Recent Advances in Analytical and Bioanalytical Methods For COVID-19 and Other Critical New Therapeutics (2022-2024): A Comprehensive Review

Pharmaceutical analysis is a dynamic field that plays a crucial role in drug discovery, development, quality assurance, and therapeutic monitoring. The recent surge in novel drug entities, such as antiviral agents, monoclonal antibodies, and RNA-based therapeutics, necessitates the development of precise and advanced analytical methods. Analytical techniques are essential in ensuring drug safety, efficacy, stability, and purity, while bio-analytical methods provide insights into drug metabolism, bioavailability, and pharmacokinetics [1][2]. The COVID-19 pandemic has underscored the importance of rapid and reliable analytical approaches. Drugs like remdesivir, molnupiravir, and nirmatrelvir/ritonavir (Paxlovid) have gained prominence, necessitating sophisticated analytical tools for their evaluation. Additionally, with novel complex therapeutic modalities including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oligonucleotides, peptides, and targeted small molecules, necessitate advanced analytical and bioanalytical methods to support R&D, PK/PD studies, QC, and therapeutic drug monitoring (TDM). This review explores the latest analytical and bio-analytical methods and advances (2022-2024) employed for these critical drugs [3][4], driven by regulatory evolution (ICH M10) [26] and technological innovation.

Analytical Techniques for Recent Small Molecule Drugs

Remdesivir

Remdesivir is a nucleotide analogue prodrug approved for the treatment of COVID-19. The analysis of remdesivir and metabolites requires sensitive and selective techniques due to its rapid metabolism. High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used [5]. A recent HPLC method utilized a C18 column with a gradient mobile phase of acetonitrile and phosphate buffer, achieving good resolution and peak symmetry. LC-MS/MS methods offer lower detection limits and better specificity, particularly for pharmacokinetic studies. These methods often use solid-phase extraction (SPE) or protein precipitation for sample preparation, followed by electrospray ionization (ESI) in positive mode [6].

HPLC and LC-MS/MS Method Parameters and Validation for Remdesivir

Figure 1. Representative HPLC chromatogram of Remdesivir using UV detection.

Case Study: Baricitinib - Analytical Evaluation in COVID-19 Therapy

Baricitinib, a JAK inhibitor, has been repurposed as a treatment option for COVID-19 due to its anti-inflammatory properties. Quantification of baricitinib in human plasma was achieved using LC-MS/MS.

Molnupiravir

Molnupiravir, another antiviral agent for COVID-19, is a prodrug of the ribonucleoside analogue N4-hydroxycytidine (NHC). Analytical challenges stem from its rapid conversion to NHC in vivo. LC-MS/MS remains the gold standard for quantifying both molnupiravir and NHC in plasma and tissues [7]. Recent studies utilized a reverse-phase C18 column and gradient elution with formic acid in water and acetonitrile. Mass spectrometric detection in positive ion mode enabled detection limits as low as 0.5 ng/mL. Sample preparation involved SPE to improve recovery and minimize matrix effects [8].

Comparative Pharmacokinetic and Analytical Profile: Molnupiravir vs. NHC

Figure 2. Overlay of LC-MS/MS chromatograms for Molnupiravir and NHC.

Lenacapavir

Lenacapavir is a long-acting capsid inhibitor approved for HIV-1 infection. Its low dosing frequency and lipophilic nature require specific analytical methods. Reverse-phase HPLC with UV detection has been developed for formulation analysis, while LC-MS/MS is used for bioanalytical applications [9].

Optimized methods include the use of acidic mobile phases to enhance retention and resolution. LC-MS/MS methods employ ESI and multiple reaction monitoring (MRM) for high specificity. These methods are validated for linearity, accuracy, precision, and robustness according to ICH guidelines [10].

Lenacapavir LC-MS/MS Method Validation Summary

Nirmatrelvir-Ritonavir (Paxlovid)

Paxlovid, a combination of nirmatrelvir and ritonavir, has been used to treat mild-to-moderate COVID-19. Nirmatrelvir is a protease inhibitor, and ritonavir boosts its plasma concentration. Analytical methods focus on simultaneous quantification of both components in biological matrices [11]. UHPLC-MS/MS methods have been developed with short run times (<5 min) and high sensitivity (LLOQ ~1 ng/mL). Sample preparation involves protein precipitation, and analysis is conducted using C18 columns with mobile phases comprising ammonium formate and acetonitrile. These methods are validated for bioequivalence and clinical studies [12].

Analytical Approaches for Biologics and RNA-based Drugs

Monoclonal Antibodies (e.g. Tocilizumab, Casirivimab, Imdevimab)

The analysis of monoclonal antibodies (mAbs) poses unique challenges due to their size, heterogeneity, and complex structure. Traditional ligand-binding assays (e.g., ELISA) are widely used but may lack specificity and quantitative precision [13]. Hybrid LC-MS/MS methods have been developed to overcome these limitations. These methods involve enzymatic digestion (e.g., trypsin) followed by targeted peptide analysis using LC-MS. Capillary electrophoresis (CE), surface plasmon resonance (SPR), and size-exclusion chromatography (SEC) are also used for assessing purity, aggregation, and binding kinetics [14].

Case Study: Sotrovimab (Monoclonal Antibody)

Sotrovimab is a monoclonal antibody developed against SARS-CoV-2. Analytical challenges include glycosylation profiling, charge variant analysis, and intact mass measurement.

