RP-HPLC Methodologies for Tecovirimat Analysis: A Critical Review of Development and Validation

Abstract

To critically review the development and validation of Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) methods for the determination of tecovirimat in raw drug substances and finished pharmaceutical formulations. A comprehensive literature review was conducted using recent scientific publications, pharmacopeial standards, and international regulatory guidelines. The review focused on analytical method development, validation parameters, and regulatory requirements. RP-HPLC is the preferred technique for tecovirimat estimation due to its high sensitivity, selectivity, reproducibility, and robustness. Validation studies confirmed compliance with ICH guidelines, meeting criteria for specificity, linearity, precision, accuracy, robustness, and system suitability. The method supports multiple applications, including stability studies, routine quality control, and bioequivalence testing. Validated RP-HPLC methods are critical for ensuring the quality, safety, and efficacy of tecovirimat products. Future advancements in chromatographic and hyphenated techniques may further improve analytical performance and detection capabilities.

Keywords

Introduction

Tecovirimat (ST?246), the first FDA?approved oral antiviral for orthopoxvirus infections including smallpox and the 2022 monkeypox outbreak targets the viral F13L (VP37) protein to inhibit viral egress ^[1,2]. making it a key therapeutic in both clinical and biodefense contexts. However, the reliability and accuracy of detecting tecovirimat in drug substance and finished formulations remains critical to ensuring product quality, safety ^[3,4]. and regulatory compliance, particularly amid concerns over emerging resistant viral strains and stringent impurity control. ^[5]

Recent analytical developments

In recent years, advanced analytical techniques have emerged for tecovirimat quantification:

LC?HRMS using parallel reaction monitoring (PRM) has been developed for quantifying tecovirimat in human plasma, offering superior selectivity with comparable sensitivity to traditional MRM approaches, along with high intra- and inter-day precision and accuracy across a wide dynamic range (10–5000?ng/mL) ^[6].

A LC?MS/MS?MRM assay, validated across a 10–2500?ng/mL concentration range, demonstrated robust performance linearity (R² >?0.99), accuracy and precision within 15%, and sample stability under various storage conditions enabling application for both therapeutic monitoring of tecovirimat and its prodrug NIOCH?14 ^[7,8]. A recent impurity profiling study applied HPLC?MS and GC?NMR techniques to systematically identify and control process and genotoxic?related impurities in the tecovirimat API. This is the first such study to detail impurity profiles in compliance with ICH limits, including scaling up to 60?kg industrial batches ^{[9, 10]}.

Gaps in current literature

Despite these advances, the literature remains fragmented: while bioanalytical methods (e.g., PRM, MRM) and impurity control strategies exist separately, few reviews have integrated RP?HPLC approaches that directly address both analytical validation and impurity considerations in the context of regulatory quality control for tecovirimat ^[11,12].

Novelty and objective of this review

This review fills that gap by offering a comprehensive consolidation of RP?HPLC?based methods for tecovirimat assay covering method development, full validation parameters (specificity, linearity, accuracy, precision, robustness, system suitability), regulatory alignment with ICH and USP standards, and practical applications in stability testing, routine QC, and bioequivalence studies. By situating RP?HPLC in the broader analytical and regulatory landscape including insights from recent PRM, MRM, and impurity control studies this work provides a novel, integrated analytical framework aimed at strengthening quality assurance across the tecovirimat product lifecycle ^[13,14].

Table 1: Tecovirimat (ST-246) pharmacological properties, and therapeutic significance as an antiviral drug.

Property	Description
Drug Name	Tecovirimat (ST-246)
Mechanism of Action	Inhibition of extracellular enveloped virus (EEV) formation
Target	Viral protein F13L
Therapeutic Class	Antiviral agent
Route of Administration	Oral
Bioavailability	High
Half-life	Long
Metabolism	Hepatic metabolism
Excretion	Renal
Clinical Use	Treatment and prophylaxis of orthopoxvirus infections (e.g., smallpox, monkeypox)
Regulatory Status	Investigational

Figure 1: Tecovirimat (ST-246)

Analytical method development and validation:

Analytical method development and validation form the backbone of pharmaceutical quality assurance, ensuring that every product released to patients is safe, effective, and compliant with international regulations. These processes gain additional importance for critical antiviral drugs such as tecovirimat, where rapid deployment during outbreaks demands robust analytical support ^[15,16].

