Rapid and validated UHPLC method for simultaneous determination of sofosbuvir, ledipasvir and paracetamol as commonly repurposed drugs for COVID-19 treatment: application in spiked human plasma

Abstract Background Sofosbuvir/ledipasvir (SOF/LDV), a combination of antiviral drugs, has been recently repurposed for COVID-19 management, according to Food and Drug Administration approval. Paracetamol (PAR) identified as a first-line antipyretic for COVID-19 symptoms' management. The use of these three drugs together has significantly influenced the management of COVID-19 by providing symptomatic relief via inhibiting viral activity. A validated ultra-high performance liquid chromatographic (UHPLC) method has been introduced for the quantification of these repurposed drugs in COVID-19 treatment. This novel chromatographic method allows the simultaneous detection of SOF, LDV, and PAR in bulk. Additionally, the method has been applied to determine the levels of SOF and LDV in human plasma samples with PAR used as an internal standard. Results A new UHPLC method was developed, using a mobile phase with a combination of acetonitrile and 0.1% orthophosphoric acid in a proportion of 42:58 (v/v).Flow rate was set at 0.4 ml/min, and UV detection was adjusted at 254 nm. The concentration of SOF, LDV, and PAR were measured by their corresponding peak areas, and showed linear relationships between concentration and peak area within the ranges of (5–60) µg/ml for SOF, (2–22) µg/ml for LDV, and (1–22) µg/ml for PAR. The presented UHPLC method was used to quantify the amounts of SOF, LDV, and PAR in both bulk samples and human plasma samples being spiked with the mentioned analytes. The elution process was completed within 4 min, with retention times of 3.28 min for SOF, 2.28 min for LDV, and 1.70 min for PAR. The method showed high separation selectivity, with an injection volume of 1µl. The precision, accuracy and repeatability of the method were found to be within acceptable limits. Conclusion The recently developed method has been successfully validated in accordance with the guidelines set by the International Council for Harmonization (ICH). This validation process ensures that the method is suitable for routine quality control analysis, making it convenient for regular use.