New Analytical Methods for the Determination of New Anti-Viral Drug Favipiravir: A Potential Therapeutic Drug Against Covid-19 Virus, in Bulk and Dosage Forms.

Simple, accurate and robust analytical methods have been developed and validated for the determination of favipiravir (FVPR) by RP-HPLC and UV spectroscopy techniques as per the ICH guidelines. In the RP-HPLC method for FVPR determination, the mobile phase was ammonium acetate buffer pH 6.5 in pump Aand methanol in pump B. The C18 (Sunfire) 5 µm, 4.6 × 250 mm column was used as a stationary phase, and the detection wavelength was at 323 nm. Under these conditions, FVPR was eluted as a sharp peak at 2.65 min and the overall time taken for each injection was 10 min. In case of the UV spectroscopy method, standard FVPR solutions were prepared with pure ethanol and scanned from 250 to 400 nm and a flourishing spectrum was obtained at 323 nm. Hence, the wavelength of 323 nm was fixed for the whole process of validation in both techniques. The limit of detection (LOD) and limit of quantification (LOQ) in the RP-HPLC method were 1.0 and 3.5 µg/mL, respectively, and the linearity was established in the 10 to 50 µg/mL range. In the UV spectroscopy method, the LOD and LOQ values were found to be 3.5 and 12 µg/mL, respectively, and the linearity was established within 20 to 60 µg/mL range. The regression coefficient was found to exceed 0.999 in both methods. The proposed RP-HPLC and UV spectroscopy techniques are simple, accurate, rugged and robust.

Evaluation of Methods and Processes for Robust Monitoring of SARS-CoV-2 in Wastewater.

The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic.

Evaluating the Benefits of Collaborative VR Review for Maintenance Documentation and Risk Assessment

Featured ApplicationBased on data from a globally operating industrial company, this study demonstrated the benefits of VR to maintenance documentation review and risk assessment processes.Technical documentation creation is a collaborative process involving several departments in R&D. Even though virtual reality (VR) has been demonstrated to facilitate industrial collaboration and advance the product development lifecycle in earlier studies, it has not been utilized for technical documentation review and risk assessment processes in industrial companies. This article presents a case study where the benefits of VR to maintenance documentation reviews and risk assessments were studied. The virtual reality environment was tested by nine domain experts from an industrial company in a user study that replicated their actual real-life industrial collaboration tasks. Both qualitative and quantitative data were collected during the study. Our findings show that collaborative VR has the potential to enhance the documentation review and risk assessment processes. Overall, the concept of using virtual reality for documentation review and risk assessment processes was rated positively by participants, and even though further development is needed for the review tools, VR was viewed as a concept that facilitates collaboration, enhances the current review practices, and increases spatial understanding. The benefits of VR are evident, especially for geographically scattered teams that rarely meet face-to-face or do not have access to the actual physical equipment. In cases where traditional means of communication are not enough, process improvements are needed for documentation review and risk assessment processes, and our proposed solution is VR.

Development and Validation of an HPLC-UV Method for the Quantification of 4'-Hydroxydiclofenac Using Salicylic Acid: Future Applications for Measurement of In Vitro Drug-Drug Interaction in Rat Liver Microsomes.

Salicylic acid is a key compound in nonsteroidal anti-inflammatory drugs that has been recently used for preventing the risk of hospitalization and death among COVID-19 patients and in preventing colorectal cancer (CRC) by suppressing two key proteins. Understanding drug-drug interaction pathways prevent the occurrence of adverse drug reactions in clinical trials. Drug-drug interactions can result in the variation of the pharmacodynamics and pharmacokinetic of the drug. Inhibition of the Cytochrome P450 enzyme activity leads to the withdrawal of the drug from the market. The aim of this paper was to develop and validate an HPLC-UV method for the quantification of 4'-hydroxydiclofenac as a CYP2C9 metabolite using salicylic acid as an inhibitor in rat liver microsomes. A CYP2C9 assay was developed and validated on the reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) using a low-pressure gradient elution programming at T = 30 °C, a wavelength of 282 nm, and a flow rate of 1 mL/min. 4'-hydroxydiclofenac demonstrated a good linearity (R2 > 0.99), good reproducibility, low detection, and quantitation limit, and the inter and intra-day precision met the ICH guidelines (<15%). 4'-hydroxydiclofenac was stable for three days and showed an acceptable accuracy and recovery (80-120%) within the ICH guidelines in a rat liver microsome sample. This method will be beneficial for future applications of the in vitro inhibitory effect of salicylic acid on the CYP2C9 enzyme activity in rat microsomes and the in vivo administration of salicylic acid in clinical trials.

Accurate Mass Identification of an Interfering Water Adduct and Strategies in Development and Validation of an LC-MS/MS Method for Quantification of MPI8, a Potent SARS-CoV-2 Main Protease Inhibitor, in Rat Plasma in Pharmacokinetic Studies.

