A Review on Development and Validation of RP-HPLC Method for Estimation of Molnupiravir in Bulk and Dosage Form

Abstract

Molnupiravir, an orally active isopropylester prodrug of ?-D-N?-hydroxycytidine (NHC), represents a significant therapeutic advancement for managing mild-to-moderate COVID-19. Precise analytical quantification of molnupiravir in bulk drug and pharmaceutical dosage forms is essential for ensuring product quality, regulatory compliance, and patient safety. Reverse-phase high-performance liquid chromatography (RP-HPLC) has emerged as the preferred analytical technique due to its exceptional resolution, sensitivity, reproducibility, and universal regulatory acceptance. This comprehensive review examines fundamental principles of RP-HPLC, including stationary phase optimization (predominantly C18 columns), mobile phase composition strategies, pH control mechanisms, and isocratic elution suitability in molnupiravir analysis. Critical sample preparation methodologies for bulk drug substance and formulated pharmaceutical products are systematically presented. Method validation requirements aligned with ICH Q2(R2) and USP guidelines encompass specificity through forced degradation studies, linearity across analytical ranges (r² ? 0.999), accuracy with mean recovery 99–101% (%RSD < 2%), and robustness with excellent system suitability. Applications for routine quality control and stability assessments are highlighted. Overall, RP-HPLC provides a robust, reliable, and cost-effective analytical platform for effective evaluation of molnupiravir throughout pharmaceutical development and commercial manufacture.

Keywords

Molnupiravir, RP-HPLC, Method development, Method validation, Bulk drug estimation

Introduction

Historical Development:

High-performance liquid chromatography (HPLC) evolved from Mikhail Tsvet's 1903 chromatographic technique separating plant pigments using packed columns. The theoretical foundation advanced in the 1940s when Martin and Synge conceptualized partition chromatography using dual liquid phases and mathematical models. Paper chromatography for amino acid identification followed in 1944. The need for rapid, efficient nonvolatile compound analysis in the early 1960s catalyzed modern liquid chromatography development. Waters Corporation introduced the first commercial HPLC system (ALC100) in 1969, marking the

transition from classical to high-performance chromatography. Subsequent innovations include high-purity type B silica materials (late 1980s), sub-2 μm microparticulate silica (early 2000s), and Ultra-High Performance Liquid Chromatography (UHPLC) systems beginning in 1997, with the first commercial UHPLC system introduced in 2004¹⁸.

Principles of Reversed-Phase HPLC:

Separation Mechanism: Reversed-phase HPLC employs a non-polar (hydrophobic) stationary phase with a polar (hydrophilic) mobile phase opposite to classical normal-phase chromatography. Separation is fundamentally based on analyte adsorption and partition according to hydrophobicity: more hydrophobic solutes exhibit greater retention on the non-polar stationary phase, while more hydrophilic solutes preferentially solubilize in the polar mobile phase and elute earlier¹⁹.

Retention Factors:

Analyte retention and selectivity in RP-HPLC are influenced by multiple physicochemical parameters²⁰:

• Octanol-water partition coefficient (logP): Quantifies inherent drug hydrophobicity
• Acid dissociation constant (pKa): Determines ionization at specific pH values
• Chromatographic parameters: Mobile phase pH, buffer concentration, organic modifier type and concentration, and additives

For ionizable compounds, mobile phase pH substantially modulates retention. Carboxylic acid groups become increasingly negatively charged above their pKa (typically pH 4–5), rendering molecules more polar and decreasing retention on non-polar stationary phases. Conversely, pH below pKa suppresses ionization and increases hydrophobicity-based retention²⁰.

Stationary Phases:

C18 and C8 Columns: The most widely utilized stationary phases are C18 (octadecyl) and C8 (octyl) columns, designated by alkyl chain length bonded to silica backbones. C18 columns provide maximum hydrophobicity and predominate in pharmaceutical applications, while C8 columns offer intermediate selectivity with different retention characteristics²¹.

