Analytical Method Development for related substances (Organic impurities) in Pharmaceuticals: A Comprehensive review of Methodologies and Regulatory Compliance

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Technical Review Article | Open Access | Published 16th December 2025

Analytical Method Development for related substances (Organic impurities) in Pharmaceuticals: A Comprehensive review of Methodologies and Regulatory Compliance

Sumeet Dwivedi¹*, Rajesh Kumar Chawla², Naresh Ambekar². | EJPPS | 304 (2025) https://doi.org/10.37521/ejpps30303

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Abstract

The terms "related substances" and "organic impurities" are fundamental and often used interchangeably within the pharmaceutical landscape, referring to any component in a drug substance or drug product that deviates from the intended active pharmaceutical ingredient (API) or its excipients. The stringent control of these entities is not merely a regulatory compliance exercise but a critical determinant of patient safety, therapeutic efficacy, and overall product quality. This comprehensive review provides an in-depth, methodical exploration of the analytical method development process specifically tailored for these impurities, with a pronounced emphasis on High-Performance Liquid Chromatography (HPLC) due to its unparalleled versatility and quantitative precision. We meticulously dissect the foundational regulatory architecture provided by key international bodies, including the International Council for Harmonisation (ICH) guidelines (Q3A, Q3B, and Q6A), in conjunction with the stringent requirements mandated by the United States Pharmacopeia (USP) and the U.S. Food and Drug Administration (FDA). The document elucidates a systematic methodology for method creation, spanning from the initial investigative literature review and meticulous sample preparation to advanced chromatographic optimization techniques. A significant portion is dedicated to the indispensable role of forced degradation studies in engineering a truly stability-indicating method, one capable of discerning and quantifying impurities arising throughout a drug's lifecycle. Through detailed practical applications and case studies for both drug substances and finished drug products, we illustrate the intricate calculations of impurity levels and the rigorous, multi-faceted process of method validation. Furthermore, the review candidly addresses prevalent challenges encountered in impurity analysis, such as co-elution, the elusive nature of unknown impurities, and matrix interferences, offering robust solutions often leveraging cutting-edge analytical technologies like LC-MS/MS. The report concludes by casting a forward-looking gaze on the future trajectory of impurity analysis, underscoring the transformative impact of Ultra-High-Performance Liquid Chromatography (UHPLC), high-resolution mass spectrometry, and the overarching principles of Quality by Design (QbD) in shaping the next generation of pharmaceutical quality control.

Keywords: Analytical method development, Related substances, Organic impurities, HPLC, ICH Q3A, ICH Q3B, ICH Q6A, USP, US-FDA, Impurity profiling, Method validation, Forced degradation, Stability-indicating method, LC-MS/MS, UHPLC, Quality by Design.

Introduction

THE CRITICALITY OF IMPURITY CONTROL IN PHARMACEUTICAL QUALITY

The journey of a pharmaceutical product, from its initial synthesis within a laboratory to its eventual administration to a patient, is underpinned by an unwavering commitment to quality, safety, and efficacy. A cornerstone of this commitment is the rigorous identification, quantification, and control of impurities. Impurities are broadly defined as any component present in a drug substance or drug product that is not the desired chemical entity, or in the case of drug products, its excipients¹. Among the various types, organic impurities are of paramount concern due to their direct relevance to the active pharmaceutical ingredient (API) and its potential degradation pathways. For all practical purposes in the realm of analytical chemistry and regulatory compliance, the terms "related substances" and "organic impurities" are used interchangeably, both referring to the array of unintended compounds associated with the drug substance or product²,³.

The genesis of these impurities can be diverse, originating from:

Starting materials: Residual unreacted precursors from the synthesis process.
Intermediates: Partially reacted compounds formed during multi-step syntheses.
By-products: Unwanted side reactions that occur during synthesis or purification.
Degradation products: Compounds formed from the breakdown of the API or excipients due to various stress factors such as light, heat, humidity, oxygen, or pH changes during manufacturing, storage, or transportation⁴.
Reagents, Ligands, and Catalysts: Residues from chemicals used in the synthetic process.
Excipient-related impurities: Interactions between the API and excipients, or impurities inherent in the excipients themselves.
Container/Closure system leachables: Substances that migrate from the packaging materials into the drug product.