MPI8, a peptidyl aldehyde, is a potent antiviral agent against coronavirus. Due to unique tri-peptide bonds and the formyl functional group, the bioassay of MPI8 in plasma was challenged by a strong interference from water MPI8. Using QTOF LC-MS/MS, we identified MPI8•H2O as the major interference form that co-existed with MPI8 in aqueous and biological media. To avoid the resolution of MPI8 and MPI8•H2O observed on reverse phase columns, we found that a Kinetex hydrophilic interaction liquid chromatography (HILIC) column provided co-elution of both MPI8 and MPI8•H2O with a good single chromatographic peak and column retention of MPI8 which is suitable for quantification. Thus, a sensitive, specific, and reproducible LC-MS/MS method for the quantification of MPI8 in rat plasma was developed and validated using a triple QUAD LC-MS/MS. The chromatographic separation was achieved on a Kinetex HILIC column with a flow rate of 0.4 mL/min under gradient elution. The calibration curves were linear (r2 > 0.99) over MPI8 concentrations from 0.5-500 ng/mL. The accuracy and precision are within acceptable guidance levels. The mean matrix effect and recovery were 139% and 73%, respectively. No significant degradation of MPI8 occurred under the experimental conditions. The method was successfully applied to a pharmacokinetic study of MPI8 after administration of MPI8 sulfonate in rats.

Distributed Asymmetric Virtual Reality in Industrial Context: Enhancing the Collaboration of Geographically Dispersed Teams in the Pipeline of Maintenance Method Development and Technical Documentation Creation

Featured ApplicationBased on data from multinational domain experts in the field of Industrial Maintenance, this study demonstrated the application cases and advantages of asymmetric VR set-up to address needs present in the industry.Virtual Reality (VR) is a critical emerging technology in industrial contexts, as it facilitates collaboration and supports the product development lifecycle. However, its broad adoption is constrained by complex and high-cost integration. The use of VR among devices with various immersion and control levels may solve this obstacle, and increase the scalability of VR technologies. This article presents a case study on applying asymmetry between the COVE-VR platform and Microsoft Teams to enable distributed collaboration of multinational departments and enhance the maintenance method and documentation creation processes. Overall, five remote collaborative sessions were held with 20 experts from four countries. Our findings suggest that asymmetry between head-mounted display and Teams users enhances the quality of communication among geographically dispersed teams and their spatial understanding, which positively influences knowledge transfer and efficiency of industrial processes. Based on qualitative evaluation of the asymmetric VR setup, we further suggest a list of guidelines on how to enhance the collaboration efficiency for low-cost distributed asymmetric VR from three perspectives: organization, collaboration and technology.

The Development and the Validation of a Novel Dissolution Method of Favipiravir Film-Coated Tablets

The aim of this study was to develop and validate a dissolution test for favipiravir release in a tablet dosage form using ultra-high performance liquid chromatography (UHPLC). The dissolution method was developed by testing the solubility of favipiravir in media with different pH values. The results demonstrated that the best dissolution was achieved in phosphate buffer with a pH of 6.8. The amount of favipiravir that was released was about 100% after 30 min. The UHPLC method presented linearity (R = 1.000) in the concentration range of 0.044–0.44 mg/mL. The recovery parameter that was achieved ranged from 102.5% to 104.2%. The system suitability, repeatability, and intermediate precision RSD% results were found to be 0.36%, 1.99%, and 2.49%, respectively. In addition to these parameters and results, an F-test was performed using the Minitab 18 Statistical Software program for the intermediate precision and repeatability results. The standard and sample solutions were found to be stable for 2 days in their respective dissolution medium. This analytical method was also found to be selective for favipiravir. In conclusion, a simple and feasible dissolution method with a short run time of 2.5 min was developed and validated successfully. The obtained results demonstrated that the dissolution test developed here is adequate for its purpose and can be applied as the dissolution method for favipiravir in film-coated tablets for release analyses.

Coordination of SARS-CoV-2 wastewater and clinical testing of university students demonstrates the importance of sampling duration and collection time.

Wastewater surveillance has been a useful tool complementing clinical testing during the COVID-19 pandemic. However, transitioning surveillance approaches to small populations, such as dormitories and assisted living facilities poses challenges including difficulties with sample collection and processing. Recently, the need for reliable and timely data has coincided with the need for precise local forecasting of the trajectory of COVID-19. This study compared wastewater and clinical data from the University of Delaware (Fall 2020 and Spring 2021 semesters), and evaluated wastewater collection practices for enhanced virus detection sensitivity. Fecal shedding of SARS-CoV-2 is known to occur in infected individuals. However, shedding concentrations and duration has been shown to vary. Therefore, three shedding periods (14, 21, and 30 days) were presumed and included for analysis of wastewater data. SARS-CoV-2 levels detected in wastewater correlated with clinical virus detection when a positive clinical test result was preceded by fecal shedding of 21 days (p< 0.05) and 30 days (p < 0.05), but not with new cases (p = 0.09) or 14 days of shedding (p = 0.17). Discretely collected wastewater samples were compared with 24-hour composite samples collected at the same site. The discrete samples (n = 99) were composited examining the influence of sampling duration and time of day on SARS-CoV-2 detection. SARS-CoV-2 detection varied among dormitory complexes and sampling durations of 3-hour, 12-hour, and 24-hour (controls). Collection times frequently showing high detection values were between the hours of 03:00 to 05:00 and 23:00 to 08:00. In each of these times of day 33% of samples (3/9) were significantly higher (p < 0.05) than the control sample. The remainder (6/9) of the collection times (3-hour and 12-hour) were not different (p > 0.05) from the control. This study provides additional framework for continued methodology development for microbiological wastewater surveillance as the COVID-19 pandemic progresses and in preparation for future epidemiological efforts.

