The development and validation of a method using high-resolution mass spectrometry (HRMS) for the qualitative detection of antiretroviral agents in human blood

Mark A. Marzinke, Autumn Breaud, Teresa L. Parsons, Myron S. Cohen, Estelle Piwowar-Manning, Susan H. Eshleman, William Clarke

Research output: Contribution to journalArticle

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. ×. 3. mm) 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 10. ng/ml for 14 drugs (9 PIs, 1 NNRTI, 4 NRTIs) and was 150. ng/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. Conclusion: Using the Exactive-MS, we developed and validated a highly selective, robust method for the multiplexed detection of 15 antiretroviral drugs.

Original languageEnglish (US)
Pages (from-to)157-168
Number of pages12
JournalClinica Chimica Acta
Volume433
DOIs
StatePublished - Jun 10 2014

Keywords

  • Antiretroviral
  • Clinical trials
  • Exactive-MS
  • High-resolution mass spectrometry
  • Validation

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

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