Modern Innovations: High-purity type B silica materials (late 1980s) with low metallic content substantially reduced silanol activity and improved lot-to-lot consistency. Hybrid organosilica particles (commercially available 1999) incorporate organic groups into inorganic silica matrices, offering superior pH stability (pH 1–12 versus pH 2–8 for conventional silica). Charged surface hybrid (CSH) technology (introduced 2010) provides significantly improved peak shapes for basic analytes under acidic, low ionic-strength conditions²².

Mobile Phase Selection:

The mobile phase comprises a polar solvent system mixing water (aqueous component) with organic solvents such as methanol or acetonitrile. The ratio of organic modifier to aqueous phase directly influences retention and selectivity. Increasing organic modifier concentration decreases retention (reduced hydrophobic interactions), while decreasing concentration increases retention. Phosphate buffers commonly adjust mobile phase pH in pharmaceutical analysis²³.

Advantages of RP-HPLC in Pharmaceutical Analysis:

Resolution and Selectivity: Exceptional chromatographic resolution distinguishes pharmaceutical ingredients from impurities and degradation products²⁴.

Broad Applicability: Suitable for analyzing compounds with diverse polarities and molecular masses (small molecules, peptides, proteins, nucleic acids), providing versatility across pharmaceutical applications²⁵

Multiple Detection Options: Interfaces readily with UV/Visible spectrophotometry, mass spectrometry, fluorescence, refractive index, and conductivity detection, enabling flexible method development²⁶.

High Reproducibility: Consistent, reliable results with excellent inter-assay and intra-assay reproducibility critical for quality control²⁶.

Sensitivity and Accuracy: With appropriate detectors (UV or MS), achieves low detection limits and exceptional quantitative precision (typically %RSD < 2%)²⁷.

Elution Flexibility: Supports both isocratic (constant composition) and gradient (time-programmed variation) elution, with gradient elution particularly powerful for complex mixtures and analytes with widely different retention characteristics.

Automation and Throughput: Modern RP-HPLC systems enable comprehensive automation, enhancing productivity through automated sampling and reducing human error.

Regulatory Acceptance: Universal regulatory acceptance by FDA, EMA, and WHO, with extensive validation literature facilitating regulatory submissions²⁷.

Stability-Indicating Capability: With appropriate method development, RP-HPLC effectively separates intact drug molecules from degradation products, making it ideal for stability-indicating development²⁷.?

METHOD DEVELOPMENT:

Stationary Phase Selection:

C18 Columns: C18 (octadecyl-bonded silica) columns are the predominant choice for molnupiravir RP-HPLC method development. The 18-carbon alkyl chain provides strong hydrophobic character, excellent retention for moderately hydrophobic compounds like molnupiravir, extensive literature precedent, broad selectivity, excellent pH stability (pH 2–8 for conventional silica, pH 1–12 for hybrid materials), and high surface area²³.

Published molnupiravir methods employ various C18 columns: Zodiac C18 (150 × 4.6 mm, 5 μm), Agilent C18 (250 × 4.6 mm, 3.6 μm), Discovery HS C18 (75 × 4.6 mm, 3 μm), Hypersil BDS C18, Phenomenex C18, and Inert Sustain C18 (250 × 4.6 mm, 5 μm). Selection depends on desired analysis time, resolution requirements, and peak symmetry. Shorter columns (75 mm) provide rapid analysis (<5 minutes), while longer columns (150–250 mm) offer superior resolution with extended retention times.

High-Purity and Hybrid Phases: High-purity type B silica materials with reduced metallic ion contamination substantially reduce silanol activity and improve reproducibility. Hybrid organosilica phases offer pH stability extending pH 1–12 (versus pH 2–8 for conventional silica), particularly beneficial for molnupiravir given its ester group susceptibility to alkaline hydrolysis²³.