Development and Validation of the Skimmed Milk Pellet Extraction Protocol for SARS-CoV-2 Wastewater Surveillance.

Wastewater surveillance for SARS-CoV-2 may serve as a useful source of data for public health departments as the virus is shed in the stool of infected individuals. However, for wastewater data to be actionable, wastewater must be collected, concentrated, and analyzed in a timely manner. This manuscript presents modifications on a skimmed milk concentration protocol to reduce processing time, increase the number of samples that can be processed at once, and enable use in resource-limited settings. Wastewater seeded with Human coronavirus OC43 (OC43) was concentrated using a skimmed milk flocculation protocol, and then pellets were directly extracted with the QIAamp Viral RNA Mini kit. This protocol has a higher average effective volume assayed (6.35 mL) than skimmed milk concentration methods, with and without Vertrel XF™, which involve resuspension of the pellets in PBS extraction prior to nucleic acid extraction (1.28 mL, 1.44 mL, respectively). OC43 was selected as a recovery control organism because both it and SARS-CoV-2 are enveloped respiratory viruses that primarily infect humans resulting in respiratory symptoms. The OC43 percent recovery for the direct extraction protocol (3.4%) is comparable to that of skimmed milk concentration with and without Vertrel XF™ extraction (4.0%, 2.6%, respectively). When comparing SARS-CoV-2 detection using McNemar's chi-square test, the pellet extraction method is not statistically different from skimmed milk concentration, with and without Vertrel XF™ extraction. This suggests that the method performs equally as well as existing methods. Added benefits include reduced time spent per sample and the ability to process more samples at a single time. Direct extraction of skimmed milk pellets is a viable method for quick turnaround of wastewater data for public health interventions.

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.

Biologically grounded scientific methods: The challenges ahead for combating epidemics.

The protracted COVID 19 pandemic may indicate failures of scientific methodologies. Hoping to facilitate the evaluation and/or update of methods relevant in Biomedicine, several aspects of scientific processes are here explored. First, the background is reviewed. In particular, eight topics are analyzed: (i) the history of Higher Education models in reference to the pursuit of science and the type of student cognition pursued, (ii) whether explanatory or actionable knowledge is emphasized depending on the well- or ill-defined nature of problems, (iii) the role of complexity and dynamics, (iv) how differences between Biology and other fields influence methodologies, (v) whether theory, hypotheses or data drive scientific research, (vi) whether Biology is reducible to one or a few factors, (vii) the fact that data, to become actionable knowledge, require structuring, and (viii) the need of inter-/trans-disciplinary knowledge integration. To illustrate how these topics interact, a second section describes four temporal stages of scientific methods: conceptualization, operationalization, validation and evaluation. They refer to the transition from abstract (non-measurable) concepts (such as 'health') to the selection of concrete (measurable) operations (such as 'quantification of ́anti-virus specific antibody titers'). Conceptualization is the process that selects concepts worth investigating, which continues as operationalization when data-producing variables viewed to reflect critical features of the concepts are chosen. Because the operations selected are not necessarily valid, informative, and may fail to solve problems, validations and evaluations are critical stages, which require inter/trans-disciplinary knowledge integration. It is suggested that data structuring can substantially improve scientific methodologies applicable in Biology, provided that other aspects here mentioned are also considered. The creation of independent bodies meant to evaluate biologically oriented scientific methods is recommended.

Adaptation of Elecsys® anti-severe acute respiratory syndrome coronavirus-2 immunoassay to dried blood spots: proof of concept.

Aim: Several automated immunoassays have been validated on serum/plasma to evaluate the presence of significant levels of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies, signs of a present or past infection, but the use of dried blood spots (DBS) would facilitate sampling, shipping and storage. Objective: The aim of this project was to give proof of concept of the possibility to use of the automatized Elecsys® anti-SARS-CoV-2 immunoassay with a volumetric DBS device. Results: Linearity and correlation were satisfactory between volumetric DBS and plasma. A cut-off value was suggested and should be validated with more samples. Conclusion: this study strongly support the possibility to work with volumetric DBS instead of serum/plasma to test for anti-SARS-CoV-2 antibodies.