Techniques used:

• Capillary Electrophoresis (CE-SDS): For molecular weight distribution
• Size Exclusion Chromatography (SEC): For aggregation profiling
• LC-MS Peptide Mapping: For sequence confirmation and post-translational modifications

Results:

• Purity: >98% (SEC)
• Glycoform distribution: G0F (~65%), G1F (~25%), G2F (~10%)
• Intact Mass Accuracy: ±2 Da

mRNA Vaccines

The development of mRNA vaccines for COVID-19 introduced new analytical challenges, including the analysis of RNA integrity, encapsulation efficiency, and lipid nanoparticle characterization. RT-qPCR and digital PCR are commonly used for quantifying mRNA content and degradation [15]. Nano LC-MS/MS has been applied to detect translated proteins in vivo. Analytical ultracentrifugation (AUC), dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA) are used to characterize lipid nanoparticles. High-resolution techniques ensure batch consistency and regulatory compliance [16].

Nano LC-MS/MS Method Performance for mRNA Vaccine Analysis

Bioanalytical Methods for Pharmacokinetic and Clinical Studies

Bioanalytical methods are essential for evaluating drug absorption, distribution, metabolism, and excretion (ADME). LC-MS/MS remains the cornerstone for small molecule analysis, offering high sensitivity and selectivity [17]. For biologics, ligand-binding assays and hybrid LC-MS approaches provide accurate quantification in plasma and tissues. Micro-sampling techniques such as dried blood spots (DBS) and volumetric absorptive micro sampling (VAMS) are gaining popularity due to minimal invasiveness and ease of transport [18].

Emerging Technologies and Recent Advances in Pharmaceutical Analysis

Innovation Drivers

Methodological advancements respond to critical challenges:

• Molecular complexity: mAbs, ADCs, and gene therapies demand multi-attribute characterization [36,37]
• Ultra-low dosing: Potent oncology/CNS drugs require exceptional sensitivity (sub-ng/mL) [38]
• Matrix interference: Analyses in CSF, tissues, and single cells [39,40]
• Real-time monitoring: Emerging point-of-care TDM needs [41]

Technological Advancements

LC-MS Innovations

• Microflow/nano-LC: 10-50x sensitivity gains for peptides (e.g., GLP-1 agonists) in biological matrices [42,43]
• Trapped Ion Mobility Spectrometry (TIMS): Resolves ADC DAR heterogeneity and isobaric metabolites [44,45]
• Hybrid immunoaffinity LC-MS: Quantifies total antibody and conjugated payload in ADCs simultaneously [46]

High-Resolution Mass Spectrometry

• Intact mass analysis: Real-time monitoring of CQAs (glycosylation, oxidation) in biologics [47]
• Multi-Attribute Methods (MAMs): Replace orthogonal assays with single LC-HRMS workflows for mAb/ADC characterization [48]

Ligand Binding Assays

• Digital ELISA: Achieves attomolar sensitivity for biomarkers and low-dose therapeutics [49]
• Multiplexed platforms: Simultaneously quantify 10+ analytes from ≤50μL samples (e.g., cytokine panels) [50]
• Cell-based assays: Functional assessment of gene therapy immunogenicity [51]

Emerging Techniques

• CE-MS: Charge-based separation for mRNA vaccines/oligonucleotides [52]
• Wearable biosensors: Continuous monitoring of antibiotics (vancomycin) and antiepileptics [53]
• Mass spectrometry imaging: Spatial drug distribution in tumor tissues [54]

Drug Class-Specific Advances

1. GLP-1 Agonists (Semaglutide, Tirzepatide)

• Challenge: Peptide stability and low plasma concentrations
• Solutions: Immunoaffinity LC-MS/MS (LLOQ 0.1 ng/mL); TIMS-enabled metabolite ID

2. ADCs (Trastuzumab Deruxtecan)

• Challenge: DAR heterogeneity and free payload toxicity
• Solutions: Hybrid LBA/LC-MS for conjugated payload; HRMS for in vivo DAR quantification

3. KRAS Inhibitors (Sotorasib)

• Challenge: Reactive metabolites and tissue distribution
• Solutions: HRMS metabolite profiling?; QWBA and MALDI imaging for tumor penetration [55,56]

4. Cell/Gene Therapies (CAR-T, AAV)

• Challenge: Vector persistence and immunogenicity
• Solutions: ddPCR for vector genomes [57]; cell-based NAD assays; capsid HRMS characterization [58]

Green Analytical Chemistry

Sustainability is gaining importance in pharmaceutical analysis. Green analytical chemistry (GAC) promotes the use of eco-friendly solvents, minimal reagent consumption, and energy-efficient instruments. Supercritical fluid chromatography (SFC) and miniaturized techniques align with these principles [19].

Artificial Intelligence and Machine Learning

AI and machine learning (ML) are transforming data interpretations in analytical chemistry. These technologies enable pattern recognition, predictive modeling, and optimization of chromatographic conditions. Integration with chemometrics enhances quality control and method development [20].

Microfluidics and Lab-on-a-Chip

Microfluidic platforms offer rapid, cost-effective, and portable analytical solutions. These systems integrate sample preparation, separation, and detection in a single device. They are particularly useful for point-of-care diagnostics and high-throughput screening [21].

Spectroscopic Techniques

Advanced spectroscopic methods such as Raman, near-infrared (NIR), and Fourier-transform infrared (FTIR) spectroscopy are employed for non-destructive analysis. Coupled with multivariate analysis, they provide real-time monitoring of critical quality attributes [22].

Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) allows spatial localization of drugs and metabolites in tissues. Techniques like matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) are used for tissue distribution studies [23].

Summary of Emerging Technologies in Pharmaceutical Analysis

Regulatory Considerations