Miniaturized on-spot protein denaturation/microwave-assisted extraction for spectrofluorimetric determination of favipiravir in dried plasma: Application to real human sample and inclusive incurred sample reanalysis study

Abstract

The development of high-throughput sample preparation methods is a key step for upgrading the throughput of classical analytical techniques. In this work, a miniaturized on-spot protein denaturation coupled with microwave-assisted extraction sample treatment method was developed for extraction and subsequent spectrofluorimetric determination of favipiravir (FAV) in human plasma spots. Dried plasma spotting was performed using only 10 µL of plasma on filter paper discs followed by on-spot protein denaturation using 10 µL methanol. Maximum extraction efficiency was achieved using water: methanol (1:1) as extraction solvent with 50 % microwave power for 60 sec. The fluorescence intensity of extracted FAV was measured at 432 nm after excitation at 361 nm. According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH -M10) guidelines for bioanalytical method validation, spiked plasma samples showed very good linearity between 100 and 1000 ng/mL. The accuracy and precision of the developed method were indicated by mean recovery (%) within 100 ± 15 % for all tested concentrations levels. The obtained results were further compared with a reported protein precipitation method using both F-test and t-test revealing no significant difference between either method. Moreover, the method was successfully applied for FAV determination in real human samples for an incurred sample reanalysis study revealing no metabolite back conversion. The developed method showed superior green performance using analytical Eco-scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness Metric Approach (AGREE) tools compared to a classical protein precipitation method. According to Blue Applicability Grade Index (BAGI), and whiteness tools, the developed method outperformed all reported methods for FAV determination in plasma in terms of practicality and analytical functionality. Compared to reported sample treatment methods, this method exhibited excellent analytical performance, increased throughput, hundred-fold reduction of sample volume (10 μL), significant reduction of organic solvent consumption (260 μL methanol) and produced negligible wastes. Thus, it can be adopted for routine work in pharmacokinetics and clinical trial studies.