Mobile Phase Optimization:

Solvent Selection: Acetonitrile is preferred over methanol in RP-HPLC methods due to superior chromatographic properties: lower viscosity (enabling higher flow rates, reduced backpressure), lower aqueous miscibility (steeper density gradients), and superior peak symmetry²³.

Published molnupiravir methods employ diverse mobile phase compositions:

• Ammonium acetate buffer (15 mM):acetonitrile:methanol (70:20:10 v/v)¹¹
• Orthophosphoric acid:acetonitrile (60:40 v/v)²⁸
• Acetonitrile:water (20:80 v/v)²⁸
• Water:acetonitrile (70:30 v/v)²⁹
• Phosphate buffer (10 mM, pH 7):acetonitrile (80:20 v/v)²⁹

Buffer System and pH Control: Mobile phase pH profoundly influences molnupiravir retention and peak characteristics by modulating ionization states. Phosphate buffers (10–25 mM) are widely employed, providing consistent buffering with minimal chromatographic interference²³.

Molnupiravir exhibits multiple pKa values (2.2, 10.2, 12.0), with lower pH values ~2 units away from typical analytical pH ranges (pH 3–7). pH variations within 3–7 produce negligible effects on retention time, peak area, or tailing factor, indicating pH primarily influences silanol ionization rather than analyte ionization. Maintaining lower pH values (pH 2.5–3.0) suppresses silanol ionization and reduces secondary interactions causing peak tailing³⁰.

Gradient Versus Isocratic Elution:

Isocratic Elution: Isocratic elution employs constant mobile phase composition throughout separation, offering advantages particularly suited to molnupiravir analysis²⁹:

• Simplicity and Speed: Single mobile phase composition reduces development cycles
• Lower Costs: Constant solvent consumption and simplified programming reduce expenses
• Excellent Reproducibility: Uniform conditions eliminate baseline drift
• Detector Stability: Constant composition eliminates detector response changes

All published molnupiravir RP-HPLC methods employ isocratic elution, achieving rapid 2–5 minute analysis times with complete resolution¹³.

Gradient Elution: Gradient elution progressively modifies mobile phase composition, offering advantages for complex mixtures:

• Superior Resolution: Time-programmed solvent strength optimizes elution across diverse polarities
• Shorter Analysis Times: Accelerates strongly retained compound elution
• Enhanced Peak Capacity: Accommodates greater compound numbers

However, for molnupiravir, gradient elution adds unnecessary complexity. Isocratic methods are the rational choice, providing rapid, simple, reproducible analysis with complete resolution³¹.

Sample Preparation:

Bulk Drug Substance: "Weigh-Dilute-Shoot" Method

1. Accurate Weighing: 10–20 mg molnupiravir bulk powder into volumetric flask¹⁰.?
2. Initial Dissolution: Add mobile phase in small portions (~30–40 mL)¹⁰
3. Sonication: Ultrasonic bath at 25°C for 5–15 minutes to ensure complete dissolution¹⁰.
4. Dilution to Volume: Cool to room temperature, dilute to final volume with mobile phase (typically achieving 100 μg/mL)¹⁰.
5. Filtration: Pass through 0.45 μm PTFE or nylon filter, discard initial 0.5 mL filtrate¹⁰.
6. HPLC Injection: Inject aliquot directly without further dilution¹⁰.

Pharmaceutical Dosage Forms: "Crush-Extract-Filter" Method:

Sample preparation for pharmaceutical dosage forms involves comminution to <100 μm particle size, with capsule contents emptied and pulverized. Accurately weighed samples (10–20 mg molnupiravir equivalent) are transferred to volumetric flasks and extracted using sonication (5–10 minutes), mechanical shaking, or automated homogenization (3–6 minutes). Following centrifugation at 3,000–5,000 rpm for 5–15 minutes, the extract is filtered sequentially through 0.45 μm (and 0.2 μm if necessary) PTFE or nylon membrane filters, with discard of the initial 0.5 mL filtrate. For capsule formulations, 10 units are weighed, emptied, and pulverized; the powder equivalent to 10 mg molnupiravir is extracted via three sequential 10 mL aliquots with 5-minute sonication each, then filtered and diluted to working concentration before HPLC injection. Critical considerations include ensuring solvent compatibility with the mobile phase to prevent precipitation, maintaining neutral to slightly acidic pH (2.5–3.0) to prevent molnupiravir degradation during storage, and validating procedures to demonstrate 95–105% quantitative recovery through matrix-spiked standards¹⁰.

