Recent Analytical Methodologies for Determination of Anti-Viral COVID-19 Drugs: A Review

----------------------


INTRODUCTION
The World Health Organization (WHO) declared the COVID-19( https://www.drugs.com/condition/covid-19.html)epidemic, which began in Wuhan, China, at the end of 2019 to be a global pandemic by March 2020 due to its rapid global spread.People in the US and around the world faced a new dilemma as a result of the extraordinary scope of COVID-19.Initially, the urgency of the issue was exacerbated by the lack of precise treatments or vaccines, which caused considerable uncertainty and fear among communities worldwide.
Many nations have enacted safety precautions, such as requiring people to wear masks while they are in public, instituting lockdowns to prevent the spread of viruses, switching to virtual work and schooling, and making outdoor meetings the new norm.Even in modern times, some social distancing practices endure, even if the virus's intensity has decreased.In order to properly manage the condition, research into new antiviral drugs and the repurposing of existing medications has increased due to the COVID-19 outbreak.The pressing need to create pharmaceutical treatments that can both fight the virus and lessen its effects on public health is reflected in the increased emphasis on research.
A wide array of drug classes, spanning antibiotics, antiviral, non-steroidal anti-inflammatory drugs (NSAIDs), anticancer agents, antimalarials, and immune suppressants, are undergoing rigorous evaluation to determine their efficacy and safety in managing symptoms associated with SARS-CoV-2 infection.Among these, antiviral drugs play a crucial role in hindering the infection and replication of SARS-CoV-2 through diverse mechanisms.(Fig. 1 ).
Medications that impede virus entry into the cell do so by blocking the virus from interacting with the host cell.This disruption typically involves interfering with the binding of the virus's spike protein to the host receptor.Among these drugs, two classes are notable: Protease Inhibitors and Angiotensin-Converting Enzyme 2 (ACE) Inhibitors: (Fig. 1A) ( Mahdavia R, Talebpoura Z;2023) Drugs that hinder the replication and dissemination of viral genomes following membrane fusion are pivotal in combating viral infections.(Fig. 1B).

Fig. -1A-1B-Mechanism of Action
As the COVID-19 pandemic progresses into its fourth year, surveillance efforts have significantly waned.Despite weekly reported cases and deaths reaching their lowest levels since the pandemic's onset, millions of individuals are still contracting or re-contracting SARS-CoV-2, with thousands succumbing to the virus each week.
Given the significance of this juncture, the World Health Organization (WHO) has revised its Strategic Preparedness and Response plan to address the evolving landscape from 2023 to 2025.The revised two-year strategy builds upon the goals outlined in the 2022 SPRP and provides assistance to countries as they endeavor to shift their crucial emergency response efforts toward sustainable, long-term prevention, control, and management of COVID-19.It is essential to read this strategy alongside pertinent WHO guidance on COVID-19, ( Wang Q, Li Z, Ho J;2022) In response to the novel coronavirus

Table 1: line of Treatment
• Paxlovid( nirmatrelvir, ritonavir) Paxlovid, a combination therapy consisting of nirmatrelvir, a SARS-CoV-2 protease inhibitor, and ritonavir, a booster, was initially authorized for use in December 2021.In May 2023, the authorization for this combination therapy was further broadened to encompass full FDA approval for adults.Emergency use authorization for this treatment in individuals at high risk with mild-to-moderate COVID-19 has been granted.This approval highlights its potential as a therapeutic solution to alleviate the effects of COVID-19, particularly among vulnerable populations.Convalescent plasma, derived from individuals who have recovered from COVID-19, contains antibodies that can aid in combating the virus in those currently infected.However, there is conflicting evidence regarding its effectiveness.While some studies suggest that convalescent plasma may reduce the risk of death in specific hospitalized individuals with severe COVID-19, other research findings have not consistently supported this conclusion.However, conflicting reports indicate it may not provide any additional benefit.According to the NIH, certain individuals with a compromised immune system may potentially benefit from it, although the supporting evidence is weak.Hern;2022).Bebtelovimab was initially granted emergency use authorization in February 2022; however, it is no longer authorized for use.This monoclonal antibody targets the receptor-binding domain of the spike protein of the SARS-CoV-2 virus.While it has been authorized for emergency use by some regulatory authorities for the treatment of COVID-19, its current authorization status may vary across regions.These monoclonal antibodies function by binding to the spike protein of the SARS-CoV-2 virus, thereby hindering its entry into human cells and replication.They have demonstrated efficacy in reducing viral load, alleviating symptoms, and mitigating the risk of hospitalization in specific high-risk patients.However, their use may be subject to specific regulatory approvals and guidelines in different regions, and treatment decisions should be made by healthcare professionals based on individual patient factors.