The presence of even minute quantities of these impurities can have profound implications. From a safety perspective, impurities can be toxic, genotoxic, carcinogenic, or allergenic, posing direct risks to patients⁵. From an efficacy standpoint, they can reduce the potency of the drug, affect its stability, or alter its bioavailability⁶. Consequently, robust analytical methods capable of accurately determining the impurity profile of a drug substance and drug product are indispensable. These methods must be "stability-indicating", meaning they can detect and quantify all potential impurities that may form over the product's shelf-life, even when present alongside the main drug component and other impurities⁷. This review aims to provide a comprehensive guide to developing such analytical methods, with a strong emphasis on High-Performance Liquid Chromatography (HPLC), the prevailing technique for this critical task.

REGULATORY FRAMEWORK: DEFINING THE SCOPE OF IMPURITY CONTROL

The global pharmaceutical industry is governed by a complex yet harmonized set of guidelines to ensure the consistent quality and safety of medicinal products. The development of analytical methods for related substances must meticulously adhere to these regulatory mandates, which dictate the types of impurities to be controlled, their acceptable limits, and the requirements for method validation and reporting.

ICH Q3A(R2): Impurities in New Drug Substances

The International Council for Harmonisation (ICH) guideline Q3A(R2) serves as the primary reference for organic impurities in new drug substances¹. It establishes a clear framework for reporting, identifying, and qualifying impurities based on their potential impact on patient safety. The guideline categorizes organic impurities as:

Process-related impurities: These include unreacted starting materials, reagents, intermediates, and by-products.
Drug-related impurities: These are degradation products of the drug substance.

ICH Q3A(R2) introduces three key thresholds, which are dependent on the Maximum Daily Dose (MDD) of the drug substance:

Reporting Threshold: This is the level (expressed as a percentage of the drug substance) above which an impurity must be reported in the certificate of analysis and included in the specification. It ensures that all significant impurities are documented.
Identification Threshold: At or above this level, an impurity must be identified, meaning its chemical structure needs to be elucidated. This often involves techniques such as LC-MS, NMR, or IR spectroscopy.
Qualification Threshold: This is the level at which the impurity needs to be qualified, typically by demonstrating its safety through non-clinical or clinical studies. If an impurity exceeds this threshold, its presence must be justified from a safety perspective.

The specific thresholds are summarized in Table 1. It is critical to note that these thresholds are general guidelines; lower thresholds may be necessary for impurities with known high toxicity or for drug substances administered in large doses or for long durations.

Table 1: ICH Q3A(R2) Impurity Thresholds for New Drug Substances¹

Maximum Daily Dose (MDD)	Reporting Threshold (%)	Identification Threshold (%)	Qualification Threshold (%)
≤ 2 g/day	0.10% or 1.0% of API (whichever is lower)	0.20% or 1.0 mg/day (whichever is lower)	0.50% or 2.0 mg/day (whichever is lower)
> 2 g/day	0.05%	0.05%	0.50% or 20 mg/day (whichever is lower)

Note: Values are expressed as percentage of the drug substance. Lower thresholds may be applied for highly toxic impurities or specific drug classes.

ICH Q3B(R2): Impurities in New Drug Products

ICH Q3B(R2) provides similar guidance, but for new drug products². This guideline acknowledges that impurities in drug products can originate from the drug substance itself, from degradation during manufacturing or storage, or from interactions with excipients and container-closure systems. The thresholds in Q3B are generally more stringent than in Q3A, reflecting the fact that the drug product is the final dosage form administered to patients and has undergone additional processing and storage.