METHOD VALIDATION:

Regulatory Guidelines (ICH, USP, EMA):

The ICH Q2(R2) guideline (adopted November 1, 2023) serves as the global gold standard for analytical method validation, establishing fitness-for-purpose validation with performance characteristics and acceptance criteria varying by intended use and technology. Validation protocols must specify intended purpose, performance characteristics, and acceptance criteria with all data and calculation formulae submitted. Data from analytical procedure development (ICH Q14) can be integrated into validation data, and platform methods can leverage prior knowledge to avoid unnecessary duplication. USP General Chapter <1225> categorizes methods into four types: Category I (qualitative identification requires specificity), Category II (quantitative assays requires specificity, linearity, range, accuracy, precision), Category III (impurity tests requires specificity, linearity, range, accuracy, precision, LOD/LOQ), and Category IV (performance tests). USP <1226> enables streamlined verification of established compendial procedures. The EMA adopts ICH guidelines through European Commission mechanisms (EMA/CHMP/ICH/82072/2006), with EMA scientific guidelines on analytical procedures aligning completely with ICH Q2(R2) requirements, ensuring uniform regulatory acceptance across European jurisdictions¹⁰.

Validation Parameters:

Specificity/Selectivity: The ability to unequivocally assess molnupiravir in the presence of impurities, degradation products, excipients, and related substances is confirmed through blank/placebo analysis, forced degradation studies (acidic: 2 M HCl/60°C, 30 min; alkaline: 0.1–5 M NaOH; oxidative: 3% H?O?; thermal: 60–100°C/24 h; photolytic: UV 254 nm), photodiode array peak purity assessment (>0.990), and baseline separation (Rs ≥ 1.5–2.0) from primary degradant NHC with 5–20% degradation under stress conditions.?

Linearity and Range: Minimum five concentration levels across analytical ranges (0.2–100 μg/mL) with 3–6 injections each demonstrate correlation coefficients (r²) ≥ 0.999 with random residual distribution. Published molnupiravir methods achieve r² = 0.9995–1.0 with %CV < 2% and %RSD of slope/intercept < 1%.?

Accuracy: Recovery studies at three concentration levels (80%, 100%, 120%) with three replicates each (9 total determinations) demonstrate mean recovery of 100% ± 2%, with individual values 95–105% and %RSD ≤ 2–5%. Published molnupiravir studies report 99–101% recovery with %RSD < 2%.?

Precision: Repeatability (%RSD ≤ 0.73–2.0%) via six consecutive injections and intermediate precision (%RSD ≤ 1–2%) through different operators/days demonstrate method reliability. Published molnupiravir methods achieve repeatability %RSD 0.30–0.45%, intra-day precision 0.51%, and inter-day precision 0.57%.?

LOD and LOQ: Calculated as LOD = 3.3σ/S and LOQ = 10σ/S (where σ = response standard deviation, S = calibration curve slope), with acceptance criteria of signal-to-noise ≥ 10:1 at LOQ. Published molnupiravir methods report LOD = 0.04–5.004 μg/mL and LOQ = 0.12–15.164 μg/mL, with exceptional methods achieving LOD = 0.013 μg/mL and LOQ = 0.043 μg/mL³⁰.?

Robustness and Ruggedness:

Robustness: Method capacity to remain unaffected by small deliberate variations in internal parameters (mobile phase composition, pH, flow rate, temperature, injection volume).