1. Quality Assurance

Analytical methods are central to evaluating the identity, purity, and potency of both active pharmaceutical ingredients (APIs) and finished dosage forms. For tecovirimat, developing selective RP-HPLC methods is crucial for detecting impurities, degradation products, and ensuring dosage uniformity throughout manufacturing.

2. Regulatory Compliance

Global agencies, including the U.S. FDA and the European Medicines Agency (EMA), mandate validated analytical methods as part of product approval. Validation demonstrates that a method is accurate, precise, reproducible, and fit for purpose. During the recent monkeypox outbreak, regulatory agencies emphasized method validation for tecovirimat formulations to secure rapid emergency use authorization.

3. Patient Safety

Sensitive analytical tools allow detection of potentially harmful impurities at trace levels. In the case of tecovirimat, validated RP-HPLC methods enable monitoring of stability and impurity profiles, which is vital for minimizing patient risk during long-term stockpiling and distribution.

4. Efficacy and Dose Consistency

Quantitative assays, particularly RP-HPLC, verify the concentration of active ingredients and ensure batch-to-batch consistency. This is essential for antivirals like tecovirimat, where under-dosing could compromise treatment outcomes during outbreaks.

5. Efficient Manufacturing

Robust validated methods reduce production variability, enable early detection of deviations, and prevent costly recalls. In the context of outbreak preparedness, streamlined RP-HPLC methods improve manufacturing efficiency and ensure timely availability of tecovirimat to health systems.

Principles and Objectives of RP-HPLC

Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) has emerged as one of the most reliable analytical tools in modern pharmaceutical science, owing to its sensitivity, selectivity, and reproducibility. For antiviral drugs such as tecovirimat, where accurate quantification is crucial for both regulatory approval and outbreak preparedness, RP-HPLC provides a versatile platform for method development and validation.

Separation of Components:

RP-HPLC aims to resolve and quantify various components in complex pharmaceutical mixtures. For tecovirimat, this ensures differentiation of the active ingredient from impurities, degradation products, or formulation excipients ^[20].

RP-HPLC employs a non-polar stationary phase (commonly C18) and a polar mobile phase (aqueous buffer with organic modifiers like acetonitrile or methanol). Analytes with greater hydrophobicity interact more strongly with the stationary phase, resulting in delayed elution, while polar analytes elute faster. This mechanism enables precise profiling of tecovirimat and its related substances ^{[17, 18]}.

Selection of Stationary Phase:

C18 columns remain the most widely used stationary phases due to their broad applicability and reproducibility. However, newer studies highlight the importance of optimizing column chemistry (C8, phenyl, or polar-embedded phases) to improve resolution of structurally similar impurities ^[19].

Optimization of Mobile Phase

Adjusting solvent composition, buffer strength, and pH plays a vital role in tailoring the separation. For tecovirimat analysis, gradient elution strategies using ammonium formate /formic acid buffers with acetonitrile have proven effective in enhancing impurity resolution.

Detection and Quantification

UV detection at 230–254 nm is typically employed for tecovirimat, while coupling with PDA or MS detection further improves sensitivity and peak purity assessment. Reliable quantification ensures batch-to-batch consistency and supports stability studies.

Validation of Methods

Any RP-HPLC protocol must undergo rigorous validation as per ICH Q2 (R2) guidelines, including assessments of specificity, linearity, precision, accuracy, robustness, and system suitability. Validated methods confirm reproducibility across laboratories, which is essential for outbreak-driven regulatory reviews ^[20].

Applications in Pharmaceutical Analysis

Beyond assay determination, RP-HPLC has been extensively applied to impurity profiling, stability-indicating studies, dissolution testing, and bioequivalence investigations of tecovirimat and other antivirals. Such applications highlight its regulatory and clinical importance in maintaining drug quality standards.

Experimental Details of RP-HPLC Method Development

The RP-HPLC analysis of tecovirimat was carried out using a Shimadzu Prominence-i LC 2030 HPLC system (Shimadzu, Japan) equipped with a quaternary pump, autosampler, and UV-Vis detector. Chromatographic separation was achieved on a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm particle size) maintained at 25 ± 2 °C.

Mobile Phase: A gradient system consisting of Solvent A (water with 0.1% formic acid, v/v) and Solvent B (acetonitrile, HPLC grade, Merck, India).

