Next-Generation Ketolides in Respiratory Infections: A Comprehensive Review of Nafithromycin in CABP

Community-acquired bacterial pneumonia (CABP) remains a major public health challenge worldwide, contributing significantly to morbidity and mortality across all age groups, particularly among elderly individuals, immunocompromised patients, and those with chronic conditions such as diabetes, chronic obstructive pulmonary disease, renal disease, and cardiovascular disorders [1,2]. Globally, CABP leads to millions of hospitalizations annually and places a substantial burden on healthcare systems due to prolonged hospital stays and intensive care requirements [1, 2].

In developing countries, this burden is exacerbated by delayed diagnosis, limited healthcare access, and suboptimal empirical antibiotic therapy. Even in well-resourced healthcare settings, rising antimicrobial resistance and safety concerns related to existing antibiotics compromise treatment outcomes [3, 4]. These challenges highlight the need for novel, effective, and safe oral antibacterial agents that can simplify therapy and improve patient outcomes.

Figure 1: Comparison of Healthy Lungs and Community-Acquired Bacterial Pneumonia (CABP) with Common Causative Pathogens.

1. Common Causative Organisms:

CABP is caused by a variety of bacterial pathogens, necessitating empirical treatment with broad-spectrum coverage. Streptococcus pneumoniae remains the predominant pathogen, accounting for a significant proportion of severe infections. Other commonly implicated bacteria include Haemophilus influenzae and Moraxella catarrhalis. Additionally, atypical pathogens such as Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila play a notable role, particularly in younger or ambulatory patients [2, 5]. The diversity of causative organisms complicates empirical therapy and underscores the importance of antibiotics active against both typical and atypical respiratory pathogens.

Figure 2: (a) Streptococcus pneumoniae (b) Haemophilus influenza (c) Moraxella catarrhalis (d) Mycoplasma pneumoniae (e) Chlamydophila pneumoniae (f) Legionella pneumophila

2.  Limitations of Existing Therapies

Currently, macrolides, fluoroquinolones, and β-lactam antibiotics form the backbone of CABP treatment. Although generally effective, these agents face limitations due to antimicrobial resistance, adverse effects, and safety concerns. Fluoroquinolones, for example, are linked to serious adverse events including tendinopathy, central nervous system effects, and dysglycemia [4]. Macrolides, while convenient for oral therapy and effective against atypical pathogens, have seen reduced susceptibility in key respiratory pathogens due to widespread use. β- Lactam antibiotics often require combination therapy to cover atypical organisms, which can increase pill burden and risk of non-compliance [3, 4].

3. Macrolide resistance

Macrolide resistance occurs primarily through target site modification (ribosomal methylation or mutation), active drug efflux, and enzymatic inactivation, which prevent the antibiotic from binding to the 50S ribosomal subunit and inhibiting protein synthesis. The most common mechanism is methylation of the 23S rRNA, reducing the drug's affinity. Genes for macrolide resistance act via three main mechanisms - target site modification, activation of efflux pumps and enzymatic modification of the drug [7].

1. Target site modification

• Erythromycin ribosome methylation (erm) genes (e.g. erm A, B, C) are transcribed and translated to produce enzymes that can methylate the 23S rRNA binding site on the 50S subunit. 
• The altered binding site results in decreased binding affinity of macrolides. 
• This results in high-level resistance.

2. Efflux pump activation

• Macrolide efflux (mef) genes (e.g. mef A, B, E) are transcribed and translated to produce transporters that pump out macrolides before they can reach their site of action on the 50S ribosomal subunit. 
• This results in relatively low to moderate resistance.

3. Enzymatic modification

• Macrolide phosphotransferase (mph) and erythromycin esterase (ere) genes are transcribed and translated to produce enzymes that can modify macrolides so they are no longer capable of binding effectively to the 50S ribosome.
• The altered binding means the macrolides are unable to exert an effect.
• This results in high-level resistance.

Figure 3: Mechanism of Macrolide Resistance [7].

4. QT Interval Prolongation:

The QT interval on an electrocardiogram (ECG) represents the total duration of ventricular depolarization and repolarization. Prolongation of the QT interval is clinically significant because it predisposes patients to life-threatening ventricular arrhythmias, particularly torsades de pointes (TdP). Drug-induced QT prolongation is a well-recognized adverse effect associated with several antimicrobial classes, including macrolides and fluoroquinolones, which are commonly used in the treatment of community-acquired bacterial pneumonia (CABP) [8,9].