Methodology: Systematically vary parameters while monitoring retention time, resolution, peak area, peak symmetry:

• Mobile phase acetonitrile ±2–5%
• pH ±0.2 units
• Flow rate ±10%
• Temperature ±5°C
• Sample preparation variables ±10%

Acceptance Criteria: Resolution ≥ 1.5 with variations; retention time ±5%; peak area ±10%; tailing factor 0.9–1.2³¹.

System Suitability Testing (SST)

Definition: Predefined criteria confirming HPLC system adequacy before analyzing unknown samples³².

Key Parameters:

Table No. 1: HPLC System Suitability Parameters33

Parameter	Acceptance Criterion
Theoretical Plates (N)	≥ 2,000
Tailing Factor (T)	0.8–1.2
Resolution (Rs)	≥ 1.5–2.0
Retention Time RSD	≤ 5% for ≥3 injections
Peak Area RSD	≤ 2% for 6 injections
Signal-to-Noise Ratio	≥ 10:1 at LOQ

Application to Dosage Forms:

Bulk Drug Analysis:

Bulk drug substance analysis represents the foundation for pharmaceutical quality assurance, ensuring that molnupiravir raw material meets predetermined specifications for purity, identity, and potency before incorporation into formulations. Bulk drug analysis provides critical information for regulatory submissions, supporting batch release decisions and long-term stability assessments¹⁰.?

Sample Preparation for Bulk Molnupiravir: Direct Dissolution Method

Molnupiravir bulk drug substance demonstrates excellent solubility in aqueous and aqueous-organic solvent mixtures, enabling straightforward sample preparation. The procedure involves:

(1) precise weighing of molnupiravir bulk powder (10–20 mg, ±0.0001 g precision) into a calibrated 100 mL volumetric flask;

(2) addition of mobile phase in portions (~30–40 mL) with dissolution occurring at room temperature within 10–15 minutes at neutral to slightly acidic pH;

(3) sonication in an ultrasonic bath at 25°C for 5–15 minutes to ensure complete dissolution and remove air bubbles;

(4) cooling to room temperature and dilution to the calibration mark with mobile phase;

(5) filtration through 0.45 μm PTFE or nylon membrane filters with discard of the initial 0.5–1.0 mL filtrate; and

(6) direct HPLC injection without further dilution. This straightforward approach ensures rapid sample preparation with minimal manipulation, reducing the risk of analyte degradation or loss during the preparation process²¹.?

Representative Validation Results for Bulk Molnupiravir:

Published studies have established comprehensive validation parameters for molnupiravir bulk drug analysis using RP-HPLC methods:

Linearity and Range:

Representative bulk drug linearity studies have established linear relationships across concentration ranges including:

• 0.2–80.0 μg/mL with correlation coefficient r² = 1.0
• 50–500 μg/mL (for high-concentration assays) with excellent linearity?

The linear regression equations typically demonstrate y-intercepts very close to zero, with slopes ranging from 10,000–15,000 (depending on specific detection wavelength and chromatographic conditions), confirming excellent proportionality between analyte concentration and detector response³⁰.

Accuracy and Recovery:

Representative recovery studies for molnupiravir bulk substance have demonstrated:

• Mean recovery: 100.29% with low variability
• Individual recovery values: Consistent recovery at 80%, 100%, and 120% concentration levels
• Overall recovery %RSD: <1–2%, indicating excellent method accuracy

These recovery results establish the method's freedom from systematic error and suitability for precise quantification of molnupiravir content in bulk material.

Precision

Comprehensive precision studies for bulk molnupiravir determination have yielded:?

• Repeatability (intra-assay precision): %RSD = 0.30–0.45% for six consecutive injections of standard solution
• Intra-day intermediate precision: %RSD = 0.51% (different operators, same day)
• Inter-day intermediate precision: %RSD = 0.57% (different days)

These precision values substantially exceed typical acceptance criteria (%RSD ≤ 2%), demonstrating excellent reliability for routine bulk drug analysis¹³.?

