title: The development and validation of a method using high-resolution mass spectrometry (HRMS) for the qualitative detection of antiretroviral agents in human blood.
abstract: BACKGROUND
  Antiretroviral drugs are used for the treatment and prevention of HIV infection. Non-adherence to antiretroviral drug regimens can compromise their clinical efficacy and lead to emergence of drug-resistant HIV. Clinical trials evaluating antiretroviral regimens for HIV treatment and prevention can also be compromised by poor adherence and non-disclosed off-study antiretroviral drug use. This report describes the development and validation of a high throughput, qualitative method for the identification of antiretroviral drugs using high-resolution mass spectrometry (HRMS) for the retrospective assessment of off-study antiretroviral drug use and the determination of potential antiretroviral therapy (ART) non-compliance.
  
  
  METHODS
  Serum standards were prepared that contained 15 antiretroviral drugs: 9 protease inhibitors (PIs), 4 nucleotide/nucleoside reverse transcriptase inhibitors (NRTIs), and 2 non-nucleoside/nucleotide reverse transcriptase inhibitors (NNRTIs). Analytical separation was achieved on a Hypersil Gold PFP (100×3mm) column and the eluent was analyzed using the Thermo Exactive Orbitrap mass spectrometer (Exactive-MS) operated in full scan mode. Limit of identification, signal intensity precision, retention time analysis, selectivity, and carryover studies were conducted. Concordance with liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods was evaluated using remnant plasma samples from a clinical trial.
  
  
  RESULTS
  The limit of identification ranged from 5 to 10ng/ml for 14 drugs (9 PIs, 1 NNRTI, 4 NRTIs) and was 150ng/ml for 1 NNRTI. Precision studies with high and low control mixtures revealed signal intensity coefficients of variation of 3.0-27.5%. The Exactive-MS method was selective for the compounds of interest. Overall, concordance ranged from 89.1% to 100% for the screening of antiretroviral drugs in clinical plasma specimens as compared to LC-MS/MS methods.
  
  
  CONCLUSIONS
  Using the Exactive-MS, we developed and validated a highly selective, robust method for the multiplexed detection of 15 antiretroviral drugs.
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Medicine, the Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Marzinke
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Autumn
authors.initials: A
authors.lastname: Breaud
authors.affiliation: Department of Medicine, the Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Teresa L
authors.initials: TL
authors.lastname: Parsons
authors.affiliation: Institute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Myron S
authors.initials: MS
authors.lastname: Cohen
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Estelle
authors.initials: E
authors.lastname: Piwowar-Manning
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Susan H
authors.initials: SH
authors.lastname: Eshleman
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: wclarke@jhmi.edu.
authors.forename: William
authors.initials: W
authors.lastname: Clarke
mesh_terms: D019380:Anti-HIV Agents / Q000097:blood* / Q000627:therapeutic use; D001774:Blood Chemical Analysis / Q000379:methods*; D015658:HIV Infections / Q000188:drug therapy; D006801:Humans; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods*; D010349:Patient Compliance
chemical_list: D019380:Anti-HIV Agents
keywords: Antiretroviral; Clinical trials; Exactive-MS; High-resolution mass spectrometry; Validation