High-sensitivity dual analysis of Baricitinib and remdesivir in serum and urine using HPLC-fluorescence and LC-MS approaches in COVID-19 therapy.

Sensitive and accurate analytical strategies for the simultaneous quantification of Baricitinib (BAR) and Remdesivir (REM) in biological fluids are essential for supporting their combined use in COVID-19 therapy. Currently, no analytical methods exist for the simultaneous quantification of both drugs in human biological fluids. To bridge this gap, we developed, optimized, and validated novel RP-HPLC-fluorescence and LC-MS methods for the concurrent assay of BAR and REM in serum and urine samples. A high-performance liquid chromatography method with fluorescence detection (HPLC-fluorescence) was optimized using an isocratic mobile phase of phosphoric acid (pH = 3) and ethanol (30:70, v/v) at a flow rate of 0.6 mL/min, with detection at excitation/emission wavelengths of 245/400 nm. Furthermore, an LC-MS approach was developed using a mobile phase of 0.1% formic acid with 0.05 M ammonium formate in water and methanol (80:20, v/v) at 0.5 mL/min in an isocratic approach. The two LC approaches were successfully validated and applied for the simultaneous analysis of spiked BAR and REM mixtures in human urine and serum samples within 5 and 6 min, respectively. In biological human samples, accuracy varied from 96.41% to 98.10% for the HPLC-fluorescence strategy and from 95.17% to 98.47% for the LC-MS approach. Additionally, the precision ranged from 0.83% to 1.23% for the HPLC-fluorescence approach and from 0.66% to 1.55% for the LC-MS strategy. The HPLC-FLD technique demonstrated higher sensitivity for quantifying both BAR and REM at trace levels, with a lower limit of quantification of 0.1 ng/mL and 0.5 ng/mL, respectively, whereas the LC-MS method offered a wider linear range, extending up to 2000 ng/mL for BAR and 1000 ng/mL for REM. The assessment of green features using the Analytical GREEnness Metric Approach (AGREE) revealed that the HPLC-fluorescence approach, particularly due to its use of ethanol, is more sustainable (AGREE score: 0.68) than the LC-MS approach (AGREE score: 0.56). The practical functionality of both novel approaches was confirmed with high Blue Applicability Grade Index (BAGI) scores (77.5 and 75, respectively). Without bio-matrices interference, the optimized LC approaches showed very high levels of sensitivity and accuracy. Therefore, these methods are highly suitable for therapeutic drug monitoring and pharmacokinetic studies of the BAR-REM combination.