Patients with CABP often have multiple risk factors for QT prolongation, such as advanced age, electrolyte imbalance, cardiovascular disease, and concomitant medications. Therefore, the cardiac safety profile of antibiotics is a critical consideration when selecting empirical and targeted therapy [9].

1. Molecular Mechanism of Drug-Induced QT Prolongation:

QT prolongation primarily occurs due to inhibition of cardiac potassium ion channels, especially the human ether-à-go-go-related gene (hERG, Kv11.1) channel. This channel mediates the rapid delayed rectifier potassium current, which is essential for phase 3 repolarization of the cardiac action potential [10].

When drugs inhibit the hERG channel:

• Potassium efflux during repolarization is reduced
• Action potential duration is prolonged
• QT interval on ECG is extended
• Electrical instability may develop, leading to TdP
• Normal Cardiac Repolarization:

Na+ influx → Ca2+ influx → K+ efflux → Normal QT

Drug-Induced QT Prolongation

Macrolide / Fluoroquinolone

↓

hERG (IKr) Channel Blockade

↓

↓ Potassium Efflux

↓

Prolonged Action Potential

↓

QT Interval Prolongation

↓

Risk of Torsades de Pointes

Figure 4: Mechanism of Drug-Induced QT Prolongation.

2. QT Prolongation Associated with Macrolide Antibiotics:

Macrolides such as erythromycin, clarithromycin, and azithromycin are known to prolong the QT interval through direct hERG channel inhibition. Additionally, many macrolides inhibit cytochrome P450 (CYP3A4), leading to increased plasma concentrations of co-administered QT-prolonging drugs [8, 11].

Clinical consequences include:

• Increased arrhythmia risk in elderly patients.
• Higher incidence of adverse events in polypharmacy.
• Restriction of use in patients with pre-existing cardiac disease.

These limitations reduce the suitability of conventional macrolides for widespread outpatient management of CABP, particularly in high-risk populations [9].

3. Relevance to CABP Treatment:

CABP patients frequently receive combination therapy and supportive medications, increasing the risk of pharmacokinetic and pharmacodynamic interactions. Antibiotics with QT-prolonging potential require ECG monitoring, which is often impractical in outpatient settings. Consequently, there is a strong clinical demand for newer oral agents that retain macrolide-like efficacy without compromising cardiac safety [12].

5. Drug–Drug Interactions in CABP Therapy:

Patients with community-acquired bacterial pneumonia (CABP) frequently belong to elderly or comorbid populations and are often prescribed multiple medications for chronic conditions such as cardiovascular disease, diabetes, and respiratory disorders. Polypharmacy substantially increases the risk of drug–drug interactions (DDIs), which may lead to reduced therapeutic efficacy, increased toxicity, or serious adverse events. Antibiotics used for CABP, particularly macrolides and fluoroquinolones, are among the most commonly implicated drug classes in clinically significant DDIs [15].

0. 1. Pharmacokinetic Mechanisms of Drug–Drug Interactions:
1. Cytochrome P450 Enzyme Inhibition-

One of the most important mechanisms underlying DDIs is inhibition of hepatic cytochrome P450 (CYP) enzymes, especially CYP3A4. Several macrolides, including erythromycin and clarithromycin, are strong CYP3A4 inhibitors. When co-administered with drugs metabolized by this pathway, plasma concentrations of the co-administered drug may increase, resulting in toxicity [16].

Commonly affected drugs include:

• Statins (risk of myopathy and rhabdomyolysis)
• Calcium channel blockers (hypotension, bradycardia)
• Oral anticoagulants (bleeding risk)
• Antiarrhythmic agents (arrhythmias)

Azithromycin has a lower CYP3A4 inhibitory effect but is still associated with clinically relevant interactions in susceptible patients [16, 17].

2. Transporter-Mediated Interactions-

Macrolides can also inhibit membrane transport proteins such as P-glycoprotein (P-gp). Inhibition of P-gp alters the absorption and elimination of drugs like digoxin, leading to elevated systemic exposure and toxicity. Transporter-based interactions further complicate antibiotic selection in CABP patients receiving long-term medications [17].