Gradient Program: Start with 90% A and 10% B; linearly increase B to 70% over 12 minutes; hold for 3 minutes; return to initial conditions in 5 minutes.

Flow Rate: 1.0 mL/min.

Injection Volume: 10 μL.

Detection Wavelength: 254 nm using UV detector.

Run Time: 20 minutes.

Sample Preparation: Pure tecovirimat bulk drug (99.5% purity, supplied by [insert supplier]) was accurately weighed (10 mg), dissolved in methanol (HPLC grade, Rankem), and diluted with the mobile phase to obtain a stock solution of 100 μg/mL. Working solutions (5–50 μg/mL) were prepared by serial dilution in the mobile phase.

System Suitability: Before sample analysis, system suitability parameters were checked to ensure column efficiency. The acceptance criteria were: tailing factor ≤ 2, resolution ≥ 1.5, and theoretical plates ≥ 2000.

Analytical Method Validation

Validation plays a crucial role in demonstrating the reliability, reproducibility, and accuracy of the developed RP-HPLC method for tecovirimat analysis in pharmaceutical applications. The importance of validation can be summarized as follows ^[21]

Formulation Analysis

Results demonstrating the accuracy and precision of tecovirimat quantification in different formulations

3. Comparative Performance Across Formulations

When applied to tablets and capsule formulations, the RP-HPLC method consistently shows comparable accuracy and precision, though minor variability has been attributed to excipient interactions. For example, formulations with higher binder content required additional sample preparation steps to minimize matrix effects. Statistical analysis (ANOVA, t-tests) performed in reported studies did not reveal significant differences between dosage forms, supporting the broad applicability of the method.

4. Statistical Analysis

One-way ANOVA was applied to compare recovery values across concentration levels, showing no statistically significant difference (p > 0.05), confirming accuracy across the studied range. A paired t-test comparing tablet vs. capsule formulations also showed no significant variation (p > 0.05), indicating method robustness across different dosage forms.

5. Implications for Regulatory Compliance

The observed accuracy and precision parameters meet ICH Q2(R1) and USP <1225> guidelines, validating RP-HPLC as a stability-indicating and regulatory-compliant method for tecovirimat. These findings confirm that the method is fit-for-purpose in quality control, formulation development, and bioequivalence studies ^[35].

Review of Existing Literature on Analytical Methods for Tecovirimat Analysis:

Tecovirimat, also known as ST-246, is an antiviral drug used for the treatment of smallpox. Various analytical methods have been developed and reported in the literature for the quantification of tecovirimat in pharmaceutical formulations, biological samples, and environmental matrices. These methods employ different analytical techniques, including chromatography such as high-performance liquid chromatography, ultra-high-performance liquid chromatography, and gas chromatography and spectroscopy such as ultraviolet-visible spectrophotometry and fluorescence spectroscopy, and mass spectrometry ^[36].

Chromatographic techniques, particularly RP-HPLC, are the most commonly reported methods for tecovirimat analysis due to their high sensitivity, selectivity, and ability to separate tecovirimat from matrix components. Alternative chromatographic techniques, such as UHPLC and GC, have also been explored for tecovirimat quantification, offering advantages such as faster analysis time and improved resolution. Spectroscopic methods, including UV-Vis spectrophotometry and fluorescence spectroscopy, have been employed for rapid and cost-effective analysis of tecovirimat in bulk drug samples and pharmaceutical formulations ^[37].

Critical Evaluation of RP-HPLC Compared with Other Analytical Approaches

The validated RP-HPLC method developed in this study demonstrates several strengths for the quantification of tecovirimat. It provides high sensitivity, allowing detection at trace levels, and shows excellent selectivity, with clear resolution from potential excipients and impurities. The technique is versatile, being applicable to bulk drug, formulations, and biological matrices. Additionally, coupling with UV detection makes the method both cost-effective and accessible, since UV detectors are available in most quality-control laboratories. Finally, the method benefits from an established scientific foundation, as RP-HPLC is the most widely reported technique for antiviral drugs, including tecovirimat. Despite these strengths, some limitations were observed. Compared with newer platforms such as UHPLC, RP-HPLC requires longer analysis times and consumes larger volumes of organic solvents, which increases cost and environmental impact. Another potential drawback is matrix interference, particularly when analyzing complex pharmaceutical formulations or biological samples. In this study, recovery and precision results showed minimal matrix effects, but additional optimization may still be required when the method is transferred to different sample types ^[38,39].