Table 2: ICH Q3B(R2) Impurity Thresholds for New Drug Products²

Maximum Daily Dose (MDD)	Reporting Threshold (%)	Identification Threshold (%)	Qualification Threshold (%)
≤ 1 mg/day	1.0%	1.0%	1.0%
> 1 mg/day to 10 mg/day	0.5%	0.5%	0.5%
> 10 mg/day to 100 mg/day	0.2%	0.2%	0.2%
> 100 mg/day to 2 g/day	0.1%	0.1%	0.1%
> 2 g/day	0.05%	0.05%	0.05%

Note: Values are expressed as percentage of the drug product. Lower thresholds may be applied for highly toxic impurities.

A key aspect of Q3B is the emphasis on stability studies (as per ICH Q1A), which are crucial for identifying degradation products that may form over the product's shelf-life. Any new impurity observed during stability studies must be evaluated against these thresholds.

ICH Q6A: Specifications for New Drug Substances and Products

ICH Q6A complements Q3A and Q3B by providing guidance on setting and justifying specifications for new drug substances and products³. It states that impurity specifications should be derived from data obtained during development, manufacture, and stability studies. Acceptance criteria for impurities should be based on safety data (qualification) and consistent with the impurity profile observed in batches used for clinical trials and stability.

USP Monographs and US-FDA Requirements

The United States Pharmacopeia (USP) sets legally recognized standards for drug substances and dosage forms in the U.S.⁴. USP monographs often include specific tests and acceptance criteria for "Organic Impurities" or "Related Substances," frequently referencing general chapters that outline chromatographic procedures. These methods, once established in a monograph, are mandatory for compliance.

The U.S. Food and Drug Administration (FDA), through its various guidance documents (e.g., Guidance for Industry: ANDAs: Impurities in Drug Substances⁸), reinforces the principles of ICH, often providing more specific recommendations for analytical method development and validation. The FDA emphasizes a comprehensive understanding of the impurity profile and the development of stability-indicating methods as a prerequisite for drug approval⁹. Regulatory submissions must include detailed information on the analytical method, its validation, and the impurity profile of the drug.

METHODOLOGIES AND ANALYTICAL TECHNIQUES: A DEEPER DIVE INTO HPLC

The development of a robust analytical method for related substances is a sophisticated process that leverages the capabilities of modern analytical chemistry. High-Performance Liquid Chromatography (HPLC) stands as the most widely employed technique for this purpose, offering unparalleled resolution, sensitivity, and quantitative precision¹⁰.

Principles of High-Performance Liquid Chromatography (HPLC)

HPLC separates components of a mixture based on their differential interaction with a stationary phase (packed within a column) and a mobile phase (a liquid solvent system). The key elements include:

Solvent Reservoir and Pump: The pump delivers the mobile phase at a constant, precise flow rate. Modern HPLC systems utilize quaternary or binary pumps for gradient elution.
Autosampler: Provides automated, reproducible injection of samples into the HPLC system, minimizing human error and increasing throughput.
Column: The heart of the separation.

○ Stationary Phase Chemistry: Reversed-phase (RP-HPLC) is the predominant mode for related substances, using a non-polar stationary phase (e.g., C18, C8, Phenyl) and a polar mobile phase. Analytes are retained based on their hydrophobicity. Other modes, such as Normal-Phase HPLC (polar stationary phase, non-polar mobile phase) or Hydrophilic Interaction Liquid Chromatography (HILIC) (polar stationary phase, high organic mobile phase for highly polar compounds), may be used for specific impurity types¹¹.

○ Column Dimensions: Standard analytical columns are typically 150-250 mm in length with an internal diameter of 4.6 mm and particle sizes ranging from 3 to 5 µm. Smaller particle sizes (sub-2 µm) are used in UHPLC for enhanced speed and resolution.

Detector: Converts the separated components into an electrical signal.

○ UV-Visible (UV-Vis) Detector: Most common. Compounds with chromophores absorb UV or visible light.

