Toward sustainable bioanalysis: HPLC analysis of Molnupiravir in rat plasma using salting and sugaring-out assisted sample cleanup methods.

The miniaturization of liquid-liquid extraction is an expanding domain of sample preparation aimed at minimizing solvent usage, safeguarding the environment, and ensuring the health of operators. In this study, salting-out and sugaring-out induced liquid-liquid micro-extraction methods have been developed as a sample pretreatment step to extract molnupiravir from rat plasma before analysis using HPLC. Factors impacting the extraction efficiency presented as enrichment factor were studied, including type of salt or sugar, its concentration, the used organic solvent and its amount. Optimum extraction efficiency was achieved using 0.5 mL acetonitrile with either 0.1 g ammonium carbonate or 0.5 g fructose, where the optimum phase separation and extraction recovery was obtained. Chromatographic separation between plasma matrix, molnupiravir, and the internal standard, linozolide (20 μg/mL) was done using an eco-friendly solvent mixture of water: ethanol (75: 25, v/v) with a flow rate of 2 mL/min. The method was then verified in accordance with FDA criteria and determined to be linear within the range of 0.5-25 μg/mL for salting-out and sugaring-out methods. Other validation parameters were found within the acceptance criteria of FDA rules. Method efficiency and sensitivity were tested by its application to analyze real plasma samples obtained from rats obtained an oral dose of 1600 mg/Kg molnupiravir taken at its Tmax. Additionally, method greenness and whiteness were evaluated and compared to a previously published one using AGREE calculator and RGB fast tools. Sample preparation was evaluated using AGREE-Prep and the novel sample preparation metric of sustainability tools. All the findings confirmed the superiority of the suggested methodology surpassing the published one concerning greenness of the extraction and analysis methods. Additionally, salting-out and sugaring-out induced liquid-liquid micro-extraction can be used as high efficient micro-extraction modes for the preparation of biological samples.