A novel LC-MS/MS method for determination of the potential antiviral candidate favipiravir for the emergency treatment of SARS-CoV-2 virus in human plasma: Application to a bioequivalence study in Egyptian human volunteers

Abstract

A novel, fast and sensitive LC-MS/MS method was developed and validated for the bioanalysis of the antiviral agent favipiravir (FAV); a promising candidate for treatment of SARS-CoV-2 (COVID-19) in human plasma using pyrazinamide as an internal standard (IS). Simple protein precipitation was adopted for plasma sample preparation using methanol. Chromatographic separation was accomplished on Eclipse plus C₁₈ column (50 × 4.6 mm, 3.5 μm) using a mobile phase composed of methanol-0.2 % acetic acid (20:80, v/v) pumped at a flow rate 0.6 mL/min in an isocratic elution mode. The API4500 triple quadrupole tandem mass spectrometer was operated with multiple-reaction monitoring (MRM) in negative electrospray ionization interface for FAV and positive for IS. The MRM function was used for quantification, with the transitions set at m/z 156.00→ 113.00 and m/z 124.80→ 81.00 for FAV and IS. The method was optimized and fully validated in accordance to US-FDA guidelines. Linearity was acquired over a concentration range of 100.0-20000.0 ng/mL by computing using weighted linear regression strategy (1/x²). The proposed method was effectively applied for the pharmacokinetic evaluation of FAV and to demonstrate the bioequivalence of a new FAV formulation (test) and reference product in healthy Egyptian human volunteers.

Keywords: Bioequivalence study; COVID-19; Favipiravir; Human plasma; LC–MS/MS; SARS-CoV-2.

Graphical abstract

Fig. 1

Chemical structure of FAV (A) and the internal standard, pyrazinamide (B).

Fig. 2

Multiple reaction monitoring (MRM) Chromatograms of: (A) blank plasma, (B) blank plasma spiked at LLOQ, (C) plasma samples of subject at 0.5 h after oral administration of one tablet containing FAV 200 mg.

Fig. 3

Representative spectra for FAV (A) and the internal standard, pyrazinamide (B).

Fig. 4

Mean plasma concentration (± SD) following administration of single oral dose of FAV 200 mg tablets; Avipiravir (test product) and Avigan® (reference product) to 26 healthy subjects.