Immuno Modulators:
According to the COVID-19 Treatment Guidelines Panel, for hospitalized patients with COVID-19, the choice of immunomodulators depends on the severity of the disease.Here are some of the recommended immunomodulators: Dexamethasone: ( Jonathan A C Sterne , Srinivas Murthy , 2020).The percentage of drug molecules bound to proteins in the blood plasma.This affects the concentration of free drugs available for pharmacological activity.These properties are vital for selecting appropriate analytical methods such as chromatography (e.g., HPLC, GC) and sample preparation techniques (e.g., liquid-liquid extraction, solid-phase extraction) for quantifying drug levels in biological samples.Additionally, these properties can inform pharmacokinetic studies and aid in understanding the drug's behavior within the body.
Considering the physicochemical characteristics of the candidate drugs for COVID-19 treatment, most compounds have molecular weights exceeding 300 Da and log P values below 5. Due to these factors, gas chromatography (GC) is limited, as it requires analytes to be volatile, thermo-stable, and either non-polar or semi-polar.Therefore, there are limited reports on the analysis of these drugs by GC or GC-MS.In contrast, liquid chromatography techniques offer more flexibility and are widely used for analyzing drugs in complex biological environments.Liquid chromatography allows for a wide range of stationary and mobile phases, no limitations in terms of polarity and volatility, and the use of various modes from normal to hydrophilic interaction chromatography (HILIC).
Additionally, liquid chromatography methods can employ suitable temperature programs.Among the liquid chromatography techniques, highperformance liquid chromatography (HPLC) stands out as the most practical choice.HPLC methods offer expedited, effective, and ecofriendly separations.Furthermore, ultra-high performance liquid chromatography (UPLC), miniaturized liquid chromatography (capillary and nano-LC), and multidimensional liquid chromatography (MD-LC) have gained prominence for their successful applications in various fields.Therefore, based on the study, an HPLC method is selected as the most suitable analytical technique for analyzing drugs used in COVID-19 treatment due to its versatility, efficiency, and wide range of applications.

HPLC Methods:
High-performance liquid chromatography (HPLC) is indeed a widely used technique in pharmaceutical analysis, including the determination of antiviral drugs used in the treatment of COVID-19.HPLC is valued for its ability to separate, identify, and quantify components within a mixture, making it particularly useful for analyzing complex pharmaceutical formulations and biological samples.While specific methods may vary depending on the drug being analyzed, here are some general considerations and techniques used in developing HPLC methods for antiviral drugs:

Selection of Chromatographic Conditions:
This includes choosing the appropriate stationary phase (reverse-phase C18 is commonly used), mobile phase (usually a mixture of water and organic solvent such as acetonitrile or methanol), and the detection wavelength based on the drug's properties.Sample Preparation: Samples may need to be prepared prior to analysis to remove interfering substances and to ensure accurate quantification.This could involve techniques such as filtration, dilution, centrifugation, or extraction.
Column Selection: The column used in HPLC plays a crucial role in separating the components of the sample.The choice of column dimensions, particle size, and chemistry can affect resolution, sensitivity, and analysis time.Optimization of Parameters: Parameters such as flow rate, column temperature, and injection volume need to be optimized to achieve the best separation and sensitivity for the target analytes.Detection Method: UV-Vis detection is commonly used in HPLC analysis.However, for some drugs, especially those with low UV absorbance, other detection methods such as mass spectrometry (MS) may be necessary for higher sensitivity and selectivity.Validation: HPLC methods need to be validated to ensure accuracy, precision, specificity, and robustness.This involves testing parameters such as linearity, range, accuracy, precision, and specificity according to regulatory guidelines.Quantification: Calibration curves are often used for quantification by plotting peak area or height against analyte concentration.Standard solutions of known concentrations are used to generate these curves.Forced Degradation Studies: In the case of stability-indicating methods, forced degradation studies may be conducted to identify and characterize degradation products and demonstrate the specificity of the method.Documentation and Reporting: Proper documentation of method development, validation, and results is essential for regulatory compliance and scientific integrity.
Compliance with Regulatory Guidelines: HPLC methods used for analyzing antiviral drugs for COVID-19 treatment need to comply with regulatory guidelines set by organizations such as the FDA (Food and Drug Administration) or EMA (European Medicines Agency).

For anti-viral Drugs :
It's important to highlight that tailored HPLC methods for each antiviral drug employed in COVID-19 treatment would need to be developed and validated based on the chemical properties of the drug and the requirements of the regulatory authorities.These methods are often published in scientific journals or provided by regulatory agencies.There are several antiviral drugs have been used or investigated for the treatment of COVID-19.
Here are some examples along with brief descriptions of their HPLC methods for analysis: Remdesivir: Description: Remdesivir is a broad-spectrum antiviral medication initially developed for Ebola virus disease.

