Development and validation of an assay to analyze atazanavir in human hair via liquid chromatography/tandem mass spectrometry

• Authors: Nhi Phung, University of California San Francisco; Karen Kuncze, University of California San Francisco; Hideaki Okochi, University of California San Francisco; Alexander Louie, University of California San Francisco; Leslie Z. Benet, University of California San Francisco; Igho Ofokotun, Emory University; David W. Haas, Vanderbilt University; Judith S. Currier, University of California Los Angeles; Tariro D. Chawana, University of Zimbabwe; Anandi Sheth, Emory University; Peter Bacchetti, University of California San Francisco; Monica Gandhi, University of California San Francisco; Howard Horng, University of California San Francisco
• Journal Title: Rapid Communications in Mass Spectrometry
• Volume: Volume 32, Number 5
• Publisher: Wiley: 12 months | 2018-03-15, Pages 431-441
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1002/rcm.8058
• Subjects: Chemistry, Analytical; Chemistry, Biochemistry; Biology, Molecular
• Keywords: Science & Technology; Life Sciences & Biomedicine; Physical Sciences; Technology; Biochemical Research Methods; Chemistry, Analytical; Spectroscopy; Biochemistry & Molecular Biology; Chemistry; HIV ANTIRETROVIRAL THERAPY; DRUG-FACILITATED CRIMES; PREEXPOSURE PROPHYLAXIS; PROTEASE INHIBITORS; HUMAN PLASMA; STRONGEST PREDICTOR; INFECTED PATIENTS; TREATMENT-NAIVE; UNITED-STATES; ADHERENCE

Abstract

Rationale: Assays to quantify antiretrovirals in hair samples are increasingly used to monitor adherence and exposure in both HIV prevention and treatment studies. Atazanavir (ATV) is a protease inhibitor used in combination antiretroviral therapy (ART). We developed and validated a liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based method to quantify ATV in human hair, per the NIH Division of AIDS Clinical Pharmacology Quality Assurance (CPQA) program and the FDA bioanalytical method validation guidelines. Methods: ATV was extracted from hair using optimized methods and the extracts were injected onto a BDS C-18 column (5 μm, 4.6 × 100 mm), followed by isocratic elution via a mobile phase composed of 55% acetonitrile, 45% water, 0.15% acetic acid, and 4 mM ammonium acetate, at a flow rate of 0.8 mL/min prior to analysis by MS/MS. Levels were quantified using positive electrospray ionization by multiple reaction monitoring (MRM) for the transitions MH+m/z 705.3 to m/z 168.0 and MH+m/z 710.2 to m/z 168.0 for ATV and ATV-d5 (internal standard), respectively. Results: Our assay demonstrated a linear standard curve (r = 0.99) over the concentration range of 0.0500 ng ATV/mg hair to 20.0 ng/mg hair. The inter- and intraday accuracy of ATV quality control (QC) samples was −1.33 to 4.00% and precision (% coefficient of variation (%CV)) was 1.75 to 6.31%. The %CV for ATV levels in hair samples from highly adherent patients (incurred samples) was less than 10%. No significant endogenous peaks or crosstalk were observed in the specificity test with other HIV drugs. The overall extraction efficiency of ATV from incurred hair samples was greater than 95%. Conclusions: This highly sensitive, highly specific and validated assay can be considered for therapeutic drug monitoring for HIV-infected patients on ATV-based ART.