Macrolide Antibiotic

↓

CYP3A4 / P-gp Inhibition

↓

↓ Drug Metabolism or Efflux

↓

↑ Plasma Drug Concentration

↓

Increased Toxicity / Adverse Effects

Figure 5:  Mechanism of action of drug-drug interaction.

1. 2. Pharmacodynamic Drug–Drug Interactions:

Pharmacodynamic interactions occur when two drugs exert additive or synergistic effects on the same physiological system. In CABP therapy, the most clinically relevant pharmacodynamic interaction involves QT interval prolongation.

When macrolides are co-administered with other QT-prolonging drugs (e.g., antiarrhythmics, antipsychotics, fluoroquinolones), the risk of torsades de pointes is significantly increased. This interaction is independent of drug concentration and can occur even at therapeutic doses [18].

1. 3. Clinical Consequences of DDIs in CABP:

The presence of DDIs can lead to:

• Increased hospitalization duration
• Need for intensive monitoring
• Restriction of antibiotic choice
• Reduced adherence due to therapy modification

In outpatient CABP management, avoiding antibiotics with high DDI potential is essential to ensure safety and treatment success [15, 19].

6. Mechanism of Action of Nafithromycin:

1. Ribosomal Binding and Protein Synthesis Inhibition:

Nafithromycin is a novel lactone ketolide antibiotic designed to overcome macrolide resistance while improving safety. It exerts its antibacterial effect by inhibiting bacterial protein synthesis through binding to the 50S ribosomal subunit. Unlike traditional macrolides, nafithromycin demonstrates dual-site ribosomal binding, which enhances its affinity and reduces susceptibility to resistance mechanisms [13].

Specifically, nafithromycin interacts with:

• Domain V of 23S rRNA
• Additional ribosomal regions involved in peptide elongation

This dual interaction leads to effective inhibition of translocation and premature termination of bacterial protein synthesis.

2. Activity against Macrolide-Resistant Pathogens:

Resistance to macrolides commonly arises through:

• Ribosomal methylation (erm genes)
• Efflux pumps (mef genes)

Nafithromycin retains activity against many resistant strains because its structural modifications allow stable ribosomal binding even in the presence of methylated rRNA. This property is particularly valuable against Streptococcus pneumoniae, a predominant CABP pathogen [13, 14].

3. Cardiac Safety Advantage of Nafithromycin:

Unlike conventional macrolides, nafithromycin exhibits minimal interaction with the hERG channel and does not significantly inhibit CYP3A4. As a result:

1. QT prolongation risk is reduced
2. Drug–drug interaction potential is minimized
3. Suitability for outpatient oral therapy is improved

These characteristics represent a major therapeutic advantage in the management of CABP [12, 14].

4. Reduced Drug–Drug Interaction Potential of Nafithromycin:

A major advantage of nafithromycin is its minimal inhibition of CYP3A4 and P-glycoprotein, significantly reducing its potential for pharmacokinetic DDIs. In vitro studies have demonstrated negligible effects on major drug-metabolizing enzymes, making nafithromycin safer for use in patients receiving multiple concomitant medications [20].

This favorable interaction profile, combined with reduced QT prolongation risk, supports its suitability as an oral monotherapy for CABP.

Nafithromycin

↓

Penetration into Bacterial Cell

↓

Binding to 50S Ribosomal Subunit

↓

Inhibition of Protein Synthesis

↓

Suppression of Bacterial Growth

↓

Clinical Cure of CABP

Figure 6: Mechanism of action Nafhithromycin.

EPIDEMIOLOGY OF COMMUNITY-ACQUIRED BACTERIAL PNEUMONIA (CABP)

Community-acquired bacterial pneumonia (CABP) remains a leading cause of infectious morbidity and mortality worldwide. Despite advances in antimicrobial therapy and vaccination, CABP continues to impose a substantial public health burden, particularly in low- and middle-income countries (LMICs) such as India. The epidemiology of CABP varies considerably across regions due to differences in population demographics, healthcare infrastructure, vaccination coverage, prevalence of comorbidities, and antimicrobial resistance patterns [21,22].

1. Global Epidemiology of CABP :

Globally, community-acquired pneumonia affects approximately 3–5% of adults annually, with bacterial pathogens accounting for a major proportion of clinically significant cases [23]. The estimated global incidence of CAP is ~4,000–4,500 cases per 100,000 population per year, with higher incidence rates observed at the extremes of age [24].

