Development and Validation of High-Performance Liquid Chromatography Method for the Quantification of Remdesivir in Intravenous Dosage Form.

Corona virus disease 2019 (COVID-19) has posed a mounting threat to public health with worldwide outbreak caused by a novel virus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recently, remdesivir (RDV) has been approved by Food and Drug Administration (FDA) for treating COVID-19 patients ≥12 years old requiring hospitalization. To the best of our knowledge, a simple method to estimate RDV in the pharmaceutical formulations using high-performance liquid chromatography (HPLC) is still unexplored, highlighting the need for a precise analytical method for its quantification. The prime purpose of the current investigation was to develop and validate a well-grounded HPLC method for quantification of RDV in pharmaceutical formulations. The best chromatogram was obtained by means of an Inertsil ODS-3V column using a mobile phase of milli-Q water modified to pH 3.0 with o-phosphoric acid and acetonitrile (50:50, % v/v) at a flow rate of 1.2 mL/min and wavelength of detector set at 246 nm with retention time being achieved at 6.0 min. The method was validated following International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 (R1) guidelines for various parameters such as specificity and selectivity, system suitability, linearity, precision, accuracy, limits of detection and quantification, and robustness. The method developed for the quantification of RDV was found to be linear in the concentration range of 25-2,500 ng/mL with limit of detection and limit of quantification of 1.95 and 6.49 ng/mL, respectively. Assay value of 102% ± 1% was achieved for marketed injectable dosage form when estimated by the validated method. Therefore, in this study a simple, rapid, sensitive, selective, accurate, precise, and robust analytical method was developed and validated for the quantification of RDV using HPLC. The established method was successfully employed for quantification of RDV in marketed pharmaceutical formulation.

Challenges and stepwise fit-for-purpose optimization for bioanalyses of remdesivir metabolites nucleotide monophosphate and triphosphate in mouse tissues using LC-MS/MS.

Remdesivir is a prodrug of the nucleotide analogue and used for COVID-19 treatment. However, the bioanalysis of the active metabolites remdesivir nucleotide triphosphate (RTP) and its precursor remdesivir nucleotide monophosphate (RMP) is very challenging. Herein, we established a novel method to separate RTP and RMP on a BioBasic AX column and quantified them by high-performance liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. Stepwise, we optimized chromatographic retention on an anion exchange column, improved stability in matrix through the addition of 5,5'-dithiobis-(2nitrobenzoic acid) and PhosSTOP EASYpack, and increased recovery by dissociation of tight protein binding with 2 % formic acid aqueous solution. The method allowed lower limit of quantification of 20 nM for RMP and 10 nM for RTP. Method validation demonstrated acceptable accuracy (93.6%-103% for RMP, 94.5%-107% for RTP) and precision (RSD < 11.9 % for RMP, RSD < 11.4 % for RTP), suggesting that it was sensitive and robust for simultaneous quantification of RMP and RTP. The method was successfully applied to analyze RMP and RTP in mouse tissues. In general, the developed method is suitable to monitor RMP and RTP, and provides a useful approach for exploring more detailed effects of remdesivir in treating diseases.

Analytical methods for the determination of remdesivir as a promising antiviral candidate drug for the COVID-19 pandemic.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is undoubtedly the most challenging pandemic in the current century. A total of 73,953,702 confirmed cases of COVID-19 and 1,644,416 deaths were reported globally up to December 17, 2020. Therefore, in the absence of a safe and effective vaccine, it is urgent to identify a novel antiviral drug to effectively treat patients with COVID-19. On October 22, the U.S. Food and Drug Administration approved remdesivir, a nucleotide analog prodrug with broad antiviral activity, for adults and children (12 years of age and older and weighing at least 40 kg) who need to be admitted to hospital for covid-19 treatment. In order to monitor the optimization of patient clinical response profile, as well as address the challenges associated with remdesivir metabolism, highly sensitive, selective and accurate analytical methods are necessary. This review clearly covers all the analytical methods developed for the identification and quantitative determination of remdesivir and its metabolites in biological matrices, which helps the researchers in developing new methods for the analysis of remdesivir by considering the pros and cons of the previously reported methods.

Development and validation of a UHPLC-MS/MS method for quantification of the prodrug remdesivir and its metabolite GS-441524: a tool for clinical pharmacokinetics of SARS-CoV-2/COVID-19 and Ebola virus disease.

BACKGROUND: Remdesivir has received significant attention for its potential application in the treatment of COVID-19, caused by SARS-CoV-2. Remdesivir has already been tested for Ebola virus disease treatment and found to have activity against SARS and MERS coronaviruses. The remdesivir core contains GS-441524, which interferes with RNA-dependent RNA polymerases alone. In non-human primates, following IV administration, remdesivir is rapidly distributed into PBMCs and converted within 2 h to the active nucleoside triphosphate form, while GS-441524 is detectable in plasma for up to 24 h. Nevertheless, remdesivir pharmacokinetics and pharmacodynamics in humans are still unexplored, highlighting the need for a precise analytical method for remdesivir and GS-441524 quantification. OBJECTIVES: The validation of a reliable UHPLC-MS/MS method for remdesivir and GS-441524 quantification in human plasma. METHODS: Remdesivir and GS-441524 standards and quality controls were prepared in plasma from healthy donors. Sample preparation consisted of protein precipitation, followed by dilution and injection into the QSight 220 UHPLC-MS/MS system. Chromatographic separation was obtained through an Acquity HSS T3 1.8 µm, 2.1 × 50 mm column, with a gradient of water and acetonitrile with 0.05% formic acid. The method was validated using EMA and FDA guidelines. RESULTS: Analyte stability has been evaluated and described in detail. The method successfully fulfilled the validation process and it was demonstrated that, when possible, sample thermal inactivation could be a good choice in order to improve biosafety. CONCLUSIONS: This method represents a useful tool for studying remdesivir and GS-441524 clinical pharmacokinetics, particularly during the current COVID-19 outbreak.