Menthol-assisted homogenous liquid-liquid microextraction for HPLC/UV determination of favipiravir as an antiviral for COVID-19 in human plasma

Abstract

Favipiravir is a promising antiviral agent that has been recently approved for treatment of COVID-19 infection. In this study, a menthol-assisted homogenous liquid-liquid microextraction method has been developed for favipiravir determination in human plasma using HPLC/UV. The different factors that could affect the extraction efficiency were studied, including extractant type, extractant volume, menthol amount and vortex time. The optimum extraction efficiency was achieved using 300 µL of tetrahydrofuran, 30 mg of menthol and vortexing for 1 min before centrifuging the sample for 5 min at 3467g. Addition of menthol does not only induce phase separation, but also helps to form reverse micelles to facilitate extraction. The highly polar favipiravir molecules would be incorporated into the hydrophilic core of the formed reverse micelle to be extracted by the non-polar organic extractant. The method was validated according to the FDA bioanalytical method guidelines. The developed method was found linear in the concentration range of 0.1 to 100 µg/mL with a coefficient of determination of 0.9992. The method accuracy and precision were studied by calculating the recovery (%) and the relative standard deviation (%), respectively. The recovery (%) was in the range of 97.1-103.9%, while the RSD (%) values ranged between 2.03 and 8.15 %. The developed method was successfully applied in a bioequivalence study of Flupirava® 200 mg versus Avigan® 200 mg, after a single oral dose of favipiravir administered to healthy adult volunteers. The proposed method was simple, cheap, more eco-friendly and sufficiently sensitive for biomedical application.

Keywords: Bioequivalence; COVID-19; FDA; Favipiravir; Menthol; Microextraction.

Fig. 1

Procedures of favipiravir sample preparation by the proposed menthol-assisted homogenous liquid–liquid microextraction method.

Fig. 2

Chromatographic separation of favipiravir using (a) chloroform-assisted homogenous liquid–liquid extraction and (b) menthol-assisted homogenous liquid–liquid extraction. Chromatographic conditions: Column: Thermo Hypersil ODS C18 (250 mm × 4.6 mm, 5 µm) at 30 °C, Mobile phase: acetonitrile: phosphate buffer (50 mM, pH 2.5) (40:60, v/v), Elution: Isocratic, Detection: DAD at 323 nm, Flow rate: 1 mL/min, Injection volume: 5 µL.

Fig. 3

The light microscope images of the extracts in chloroform-assisted homogenous liquid–liquid microextraction (a), and menthol-assisted homogenous liquid–liquid microextraction (b). Chemical structures of favipiravir, propranolol as an internal standard and menthol (c).

Fig. 4

Effect of extractant type (a), THF volume (b), and menthol amount (c) on the efficiency of menthol-assisted homogeneous liquid–liquid microextraction of favipiravir. Favipiravir concentration: 10 µg/mL, Centrifugation time: 5 min, Chromatographic conditions: Column: Thermo Hypersil ODS C18 (250 mm × 4.6 mm, 5 µm) at 30 °C, Mobile phase: acetonitrile: phosphate buffer (50 mM, pH 2.5) (40:60, v/v), Elution: Isocratic, Detection: DAD at 323 nm, Flow rate: 1 mL/min, Injection volume: 5 µL. The error bar represents the standard deviation of three readings.

Fig. 5

Representative HPLC chromatograms for favipiravir in spiked plasma samples (a), and in real plasma sample (b) from a healthy volunteer administered one tablet of Flupirava® 200 mg under fasting conditions. The real sample was collected at Tmax (0.5 h). Chromatographic conditions: Column: Thermo Hypersil ODS C18 (250 mm × 4.6 mm, 5 µm) at 30 °C, Mobile phase: acetonitrile: phosphate buffer (50 mM, pH 2.5) (40:60, v/v), Elution: Isocratic, Detection: DAD at 323 nm, Flow rate: 1 mL/min, Injection volume: 5 µL.

Fig. 6

Favipiravir plasma concentration – time profile of four subjects after a single oral administration of Avigan® 200 mg (Reference product) versus Flupirava® 200 mg (Test product). The error bar represents the standard deviation of the different subjects.