Therapeutic drug monitoring, which is concerned with the measurement of drug concentrations in biological fluids to optimize the drug regimen and evade toxic properties or therapeutic letdowns, is already well known in numerous areas, such as in HIV treatment, and may be valuable in the issue of COVID-19 therapy [21
]. Therefore, both for pharmacokinetics studies and probable future therapeutic drug monitoring, there is an urgent need for accurate and precise analytical methods to quantify remdesivir and/or its metabolite GS-441524 in human plasma. Nevertheless, in literature the information about the pharmacokinetics and pharmacodynamics of remdesivir in humans is insufficient, as well as no therapeutic and toxic ranges have been reported. The reason for such insufficiency is the few cases treated with remdesivir. Surprisingly, a remdesivir qualitative and quantitative analysis was discussed only in a very small number of studies. In the first study, Avataneo and her colleagues [24
] studied the analysis of remdesivir and its metabolite GS-441524 in human plasma by using the UHPLC-MS-MS technique. Their research team used the UHPLC system coupled with a Triple Quadrupole for a chromatographic analysis. A chromatographic separation was obtained on an Acquity®
HSS T3 1.8 µm, 2.1 × 50 mm column and a physical filter (‘Frit’, 0.2 µm, 2.1 mm) with a pre-column set at 40 °C by using a column thermostat. The optimum separation was achieved by using a mobile phase consisting of H2O + 0.05% formic acid and acetonitrile +0.05% formic acid in gradient elution mode. 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline was used as an internal standard in this study. Positive electrospray ionization (ESI+) was used for all the analytes. Multiple reaction monitoring (MRM) traces (m/z) were quantified as 603.15 > 200 for remdesivir, 292,163 for GS-441524, and 313.2 > 78.05 for the internal standard. The rapid protein precipitation was performed using methanol: acetonitrile (50:50 v
) for the extraction of remdesivir and its metabolite GS-441524. The established method was shown to be accurate, precise, sensitive, and linear. Moreover, the developed method is shown to have a lower limit of quantitation (LLOQ) for remdesivir, and GS-441524 was 0.98 ng/mL, while the LOD values were 0.24 ng/mL for remdesivir and 0.98 ng/mL for GS-441524. Both remdesivir and GS-441524 remained as stable in-stock solutions when stored at −80 °C for over 4 months. Additionally, remdesivir was revealed to be stable in the stock solution for at least 10 months, while the stability of the GS-441524 stock solution in the same conditions had not been tested yet. Although remdesivir was found to be unstable at room temperature and at 4 °C when dissolved in plasma for 24 h, remdesivir was stable for 7 days in the extracted plasma samples in the auto-sampler (10 °C). This method was not tested on real-life samples but will be very useful in studies of the pharmacokinetics of remdesivir [24
]. In the second study, high-performance liquid chromatography was used to assess the remdesivir purity during the whole synthesis process. In this study, Kinetex®
(2.6 μm, 100 × 4.6 mm) was used as a stationary phase, and 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile was used as a mobile phase. The mobile phase was run in a gradient mode at a flow rate of 1.5 mL/min. Moreover, the progress of the reaction during synthesis was monitored by using LC-MS equipped with a Gemini®
5 μm × 30 × 4.6 mm column. The study pointed out the role of HPLC in evaluating remdesivir synthesis, screening, separation, and purification during the synthesis process as well as other antiviral drugs [25
In 2018 Murphy and his colleagues examined remdesivir and its metabolite, C-nucleoside ribose analog, on domestic cats infected with feline infectious peritonitis (FIP) [26
]. FIP is caused by a coronavirus that tends to attack the cells of the intestinal wall in cats. It was demonstrated that the remdesivir metabolite GS-441524 is effective, safe, and inhibits FIP virus replication. Murphy with his team evaluated the effectiveness, safety, and therapeutic doses of the nucleoside. During the study, the analysis of cells and body fluids for the concentration of C-nucleoside that is created from remdesivir and also for its triphosphate was performed in plasma, aqueous humor, and cerebrospinal fluid after precipitating the proteins by acetonitrile in the presence of an internal standard of 5-(2-aminopropyl)indole at the concentration of 20 nM. Following the filtration for protein removal, drying under a stream of nitrogen, and reconstitution with 0.2% formic acid and 1% acetonitrile, LC/MS-MS analyses for the GS-441524 concentrations were performed. The level of phosphorylation of GS-441524 to its triphosphate was determined in the frozen samples of cultured cells and peripheral blood mononuclear cells. The frozen cells were resuspended in 0.5 mL of 70% methanol with an internal standard 2-chloro-adenosine triphosphate (500 nM) and kept for 30 min at −80 °C. After drying and evaporating, the samples were reconstituted with an aqueous 1 mM ammonium phosphate. The pH of these reconstituted samples was 7.
A 50 × 2 mm, 2.5 µm Luna C18(2) HST column (Phenomenex, Torrance, CA, USA) connected to an LC-20ADXR (Shimadzu, Columbia, MD, USA) pump system and autosampler was used for the separation of analytes in a multi-stage linear gradient from 10% to 50% acetonitrile in a mobile phase containing 3 mM of ammonium formate (pH 5.0) with 10 mM of dimethylhexylamine at a flow rate of 150 µL/min. The MS/MS was operated in positive ion and multiple reaction-monitoring modes. The used instrumental method [26
] seems to be very efficient and has enough precision.