MSACL 2019 EU Abstract

A UHPLC-MS/MS Method for the Simultaneous Quantification of Ten Directly Acting Anti-HCV Drugs in Patient Plasma

Amedeo De Nicolò, Luca Paglietti, Valeria Avataneo, Miriam Antonucci, Elisa De Vivo, Alice Ianniello, Jessica Cusato, Giovanni Di Perri and Antonio D’Avolio.
1.Laboratory of Clinical Pharmacology and Pharmacogenetics #, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy. 2.Division of Internal Medicine and Hypertension Unit, University of Turin, Department of Medical Sciences, AOU “Città della Salute e della Scienza”, Turin, Italy.

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Amedeo De Nicolò (Presenter) University of Turin >> POSTER (PDF)

Presenter Bio: I graduated and achieved a PhD in Medicine and Experimental Therapy at the University of Turin. My main research field consists in the development and validation of chromatographic methods for drugs quantification at the Laboratory of Clinical Pharmacology and Pharmacogenetics of “Amedeo di Savoia” Hospital, Turin, Italy.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction: To date, the infection by Hepatitis C Virus (HCV) affects more than 130 million patients worldwide and causes hepatic cirrhosis, hepatocarcinoma and liver transplantation. In the recent years, Directly Acting Antivirals (DAAs) greatly improved the effectiveness of anti-HCV treatments. Despite this, some issues concerning patients with comorbidities and polytherapies (eg. drug-drug interactions) are still present. For this reason, measurement of DAAs concentrations in patients plasma could be useful for the pharmacokinetic research or Therapeutic Drug Monitoring (TDM).
Nevertheless, no method is available in literature to quantify all the new DAAs.
Aim: The aim of this work was the validation of a UHPLC-MS/MS multiplexed method to quantify 10 currently administered anti-HCV drugs.
Materials and Methods: Briefly, 200 μL of plasma (both standards, quality controls and patients samples) were added with 50 μL of Internal Standard (1 μg/mL of thymidine, 100 ng/mL of 6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline, D4-Daclatasvir and D3-Omditasvir) and 400 μL of 1% Phosphoric Acid. After mixing and centrifugation, the supernatant was loaded on Oasis ® HLB SPE 30 mg plates (Waters, Milan), previously activated and equilibrated.
After a washing step with 200 μL of pure water, elution was obtained with 350 μL of a mixture of acetonitrile:methanol 60:40 v:v, diluted with 400 μL of water + 0.05% formic acid and 4 µL were injected in the LX-50 ® chromatographic system (Perkin Elmer). Chromatographic separation was obtained with a 7 minute gradient of water and acetonitrile, both acidified with 0.05% of formic acid, on an Acquity ® HSS T3 1.8 μm 2.1 x 100 mm (Waters, Milan), maintained at 45°C. The mass instrument was a QSight 220 ® (Perkin Elmer) and the quantification was performed by multiple reaction monitoring (MRM) with ESI positive, except for glecaprevir and dasabuvir, which were ionized in negative mode.
Results: The drugs were successfully recovered by SPE and chromatographically separated. All extraction recoveries and matrix effect data resulted consistent (>80%) and contained (<20%), respectively, and reproducible (both CV% < 15%). Accuracy, intra- and inter-day precision values resulted within the limits indicated by EMA and FDA guidelines. Calibration curves were linear (R2 > 0.996) for all the considered drugs. Stability studies are still ongoing. The method is now being applied to a clinical pharmacokinetic study (KINETI-C), resulting useful for the identification of clinically relevant drug-drug interactions.
Conclusion: The described method is the first capable of simultaneously quantifying all the most used anti-HCV drugs, resulting widely applicable in both the pharmacokinetic research and clinical TDM context.