title: Development and validation of a liquid chromatographic-tandem mass spectrometric method for the multiplexed quantification of etravirine, maraviroc, raltegravir, and rilpivirine in human plasma and tissue.
abstract: BACKGROUND
  Analytical methodologies for antiretroviral (ARV) quantification are important in determining both systemic and localized drug concentrations. The CCR5-antagonist maraviroc (MVC), the non-nucleoside reverse transcriptase inhibitors (NNRTIs) etravirine (ETV) and rilpivirine (RPV), as well as the integrase strand transfer inhibitor (INSTI) raltegravir (RAL), have all been evaluated using both oral and non-oral dosing regimens, demonstrating a need for dynamic and sensitive bioanalytical tools for drug quantification in plasma and tissue.
  
  
  METHODS
  K2EDTA plasma or blank luminal tissue lysate were spiked with ETV, MVC, RAL, and RPV. Following the addition of isotopically-labeled internal standards and sample extraction via protein precipitation or solid phase extraction, respectively, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Chromatographic separation was performed using a Waters BEH C8, 50×2.1mm, 1.7μm particle size column, and detected on an API 5000 mass analyzer operated in selective reaction monitoring mode. The method was validated according to FDA Bioanalytical Method Validation guidelines.
  
  
  RESULTS
  Analytical methods were optimized for the multiplexed monitoring of ETV, MVC, RAL, and RPV in plasma and homogenized tissue lysate. The lower limits of quantification (LLOQs) for ETV, RAL, and RPV were 1ng/mL and the LLOQ for MVC was 0.1ng/mL in plasma; the LLOQs for all ARVs in homogenized tissue lysate was 0.05ng/sample. Standard curves were generated via weighted quadratic (plasma) or linear (tissue) regression of calibrators. Intra- and inter-assay precision and accuracy studies demonstrated %CVs≤15.93% and %DEVs ≤±13.52%, respectively. Stability and matrix effects studies, as well as external proficiency testing assessment, were also performed. All results were acceptable and in accordance with the guidelines recommended by the FDA, Guidance for Industry: Bioanalytical Method Validation document.
  
  
  CONCLUSIONS
  LC-MS/MS assays that are sensitive, specific, and dynamic have been developed and validated for the multiplexed quantification of ETV, MVC, RAL, and RPV in plasma and homogenized tissue lysate. The described methods meet sufficient throughput criteria to support large research trials.
authors.affiliation: Department of Medicine, Johns Hopkins University, Bayview Medical Center, 4940 Eastern Ave, Mason F. Lord Tower, Suite 6000, Room 606, Baltimore, MD, 21224, USA.
authors.forename: Teresa L
authors.initials: TL
authors.lastname: Parsons
authors.affiliation: Departments of Medicine and Pathology, Johns Hopkins University, 1800 Orleans St., 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: D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D003510:Cyclohexanes / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D000077592:Maraviroc; D009570:Nitriles; D011724:Pyridazines / Q000032:analysis / Q000378:metabolism*; D011743:Pyrimidines; D000068898:Raltegravir Potassium / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D000068696:Rilpivirine / Q000032:analysis / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*; D014230:Triazoles / Q000032:analysis / Q000378:metabolism*
chemical_list: D003510:Cyclohexanes; D009570:Nitriles; D011724:Pyridazines; D011743:Pyrimidines; D014230:Triazoles; C451734:etravirine; D000068898:Raltegravir Potassium; D000068696:Rilpivirine; D000077592:Maraviroc
keywords: Antiretroviral; HIV; LC–MS/MS; Tissue; Validation