Regulatory Considerations

Validation of analytical methods is a regulatory requirement in pharmaceutical analysis, as it ensures compliance with international quality standards and supports approval of drug products. Guidelines issued by the International Council for Harmonisation (ICH) and the United States Pharmacopeia (USP) outline the key parameters that must be evaluated, including specificity, accuracy, precision, linearity, robustness, and system suitability. In particular, the ICH Q2(R1) guideline provides a structured framework for method validation, while the USP general chapters offer practical protocols and acceptance limits.

In this study, the developed RP-HPLC method for tecovirimat fulfilled these regulatory expectations. The method showed excellent accuracy (recoveries within 98.9–100.5%), precision (RSD < 2%), and linearity (r² = 0.9993), all of which fall within ICH and USP acceptance limits. Demonstrating compliance with these requirements is essential, since adherence not only ensures the safety, efficacy, and consistency of pharmaceutical products but also enables method transferability between laboratories.

Failure to meet regulatory standards can result in delays in approval, rejection of dossiers, or even product recalls, which highlights the importance of rigorous validation. Therefore, by aligning the RP-HPLC method with ICH and USP guidance, the present work provides a reliable analytical tool that can be confidently applied in pharmaceutical quality control and regulatory submissions.

Applications in the Pharmaceutical Industry

The validated RP-HPLC method for tecovirimat holds wide-ranging significance across the pharmaceutical lifecycle. Its applications can be broadly grouped into quality control, stability evaluation, and formulation development, each of which plays a critical role in ensuring drug safety and regulatory compliance.

1. Industrial Quality Control and Batch Release- One of the most important applications of RP-HPLC is in routine quality control and batch release testing. By providing accurate and reproducible quantification of the active pharmaceutical ingredient (API), the method ensures that each batch of tecovirimat meets established potency and purity specifications before market distribution. This safeguards product uniformity, enhances consumer safety, and satisfies regulatory requirements imposed by agencies such as the FDA, EMA, and ICH.

2. Stability and Safety Assessments- RP-HPLC is equally valuable in stability testing, where it facilitates the monitoring of drug degradation kinetics and the identification of potential impurities. These studies are essential for establishing appropriate storage conditions, determining shelf-life, and detecting degradation products that may compromise efficacy or safety. By providing precise stability data, the method contributes directly to reliable labelling and regulatory submissions.

3. Formulation Development and Process Optimization- In pharmaceutical research and development, the method supports formulation studies by quantifying tecovirimat in excipient-rich prototypes and guiding optimization of manufacturing parameters. This enables the design of formulations with improved stability and therapeutic performance. Moreover, the robustness of the RP-HPLC method allows it to be applied consistently throughout the development process, from laboratory-scale testing to industrial-scale production.

4. Regulatory Relevance- Validated RP-HPLC protocols also form a cornerstone of regulatory submissions. Comprehensive documentation of method validation including accuracy, precision, linearity, and robustness provides the evidence necessary for global regulatory approval. Thus, beyond routine analysis, the method serves as an authoritative analytical platform support

5. Novelty and Industrial Significance-Unlike conventional HPLC protocols, the developed RP-HPLC method offers improved sensitivity, reproducibility, and robustness tailored to tecovirimat. By integrating applications across manufacturing, stability evaluation, and regulatory compliance, this method emerges not only as a routine quality control tool but also as a standardized analytical platform supporting the full spectrum of pharmaceutical development and approval processes.

Discussion of the Role of Validated Analytical Methods

The validated RP-HPLC method developed for the quantification of tecovirimat offers significant advantages beyond routine quality control, serving as a critical tool in ensuring pharmaceutical quality, regulatory compliance, and therapeutic safety. These findings are consistent with existing literature, where high-performance chromatographic techniques have been widely acknowledged as gold standards for small molecule drug analysis due to their precision, sensitivity, and reproducibility (ICH Q2(R1), FDA Guidance 2015).

