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

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.

Measuring Adherence to Antiretroviral Therapy via Hair Concentrations in India

“…This paper examines hair levels as objective markers of antiretroviral adherence among people living with HIV on ART in India for the first time, further exploring the transfer of the hair testing methodology to an India-based laboratory. We found moderate (higher for efavirenz) levels of agreement and correlation between the hair ARV concentrations measured in the India-based laboratory to those measured in the U.S.-based laboratory (the UCSF HAL 26 ), whose assays are reviewed by the Clinical Pharmacology and Quality Assurance program. 22 Moreover, as in previous studies, we found that self-reported adherence was not well-correlated with an objective metric of adherence using hair levels.…”

Tenofovir and emtricitabine concentrations in hair are comparable between individuals on tenofovir disoproxil fumarate versus tenofovir alafenamide‐based ART

“…Blank hair samples were used as a matrix for standards and controls and were acquired from healthy volunteers, who were HIV‐negative and had never taken TDF, TAF, and FTC. Positive control hair samples were acquired from participants living with HIV, who were recruited from the “Shaved Heads Study” from the UCSF HIV clinic (“Ward 86”) at Zuckerberg San Francisco General Hospital 19 . The “Shaved Heads Study” procedures were approved by the UCSF Institutional Review Board (IRB#14‐13687) and all participants provided written informed consent prior to participation.…”

“…Positive control hair samples were acquired from participants living with HIV, who were recruited from the "Shaved Heads Study" from the UCSF HIV clinic ("Ward 86") at Zuckerberg San Francisco General Hospital. 19 The "Shaved Heads Study" procedures were approved by the UCSF Institutional Review Board (IRB#14-13687) and all participants provided written informed consent prior to participation. Participants enrolled in the current study were required to be on TDF-or TAF-containing ART for at least 6 months with good adherence as reported by the provider, the participant, and sustained documented virologic suppression (HIV RNA < 40 copies/ml).…”

“…Hair samples are collected non‐invasively and can be shipped and stored at room temperature 17 . The University of California San Francisco‐Hair Analytical Laboratory (UCSF‐HAL) has quantified a number of ARVs in hair (e.g., atazanavir, 19 lopinavir, 20 ritonavir, 20 efavirenz, 20 nevirapine, 21 darunavir, raltegravir, dolutegravir, TFV/FTC, 22–29 ) for research projects over the past 15 years. Hair concentrations of ARVs have been shown to be strong predictors of virologic response for a variety of ARVs, including TFV/FTC 30–41 …”

Development and Validation of an Immunoassay for Tenofovir in Urine as a Real-Time Metric of Antiretroviral Adherence

“…For the development of a TFV-specific immunoassay ( Fig. 1 illustrates a schema of immunoassay development) [17] , the UCSF Hair Analytical Laboratory (HAL), with expertise in LC/MS–MS and analyzing ARV concentrations in hair in the context of HIV treatment and prevention [18] , [19] , formed a collaboration with Alere™ Rapid Diagnostics, a company with expertise in developing POC immunoassays [20] , [21] . Scientists from the UCSF HAL and Alere™ scrutinized the molecular structure of TFV to identify unique derivatives with structural distinction from endogenous nucleotides.…”