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Abstract Introduction
The development of combination antiretroviral (ARV) therapy had a major impact on survival rate and quality of life of HIV-infected patients. The long-term drug regimens raise some specific issues, among other adherence, virus resistance, and associated metabolic syndrome. In order to improve the treatment, these patients need close clinical and laboratory evaluations, including therapeutic drug monitoring (TDM).
Objective
The aim of our study was to develop a novel method for simultaneously determination of 14 ARV drugs currently used in our hospital (atazanavir, darunavir, lopinavir, ritonavir, abacavir, emtricitabine, lamivudine, zidovudine, raltegravir, dolutegravir, efavirenz, nevirapine, etravirine and maraviroc) in plasma samples of treated patients by micro-liquid-chromatography coupled with mass spectrometry (micro-LC-MS/MS).
Methods
Quantitative determination of ARVs was performed in Multiple Reaction Monitoring, positive electrospray ionization, using deuterated analogues as internal standards. Analytes were isolated from biological samples by protein precipitation. Because of the very different polarity of the target analytes, the chromatographic separation was challenging, and after testing several stationary phases, a PFP (50x0.5 mm, 2.7 μm, 90A) column was selected. Calibration curves were built in plasma enriched with analyzed drugs at different concentrations, according to their pharmacokinetics. The overall range was from 1 to 15000 ng/mL. Quality control (QC) samples were prepared at 4 concentrations (3xLLOQ, 2 mid-range QCs and 1 high-range). Method was validated according to guidelines, for selectivity, linearity, accuracy, precision, samples stability. The clinical samples were collected with informed consent from 50 HIV positive patients of the National Institute of Infectious Diseases “Prof. Dr. Matei Bals”.
Results
Calibration curves with correlation coefficients >0.995 were obtained for all analytes. Adequate accuracy (85-115%) was also obtained, with good precision (within-run and between-run CVs <10%) in the whole measured range for most analytes, except zidovudine (CV>15% at low concentrations due to sensitivity issues). Maraviroc, efavirenz, atazanavir and raltegravir also showed higher imprecision (CVs 10-15%).
The plasma samples from 50 treated patients were collected before ARV administration (for Cthrough), but also at 2, 4 and 8 hours after administration. ARVs concentrations showed great variability among patients with the same therapy regimen. Adherence could be estimated by examining similar TDM results for the same patient in different sampling days. Three of the patients had no plasma concentrations of ARVs in the collected samples and were considered non-adherent.
Conclusion
The new method was applied in our hospital for monitoring patients with long therapeutic experience and multi-drug resistance. TDM results, together with viral load and resistance data were used for the therapeutic decisions.
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= Discovery stage.
= Translation stage.
= Clinically available.