Lower respiratory tract infections, including CABP, are consistently ranked among the top three causes of infectious disease–related mortality worldwide, contributing to approximately 2–2.5 million deaths annually [24,25].

Figure 7: Marked regional differences in the incidence and mortality of CABP are observed worldwide, with the highest burden reported in South-East Asia and Africa.

1. Mortality and Case Fatality Rates:

CABP-associated mortality varies according to disease severity and healthcare setting:

• Outpatient CABP: <2%
• Hospitalized CABP: 5–20%
• Severe CABP (ICU-admitted): up to 40–50%

Elderly patients (≥65 years), immune compromised individuals, and those with chronic cardiopulmonary diseases experience disproportionately higher mortality rates [22, 26].

Figure 8: Mortality increases substantially with disease severity, particularly among ICU-admitted patients.

2. Age-Specific Distribution:

Epidemiological studies demonstrate a U-shaped age distribution, with the highest incidence and mortality observed in:

1. Children <5 years (especially in LMICs)
2. Adults ≥65 years

In older adults, incidence rates may exceed 25–40 cases per 1,000 persons per year, reflecting immunosenescence and increased comorbidity burden [23, 24].

3. Global Etiological Pattern:

Streptococcus pneumoniae remains the predominant bacterial cause of CABP globally, followed by Haemophilus influenzae, Staphylococcus aureus, and atypical pathogens such as Mycoplasma pneumoniae [27]. Increasing antimicrobial resistance, particularly macrolide and β-lactam resistance, has significantly influenced contemporary CABP epidemiology and treatment strategies [26, 27].

2. Epidemiology of CABP in India:

India bears a disproportionately high burden of pneumonia, contributing nearly 20–25% of global pneumonia cases. Epidemiological estimates suggest an annual pneumonia incidence of 5–11 cases per 1,000 population, corresponding to approximately 4–5 million adult CABP cases per year. Under-diagnosis, limited access to microbiological testing, and under-reporting—particularly in rural regions—suggest that the true burden may be substantially higher [28, 29].

Figure 9: WHO-Defined Clinical pneumonia incidence in children younger than 5 years (per 1000).

1. 1. Mortality Patterns in India:

Mortality rates associated with CABP in India remain significantly higher than those reported in high-income countries:

1. Overall hospital mortality: 14–30%
2. Severe CABP (ICU): up to 45–47%

Delayed healthcare presentation, inappropriate empirical therapy, limited ICU capacity, and high prevalence of multidrug-resistant organisms contribute to adverse outcomes [21, 29].

1. 2. Etiological Distribution in Indian CABP:

In contrast to Western populations, Indian studies report a higher prevalence of Gram-negative pathogens, particularly in hospitalized and severe cases. This pathogen distribution has direct implications for empirical antibiotic selection and antimicrobial stewardship in India [28, 29]. The relative contribution of major bacterial pathogens causing CABP in India is illustrated in Fig1.9.

Table 1: Major bacterial pathogens causing CABP in India

Figure 10: Etiological Distribution of CABP in India.

Distribution of major bacterial pathogens causing community-acquired bacterial pneumonia in India. Streptococcus pneumoniae remains the predominant pathogen, while Gram-negative organisms, particularly Klebsiella pneumoniae, contribute significantly to hospitalized and severe cases. CABP incidence in India demonstrates seasonal peaks during winter and post-monsoon months, likely related to increased viral respiratory infections, air pollution, and overcrowding.

OVERVIEW OF THE NOVEL LACTONE KETOLIDE NAFHITHROMYCIN:

Nafithromycin (also known as WCK 4873) is a next-generation ketolide antibiotic developed as an advanced derivative of the macrolide class. Chemically, it belongs to the lactone ketolide subclass, which represents structurally modified macrolides designed to overcome resistance mechanisms associated with conventional agents such as azithromycin and clarithromycin. Ketolides differ from classical macrolides primarily by removal of the cladinose sugar at the C3 position, replacement with a keto functional group, and the introduction of an extended aryl–alkyl side chain. These modifications confer enhanced ribosomal binding, improved antibacterial potency, and reduced susceptibility to macrolide resistance mechanisms [30, 31].