○ Diode Array Detector (DAD): A highly recommended UV-Vis variant that acquires a full UV spectrum of each eluting peak. This is crucial for peak purity assessment (confirming that a peak represents a single compound, not co-eluting ones) and for identifying unknown peaks based on their spectral characteristics¹²

○ Evaporative Light Scattering Detector (ELSD): Useful for compounds lacking chromophores (e.g., sugars, lipids, some excipients).

○ Refractive Index (RI) Detector: A universal detector, but less sensitive and incompatible with gradient elution, limiting its use for impurities.

○ Mass Spectrometry (MS) Detector: Provides molecular weight information and fragmentation patterns, invaluable for identifying unknown impurities and confirming the identity of known ones. Often coupled directly with HPLC (LC-MS)¹³.

Systematic Method Development Strategy

A systematic, science-driven approach is essential for developing a robust and reliable related substances method¹⁴.

1. Understanding the API and its Impurities:

○ API Chemistry: Comprehensive knowledge of the API's chemical structure, pKa, logP, solubility, and potential reactive functional groups is crucial.

○ Synthesis Route Analysis: Identify potential process impurities from the manufacturing route, including unreacted starting materials, intermediates, and known by-products.

○ Degradation Pathways: Predict likely degradation pathways based on functional groups (e.g., hydrolysis of esters/amides, oxidation of thiols/amines, photolysis of conjugated systems).

2. Forced Degradation Studies (Stress Testing):

These studies are paramount for demonstrating the stability-indicating nature of the method⁷. The API (and optionally the drug product) is intentionally subjected to exaggerated stress conditions to generate potential degradation products.

○ Acidic Hydrolysis: Exposure to strong acid (e.g., 0.1 N to 1 N HCl) at elevated temperatures (e.g., 60-80°C).

○ Basic Hydrolysis: Exposure to strong base (e.g., 0.1 N to 1 N NaOH) at elevated temperatures.

○ Oxidative Degradation: Treatment with oxidizing agents (e.g., 3% H2O2, typically at room temperature or slightly elevated).

○ Thermal Degradation: Heating the solid drug substance (e.g., 80-105°C) or solution.

○ Photolytic Degradation: Exposure to intense UV/Visible light (e.g., using a photostability chamber).

○ Humidity/Hydrolysis: Exposure to high humidity or water.

○ The goal is to generate sufficient degradation products (typically 5-20% degradation of the main peak) that can be detected and separated by the analytical method.

3. Initial Chromatographic Screening and Optimization:

○ Column Selection: Begin with a standard C18 column. If separation is challenging, screen different C18 columns (different manufacturers, bonding chemistries), or alternative stationary phases (e.g., C8, Phenyl-Hexyl for aromatic interactions, HILIC for very polar compounds)¹⁵.

○ Mobile Phase Selection:

■ Aqueous Component: Buffers (e.g., phosphate, acetate, formate, trifluoroacetic acid) are used to control pH and provide ionic strength. pH is critical for separating ionizable compounds, affecting their ionization state and thus retention.

■ Organic Modifier: Acetonitrile and methanol are most common. Acetonitrile typically provides lower viscosity and higher elution strength compared to methanol.

■ Gradient Elution: Essential for separating a wide range of polarities. Optimize the gradient steepness, duration, and number of steps to achieve optimal resolution of all impurities from each other and from the main peak.

○ Detection Wavelength: Select a wavelength where both the main drug and potential impurities exhibit good absorption. DAD can help identify optimal wavelengths.

○ Column Temperature: Temperature influences retention, selectivity, and peak shape. Optimization (e.g., 25-40°C) can improve separation and reduce the run time.

○ Flow Rate: Adjust to optimize efficiency and run time without exceeding column pressure limits.

4. System Suitability Tests (SST):

Before performing sample analysis, a SST ensures the chromatographic system is functioning correctly and delivering results of acceptable quality. Typical SST parameters include⁶:

○ Resolution (Rs): The measure of separation between two adjacent peaks (Rs ≥ 1.5 is generally desired).

