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Development and Validation of an Assay to Analyze Atazanavir in Human Hair Via Liquid Chromatography‐Tandem Mass Spectrometry (LC‐MS/MS)

Abstract

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. ATV was extracted from hair using optimized methods and the extracts were injected onto a BDS C-18 column (5 μm, 4.6 x 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. 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 intra-day 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%. This highly sensitive, highly specific and validated assay can be considered for therapeutic drug monitoring for HIV-infected patients on ATV-based ART.

Authors:   Nhi Phung, Karen Kuncze, Hideaki Okochi, Alexander Louie, Leslie Z. Benet, Igho Ofokotun, David W. Haas, Judith S. Currier, Tariro D. Chawana, Anandi N. Sheth, Peter Bacchetti, Monica Gandhi, Howard Horng
Journal:   Rapid Communications in Mass Spectrometry
Year:   2018
Pages:   n/a
DOI:   10.1002/rcm.8058
Publication date:   09-Jan-2018
Facts, background information, dossiers
  • hair
  • HIV
  • samples
  • Art
  • water
  • validation
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