LOD and LOQ

Published bulk drug validation studies have established exceptional sensitivity for molnupiravir RP-HPLC methods:

• Limit of Detection (LOD): 0.04–0.06 μg/mL
• Limit of Quantification (LOQ): 0.12–0.20 μg/mL

These detection limits provide adequate sensitivity for impurity determinations and trace-level analysis beyond the typical assay range³⁴.

Bulk Drug Assay Results

Representative bulk drug assay results demonstrate the practical applicability of RP-HPLC methods:

• Molnupiravir bulk powder (98% purity reference standard): Assay = 99.5–100.8%
• Commercial bulk samples: Assay values within 98–102% of label claim³⁴.?

System suitability parameters for bulk analysis consistently demonstrate:

• Retention time: 4.0–4.9 minutes (depending on method parameters)
• Tailing factor: 0.95–1.10 (excellent peak symmetry)
• Theoretical plates: >3,000 (exceptional column efficiency)
• Resolution: >2.0 from any nearby peaks³⁴.

Limitations of RP-HPLC for Molnupiravir Analysis

Moderate Sensitivity for Trace Impurities

While adequate for assay (LOQ 0.1–0.2 μg/mL), RP-HPLC/UV lacks sensitivity for trace-level impurity detection at parts-per-million levels achievable by LC-MS/MS (10–100 fold superior sensitivity)¹⁶.

Lack of Structural Information

RP-HPLC with UV detection provides only indirect characterization through retention time comparison. Peak identification relies entirely on authentic standards, creating challenges for characterizing novel degradation products lacking commercial reference standards.

Limited Multiplexing for Multi-drug Analysis

Simultaneous quantification of molnupiravir with multiple co-administered antivirals (favipiravir, remdesivir) requires method reconfiguration for each combination, whereas LC-MS/MS enables inherent multiplexing without modification¹⁰.

Matrix Interference in Biological Samples

In complex matrices (plasma, serum), RP-HPLC/UV exhibits reduced reliability due to protein, lipid, and endogenous metabolite interference causing response suppression or enhancement. LC-MS/MS provides superior selectivity through mass spectrometric discrimination.

Dependence on Reference Standards

External calibration requires authentic reference standards; unavailability, high cost, or purity uncertainties substantially impact analysis reliability and create supply chain vulnerabilities absent in mass spectrometry approaches.

CONCLUSION:

Reverse-phase high-performance liquid chromatography has established itself as the gold standard analytical technique for precise quantification and quality assessment of molnupiravir in pharmaceutical applications. The comprehensive review demonstrates that RP-HPLC methods provide excellent specificity, sensitivity, linearity, accuracy, and reproducibility across bulk drug and formulated dosage forms when developed and validated in compliance with ICH Q2(R2), USP, and EMA regulatory frameworks. Systematic optimization of stationary phases (predominantly C18), mobile phase composition, pH control (typically 2.5–3.0), and isocratic elution conditions enables rapid, cost-effective analysis with detection limits adequate for routine quality control and stability monitoring. Validated RP-HPLC protocols consistently achieve >99% recovery with relative standard deviations <2%, system suitability parameters exceeding acceptance criteria, and excellent inter-day reproducibility. While acknowledging inherent limitations including moderate sensitivity for trace impurities, lack of structural characterization, and dependence on reference standards, RP-HPLC remains the preferred technique for pharmaceutical lifecycle assessment. Future developments incorporating hybrid stationary phases, enhanced detectors, and automated sample preparation systems will further strengthen its utility in molnupiravir quality assurance. Continued standardization and broader adoption of validated RP-HPLC methods across manufacturing facilities will ensure consistent product quality, regulatory compliance, and patient safety throughout molnupiravir's therapeutic application in pandemic and post-pandemic scenarios.

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