Figure 11: Chemical Structure of Nafhithromycin

Formula: C₄₂H₆₂N₆O₁₁S

Molecular Weight: 859.05 g/mol

Structure: It is a 16-membered lactone-ketolide featuring a 2-pyridine-1,3,4-thiadiazole side chain connected via a four-atom spacer. 

1. Structural Features of Nafithromycin:
   1. Macrolactone Ring System-

Nafithromycin retains a 14-membered macrolactone ring, a core structural feature essential for binding to the bacterial ribosome. This lactone ring acts as the scaffold upon which additional functional groups are positioned to optimize antimicrobial activity. Compared with earlier macrolides, the ring system in nafithromycin is chemically stabilized to enhance acid resistance and metabolic durability [32].

1. 2. C3 Keto Group (Absence of Cladinose Sugar)-

A defining feature of nafithromycin is the replacement of the cladinose sugar at the C3 position with a keto group. In classical macrolides, the cladinose sugar plays a key role in triggering inducible MLS resistance via erm-mediated methylation of the 23S rRNA. Removal of this sugar significantly reduces resistance induction and enhances activity against macrolide-resistant Streptococcus pneumoniae. This modification is fundamental to the ketolide class and directly contributes to the superior resistance profile of nafithromycin [31].

1. 3. Extended Aryl–Alkyl Side Chain-

Nafithromycin contains a rigid aromatic side chain attached to the macrolactone ring, which is absent in azithromycin and clarithromycin. This side chain extends into domain II of the 23S rRNA, enabling a second anchoring point within the ribosomal exit tunnel. As a result, nafithromycin exhibits dual ribosomal binding, significantly increasing binding affinity and reducing the likelihood of resistance due to target site modification [30].

2. Structural Comparison with Classical Macrolides:

1. 1. 1. Comparison with Azithromycin:

Azithromycin is a 15-membered azalide macrolide, characterized by the insertion of a nitrogen atom into the lactone ring and retention of the cladinose sugar. While azithromycin demonstrates broad-spectrum activity, its reliance on a single ribosomal binding site (domain V) makes it vulnerable to methylation-mediated resistance. In contrast, nafithromycin:

• Lacks cladinose sugar
• Possesses a keto group at C3
• Exhibits dual binding to ribosomal domains II and V

These differences translate into higher potency against resistant pneumococcal strains and improved pharmacodynamic performance [33, 34].

1. 1. 2. Comparison with Clarithromycin:

Clarithromycin is a 14-membered macrolide derived from erythromycin with enhanced acid stability due to methoxy substitution. However, it still retains the cladinose sugar and binds predominantly to domain V of 23S rRNA, making it susceptible to MLS_B resistance. Nafithromycin overcomes these limitations through:

• Structural elimination of cladinose
• Addition of a ribosome-anchoring side chain
• Reduced induction of resistance genes

As a result, nafithromycin demonstrates greater in vitro and in vivo activity against macrolide-resistant respiratory pathogens [32, 35].

3. Structural–Functional Correlation:

Table 2: Structural Comparison of Nafithromycin with Azithromycin and Clarithromycin.

The collective structural modifications in nafithromycin lead to:

• Improved antibacterial potency
• Enhanced lung tissue penetration
• Prolonged epithelial lining fluid exposure
• Reduced cardiac safety concerns relative to older macrolides

These properties make nafithromycin particularly suitable for community-acquired bacterial pneumonia (CABP), where high pulmonary concentrations and resistance coverage are critical. Nafithromycin represents a rationally designed ketolide, in which specific chemical modifications—namely cladinose removal, keto substitution, and an extended aryl–alkyl side chain—directly translate into improved antibacterial efficacy and resistance suppression.

Its dual ribosomal binding mechanism distinguishes it structurally and functionally from azithromycin and clarithromycin, positioning nafithromycin as a promising next-generation therapy for respiratory infections [34, 35].

4. Dual Ribosomal Binding Mechanism:

Unlike classical macrolides that bind primarily to domain V of the 50S ribosomal subunit, nafithromycin interacts simultaneously with domain V and domain II of 23S rRNA. This dual interaction stabilizes the drug–ribosome complex and prevents displacement caused by methylation or point mutations.

