: Department of Medicine, Johns Hopkins University, 600 North Wolfe Street, Osler 500, Baltimore, MD 21287, USA.
authors.forename: Teresa L
authors.initials: TL
authors.lastname: Parsons
authors.affiliation: Department of Medicine, Johns Hopkins University, 600 North Wolfe Street, Osler 500, Baltimore, MD 21287, USA.
authors.forename: Joshua F
authors.initials: JF
authors.lastname: Emory
authors.affiliation: Department of Medicine, Johns Hopkins University, 600 North Wolfe Street, Osler 500, Baltimore, MD 21287, USA.
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Seserko
authors.affiliation: Department of Medicine, Johns Hopkins University, 600 North Wolfe Street, Osler 500, Baltimore, MD 21287, USA.
authors.forename: Wutyi S
authors.initials: WS
authors.lastname: Aung
authors.affiliation: Department of Medicine, Johns Hopkins University, 600 North Wolfe Street, Osler 500, Baltimore, MD 21287, USA; Department of Pathology, Johns Hopkins University, 1800 Orleans Street, Sheikh Zayed Tower, B1020-G, Baltimore, MD 21287, USA. Electronic address: mmarzin1@jhmi.edu.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Marzinke
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry; D002138:Calibration; D002584:Cervix Uteri / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D003510:Cyclohexanes / Q000737:chemistry*; D005260:Female; D006801:Humans; D000077592:Maraviroc; D011091:Polyesters / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D011934:Reagent Strips / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014230:Triazoles / Q000737:chemistry*; D014621:Vagina
chemical_list: D000998:Antiviral Agents; D003510:Cyclohexanes; D011091:Polyesters; D011743:Pyrimidines; D011934:Reagent Strips; D014230:Triazoles; D000077592:Maraviroc; C481671:Dapivirine
keywords: Antiretroviral; Cervicovaginal fluid; Dapivirine; Maraviroc; Validation