MSACL 2019 EU Abstract

Development and Validation of the First UHPLC-MS/MS Method for the Quantification of the New Anti-Ebola Drug Remdesivir: Application to Healthy Volunteers

Valeria Avataneo 1, Amedeo De Nicolò 1, Miriam Antonucci 1, Elisa De Vivo 1, Jessica Cusato 1, Mohammed Lamorde 2, Giovanni Di Perri 1 and Antonio D’Avolio 1.
1: Laboratory of Clinical Pharmacology and Pharmacogenetics, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy. 2: Infectious Diseases Institute, Makerere University College of Health Sciences, P.O. Box 22418, Kampala, Uganda.

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Antonio D'Avolio (Presenter) University of Turin >> POSTER (PDF)

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

Aim of the work. Ebola virus disease shows a very high death rate (up to 90%) and the 2014-16 outbreak has been one of the deadliest since 1976, year in which ebola virus was identified. Nevertheless, up to now, any effective pharmacological treatment has been discovered. Some molecules are under study and, among all, remdesivir (RDV) revealed really promising and is now on fase II/III studies. Unfortunately, detailed information about RDV pharmacokinetics are still lacking and no methods for its quantification in patient’s plasma have been reported in literature.
The aim of this work is the development and validation of a method for the Therapeutic Drug Monitoring (TDM) of RDV using liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), in order to describe its pharmacokinetics in healthy volunteers.
Materials and Methods. Briefly, to a volume of 50μl of plasma sample/calibration standard/quality control (QC) are added 100μl of internal standard (IS, a mixture of 6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline –QX– and tenofovir-alafenamide –TAF–) and 600μl of methanol:acetonitrile 50:50 (v:v). After vortex and centrifuge steps, 300µL of the supernatant are diluted with 600μl of water and injected on a UHPLC system, coupled with Q-Sight 220 MS/MS detector. Chromatographic separation is obtained on a HSS T3 1.8μm 2.1x50 mm column, with a binary gradient separation of water and acetonitrile, both added with formic acid 0.05%. Positive electrospray ionization (ESI+) is used for both RDV and its ISs. The quantification MRM traces (m/z) are: 603.15>200 for RDV, 313.2>78.05 for QX and 477.20>346.05 for TAF. Validation process, according to FDA and EMA guidelines, is still ongoing: recovery (REC), matrix effect (ME), IS-normalized matrix effect, extraction efficiency (EE), accuracy, intra- and inter-day precision are being evaluated in 6 different analytical sessions. The validated method will be tested on real samples from healthy patients, enrolled in the CAPA-CT-II study, all giving informed consent.
Results. All analytes are successfully retained by the column: TAF retention time is 0.95 min (column “dead-time” is 0.32 min) while QX and RDV co-elute at 1.57 min. Preliminary data from the first four sessions are very encouraging: for remdesivir, REC resulted among 70 and 80%, ME ranged among -3 and +10%, EE was approximately 75%; accuracy and precision data are also within the limits indicated by the guidelines. RDV calibration curves resulted linear (r2 > 0.996) with 1/X weighting.
Conclusions. This method is currently being validated according to FDA and EMA guidelines, and results the first for RDV quantification. Its main features are the very short analytical run (4 min) and the very small amount of plasma required (50μl). Precipitation and strong dilution of samples (45-folds) contribute to a low instrumental contamination and low matrix effect.