This mechanism:

• Enhances inhibition of peptide elongation
• Maintains activity against erm-positive strains
• Prolongs ribosomal occupancy time

Dual binding is considered the principal structural reason for nafithromycin’s enhanced antibacterial spectrum and lower resistance potential [31].

Figure 12: Dual ribosomal binding mechanism of nafithromycin at domains II and V of 23S rRNA.

5. Side effect of Nafhitromycin:

Nafithromycin (marketed as Miqnaf) is a well-tolerated novel macrolide antibiotic generally causing mild side effects like dysgeusia (taste change), nausea, diarrhea, headache, and dizziness. It shows minimal gastrointestinal issues and is considered a safer alternative to older antibiotics for treating Community-Acquired Bacterial Pneumonia (CABP). 

Common Side Effects:

Based on clinical trial data, the most frequently reported treatment-emergent side effects include: 

1. Dysgeusia (Taste Distortion): Reported in up to 67% of subjects, making it the most common effect.
2. Gastrointestinal Issues: Nausea, diarrhea, vomiting, and flatulence.
3. Neurological: Headache and dizziness.
4. Other: Temporary Skin Rashes [36].

ANALYTICAL METHODS DEVELOPED FOR NAFITHROMYCIN (WCK 4873)

Analytical methods are crucial in drug development for quantification, pharmacokinetic studies, impurity profiling, quality control, and microbiological activity assessment. Nafithromycin, a novel lactone ketolide antibiotic under development for community-acquired bacterial pneumonia (CABP), has prompted the development of several analytical techniques to support its bioanalysis, quality control, and microbiological evaluation.

1. Bioanalytical Quantification Methods:
   1. 1. LC-MS/MS for Plasma and Tissue Quantification:

One of the most widely used methods for nafithromycin quantification in biological matrices is liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method enables sensitive, precise, and selective measurement of nafithromycin and its major metabolite in human plasma, urine, and other samples, which is essential for pharmacokinetic and bioavailability studies.

A validated LC-MS/MS assay was developed for simultaneous quantification of nafithromycin and its N-desmethyl metabolite in human plasma. Protein precipitation was used for sample preparation, and clarithromycin served as an internal standard. The calibration curve was linear (r ≥ 0.99) over a wide range (10–5000 ng/mL), with acceptable precision and accuracy according to regulatory bioanalytical guidelines [37].

A similar LC-MS/MS method was applied in pharmacokinetic studies of oral nafithromycin, using protein precipitation with acetonitrile followed by LC-MS/MS analysis. Nafithromycin concentrations in plasma and in bronchoalveolar lavage fluids were quantified with linear standard curves and low limits of quantification, supporting in vivo PK/PD research [38].

Figure 13: Bio analytical Workflow

Table 3: Key LC-MS/MS Bio analytical Parameters

0. 1. 2. Chromatographic Methods for Drug and Impurity Analysis:
1. RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography)

Classic chromatographic analysis of nafithromycin using RP-HPLC has been used for quantitative estimation and detection of related impurities. One analytical investigation employed an RP-HPLC system with a C18 column, buffer–acetonitrile mobile phase, and UV detection to quantify nafithromycin in pharmaceutical samples [39].

2. RP-UPLC (Reversed-Phase Ultra Performance Liquid Chromatography)

A refined method using RP-UPLC (BEH C18 column with a gradient mobile phase and PDA detection) offers faster run times and improved resolution than traditional HPLC. This is useful for routine quality control during drug substance and formulation analysis and provides robust analytical data for regulatory compliance [39].

3. UHPLC-MS:

Ultra-High-Performance Liquid Chromatography coupled with mass spectrometry (UHPLC-MS) has also been developed for quantifying nafithromycin with high specificity. This technique integrates chromatographic separation with mass detection, enhancing sensitivity and structural information, and is particularly useful for metabolite profiling and trace level quantitation [39].

Table 4: Chromatographic Methods Summary

2. Impurity Profiling and Degradation Products:
   1. 1. 1. RP-LC with MS for Impurity Identification

Another key analytical advancement is the identification and quantification of related impurities of nafithromycin using reversed-phase liquid chromatography compatible with mass spectrometry. Forced degradation studies revealed process impurities and degradation products. Chromatographic separation using XTerra C18 and gradient elution, followed by MS identification, allowed structural characterization of impurities, supporting stability studies and quality control [40].

Figure 14: Impurity Analysis Framework