Comparative insights to microwave and ultrasound as extraction tools for on-spot protein denaturation and HPLC-UV determination of favipiravir in human plasma: Application to real sample and storage stability.

Miniaturized sample preparation methods are becoming a growing trend in analytical chemistry to reduce solvent/ sample consumption and enhance analytical performance. This study encompasses evaluating the performance of microwave-assisted extraction and ultrasound-assisted extraction for extraction of favipiravir from dried plasma spots after on-spot protein denaturation. Each extraction method was combined with high-performance liquid chromatography with UV detection. Microwave-assisted extraction was performed using methanol (10 µL) as an on-spot plasma protein denaturant, the mobile phase as extraction solvent, and microwave power of 50 % for 40 s. Ultrasound-assisted extraction was accomplished using ethanol (10 µL) as a denaturant and mobile phase as a solvent of extraction for 40 min. The developed methods were validated as per the International Council for Harmonization guidelines M10. Both methods showed very good linearity in the concentration range of 10-50 μg mL-1, negligible matrix effect and carryover, and good selectivity and specificity. The recovery results were found to be between 87 - 99 % and 93 - 103 %, for microwave and ultrasound methods, respectively. The methods were successfully applied for favipiravir determination in real human plasma. An incurred real sample reanalysis study was performed to investigate dried sample stability, revealing satisfactory sample stability for up to one month in both freezer and refrigerator as indicated by % differences <13 %. The comparative analysis of methods' profiles, using different greenness and whiteness metric tools, revealed superior greenness and analytical performance of ultrasound-assisted extraction with similar practicality. The developed methods exhibited excellent analytical performance, increased throughput, reduced volumes of sample (10 μL) and organic solvents (210 μL), and negligible waste. Thus, it can be adopted for routine work in pharmacokinetics and clinical trial studies.