Nhi Phung1, Karen Kuncze1, Hideaki Okochi2, Alexander Louie1, Leslie Z Benet2, Igho Ofokotun3, David W Haas4, Judith S Currier5, Tariro D Chawana6, Anandi N Sheth3, Peter Bacchetti7, Monica Gandhi1, Howard Horng1. 1. Division of HIV, Infectious Diseases and Global Medicine, Department of Medicine, University of California, San Francisco, San Francisco, CA, 94143, USA. 2. Department of Bioengineering and Therapeutic Sciences and Department of Medicine, University of California, San Francisco, San Francisco, CA, 94143, USA. 3. Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia GA, USA; and Grady Healthcare System, Atlanta, Georgia GA, USA. 4. Division of Infectious Diseases, Department of Medicine, Vanderbilt School of Medicine and Department of Internal Medicine, Meharry Medical College, Nashville, TN, USA. 5. Division of Infectious Diseases, Department of Medicine, University of California, Los Angeles, CA, USA. 6. Department of Clinical Pharmacology, University of Zimbabwe, Harare, Zimbabwe. 7. Department of Epidemiology and Biostatistics, University of California, San Francisco (UCSF), San Francisco, CA, 94143, USA.
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.
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-infectedpatients on ATV-based ART.
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