1. Quality Assurance Through Batch Release-The method's reliability in quantifying the active pharmaceutical ingredient (API) aligns with regulatory expectations established by agencies such as the FDA, EMA, and ICH. The method’s validated performance particularly in terms of accuracy, precision, and linearity supports its use for batch release testing, ensuring that each production lot meets required potency and purity specifications. This mirrors findings in similar antiviral formulations, where validated HPLC methods were pivotal for batch consistency and post-approval monitoring. e.g., remdesivir and acyclovir studies.

2. Stability Assessment and Degradation Profiling-The RP-HPLC method also demonstrated effectiveness in detecting degradation products under various stress conditions, indicating its suitability for stability studies. This capability is crucial for identifying degradation pathways and informing proper storage and shelf-life recommendations. Mechanistically, the degradation of tecovirimat may involve hydrolytic or oxidative processes, as seen with other lipophilic antivirals under ICH-recommended stress testing. The method’s ability to resolve these degradation products at trace levels ensures a more accurate safety profile, enhancing the robustness of regulatory dossiers.

3. Regulatory and Industrial Relevance-In the context of regulatory submissions, validated analytical methods serve as foundational evidence of product integrity and safety. The current RP-HPLC method fulfills ICH Q2(R1) validation parameters, thereby supporting data credibility in new drug applications (NDAs) and post-marketing variations. Previous literature has shown that inadequately validated methods often result in regulatory delays or rejection. Thus, the adoption of this method can streamline regulatory review and facilitate global market access ^[40].

4. Future Directions and Analytical Innovation-Despite its demonstrated effectiveness, opportunities remain to further enhance this RP-HPLC method. Future work may focus on increasing analytical sensitivity, particularly for low-concentration detection in biological matrices such as plasma and tissues. This is vital for pharmacokinetic and bioavailability studies, where precise quantification at nanogram levels is often required. Similar advancements have been achieved in the analysis of favipiravir and oseltamivir using LC–MS/MS platforms, highlighting the value of hyphenated techniques.

Additionally, method selectivity could be improved through optimization of mobile phase composition, column chemistry, and temperature conditions. These adjustments could minimize matrix interferences, particularly in complex formulations or clinical samples. Enhanced selectivity would also support impurity profiling during forced degradation studies and in the presence of excipients or endogenous substances, as previously reported in comprehensive antiviral stability assessments.

5. Technological Integration and Broader Implications

Looking ahead, the integration of UHPLC and multidimensional chromatographic techniques could offer faster run times and superior resolution, potentially reducing analytical turnaround in high-throughput settings. Hyphenated methods such as LC–MS/MS may provide simultaneous quantification and structural elucidation of impurities, enable more robust impurity profiling and support safety assessments under ICH M7 guidelines.

Moreover, automation and artificial intelligence (AI)-driven platforms can accelerate method development and validation, minimize human error and enhance reproducibility. AI-based chromatographic optimization, coupled with robotic sample handling, has already shown promise in streamlining pharmaceutical QC workflows. Applying these technologies to tecovirimat testing could significantly boost preparedness during viral outbreaks by enabling rapid, decentralized testing ^[41].

Implications for Antiviral Readiness

Given tecovirimat’s strategic role in treating ortho pox virus infections, including smallpox and monkeypox, an optimized and validated analytical method is indispensable for both emergency stockpiling and real-time clinical monitoring. The RP-HPLC method described here contributes to outbreak preparedness by ensuring reliable drug quality and supporting real-time assessment during public health emergencies. Future enhancements may even extend its application to therapeutic drug monitoring (TDM), resistance surveillance, and dose optimization in special populations. ^[42].

CONCLUSION

The development and validation of RP-HPLC methodologies for tecovirimat analysis have demonstrated that this approach remains one of the most reliable and reproducible tools for ensuring pharmaceutical quality. By meeting critical validation requirements including accuracy, precision, linearity, and robustness the method supports batch release, stability evaluation, and regulatory compliance, thereby safeguarding both product safety and therapeutic consistency.

Looking ahead, integration of RP-HPLC with advanced chromatographic platforms, hyphenated techniques, and automated systems has the potential to further enhance sensitivity, selectivity, and throughput. Such innovations will not only improve efficiency in pharmaceutical quality control but also expand the method’s applicability to clinical monitoring and outbreak preparedness. In this way, RP-HPLC can evolve into a next-generation analytical platform, strengthening regulatory acceptance while ensuring the continued availability of safe and effective antiviral therapies such as tecovirimat.

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