HPLC
Method: (Padhye Hemangi, Sonawane B;2022, Valeria Avataneo , Amedeo de Nicolò;2020)HPLC methods for the analysis of remdesivir typically involve reverse-phase chromatography using C18 columns and a mobile phase Ammonium acetate buffer pH (4.6) and acetonitrile (60:40) %v/v and it is also performed by using a mixture of buffer and organic solvents such as acetonitrile or methanol.Detection is often performed using UV-Vis detection at wavelengths around 260 nm.Favipiravir: Description: Favipiravir is a broad-spectrum antiviral medication that has shown some efficacy against RNA viruses.HPLC Method: (13) ( Furuta Y, Gowen BB;2013) Reverse-phase chromatography using a C18 column and a mobile phase comprising a buffer and organic solvent mixture is commonly used in HPLC analysis of favipiravir.Quantification frequently uses UV detection.Paxlovid :( Nirmatrelvir/Ritonavir): Description: Nirmatrelvir/ritonavir is a combination drug utilized in the management of mild to moderate COVID-19 HPLCMethod:(https://www.ema.europa.eu/en/medicines/human/EPAR/paxlovid;2022) HPLC methods for nirmatrelvir and ritonavir analysis involve reverse-phase chromatography with C18 columns.The mobile phase can be made up of ethanol, buffer, and organic solvent mixed with water; UV-Vis is used for detection.

Molnupiravir: Description:
In preclinical trials, molnupiravir has shown broad-spectrum antiviral effectiveness against a number of RNA viruses, including coronaviruses like SARS-CoV-2.It functions by inducing mistakes in the replication of viral RNA, leading to the introduction of mutations in the viral genome and ultimately inhibiting viral replication.HPLC Method: ( Reçber T, Timur SS;2022) Molnupiravir analysis using HPLC methods involves reverse-phase chromatography using a C18 column.The mobile phase, which is detectable by UV-Vis detection, may be composed of methanol, water, and buffer containing 0.1% OPA and acetonitrile.

CONCLUSION:
While the pandemic continues to spread worldwide, there is an immediate necessity to comprehend the advantages and drawbacks of each treatment.The current review is based on the overview of COVID-19 drugs that are effectively used for the treatment.The review also focused on various RP-HPLC Methods for the determination of anti-viral drugs such as Remdesivir, Paxlovid, Molnupiravir, Favipiravir and the chromatographic conditions which were adopted based on their ionic interactions and physicochemical properties the detection wavelength, buffer pH ratio of organic solvent, flow rate and elution time were studied.Linearity, Specificity, Accuracy, Precision, LOD, and LOQ were researched as validation metrics.
The selected few analytical methods that were reported in the article were based on the usage of different columns such as C 8, C18 and cyano columns studied the review method was focused on the different modes of elutions such as isocratic as well as gradient modes.The drugs such as Nirmatrelvir and ritonavir were recently approved and recommended and these drugs belong to anti-viral agents hence there is a need to study the drugs coming under the category such as Remdesivir, Molnupiravir, Favipiravir.Based on this review the objective is to develop RP-HPLC Methods for these selected drugs using solvents that are eco-friendly and optimize the methods based on DOE and factorial designs which can be an economical and reliable approach.Future Prospective: The author's aim is to evaluate the chromatographic conditions and optimize the method based on system suitability further the author is interested in focusing on the ionic interactions and physicochemical properties of recently approved drugs for their estimation using the mobile phase which is eco friendly and adoption of DOE to minimize time and expenditure.

FigFig. 11 :
Fig. 10: Structure of Dexamethasone.The COVID-19 Treatment Guidelines Panel recommends the use of dexamethasone, a corticosteroid, in hospitalized patients with COVID-19 who require supplemental oxygen.This recommendation is supported by a rating of B-IIa, indicating moderate certainty in the evidence supporting its efficacy and safety for this indication.Dexamethasone is considered a cornerstone of COVID-19 treatment protocols due to its ability to suppress the hyperactive immune response associated with severe disease and reduce inflammation in the lungs, potentially improving clinical outcomes.Tocilizumab (or sarilumab):( Obbina Abani, Ali Abbas, Fatima Abbas;Lancet,2022) Tocilizumab was initially authorized for use in June 2021 and later received full approval in December 2022.This monoclonal antibody targets the interleukin-6 (IL-6) receptor, a key player in the inflammatory response.By inhibiting IL-6 signaling, tocilizumab aims to dampen the excessive inflammation observed in severe cases of COVID-19, potentially improving patient outcomes.Baricitinib (or tofacitinib) :( Obbina Abani, Ali Abbas, Fatima Abbas; Lancet, 2022) outbreak in March 2020, In response to the urgent need for effective treatments, the FDA swiftly initiated the Coronavirus Treatment Acceleration Program (CTAP).
This article does not contain any studies with human participants or animals performed by any of the authors.