○ Tailing Factor (Tf) or Asymmetry Factor (As): Indicates peak symmetry (Tf < 2.0, ideally 0.9-1.5).

○ Theoretical Plates (N): A measure of column efficiency.

○ Relative Standard Deviation (RSD) of peak areas/retention times: For replicate injections, ensuring reproducibility.

Impurity Quantification

Accurate quantification of impurities is critical. Common approaches include:

1. External Standard Method: The simplest method. A calibration curve is generated using a known concentration range of each impurity standard. The peak area of the impurity in the sample is then interpolated from this curve. This requires obtaining and characterizing each impurity standard, which can be challenging for all impurities.

2. Relative Response Factor (RRF) Method: This is widely used when impurity standards are available. The RRF corrects for the difference in detector response between the impurity and the main drug substance¹⁶.

3. Area Normalization Method (Peak Area Percent): This approach assumes all impurities have the same detector response as the main drug. The percentage of an impurity is calculated as its peak area divided by the total area of all peaks (including the main peak) multiplied by 100.

This method is simple but less accurate if RRFs vary significantly. It is generally used for unknown impurities where standards are unavailable, or for reporting purposes as a general indication of impurity levels. Regulatory guidelines often permit the use of area normalization for impurities below the identification threshold where no RRF is available¹,².

PRACTICAL APPLICATIONS AND CASE STUDIES

Developing and validating related substances methods requires a meticulous approach, integrating regulatory requirements with scientific principles.

Case Study: Impurity Profiling of an Antiviral Drug Substance

Objective: To develop a stability-indicating HPLC method for the determination of organic impurities in a novel antiviral drug substance, ensuring compliance with ICH Q3A(R2) guidelines.

Methodology:

Initial Characterization: The antiviral drug (Mol. Wt. ~350 Da, weakly basic, pKa ~8.5) was highly susceptible to hydrolysis and oxidation based on its functional groups.
Forced Degradation Studies:

○ Acidic: 0.5 N HCl, 60°C, 8 hrs. Result: ~15% degradation, one major impurity (Imp-1) identified as a hydrolyzed product.

○ Basic: 0.1 N NaOH, 60°C, 8 hrs. Result: ~20% degradation, two new impurities (Imp-2, Imp-3) identified as base-catalyzed degradation products.

○ Oxidative: 3% H_2O_2, room temp, 24 hrs. Result: ~10% degradation, one distinct impurity (Imp-4) identified as an oxidative product.

○ Thermal: 105°C, 72 hrs. Result: negligible degradation.

○ Photolytic: Exposed to 1.2 million lux-hr white light and 200 W-hr/m2 UV light. Result: ~5% degradation, one impurity (Imp-5) identified as a photo-degradation product.

Chromatographic Optimization:

○ Column: Initially screened C18 columns from different vendors. Optimal separation achieved on a specific C18 (250 mm x 4.6 mm, 5 µm) column.

○ Mobile Phase: Optimized to a gradient system using 0.1% formic acid in water (pH 3.0) as Mobile Phase A and acetonitrile as Mobile Phase B. The gradient was carefully refined to separate all five degradation products and any process impurities from the main drug peak.

○ Detection: UV detection at 270 nm, chosen after scanning the UV spectra of the drug and identified impurities using a DAD. DAD confirmed the peak purity of the main peak, and all identified impurities.

Impurity Quantification and Reporting: Impurities were quantified using RRFs, which were experimentally determined for Imp-1, Imp-2, Imp-3, and Imp-4. Imp-5 was quantified using area normalization as no standard was readily available and its level remained below the identification threshold. The total impurity level was calculated as the sum of all individual impurities.

○ For a batch of drug substance with an MDD of 100 mg/day, the reporting threshold is 0.10%, and the identification threshold is 0.20% as per ICH Q3A¹. If Imp-1 was found at 0.18%, it would be reported. If it was found at 0.25%, it would need to be identified (which it was).

Case Study: Related Substances in a Sustained-Release Tablet Formulation

Objective: To develop and validate a stability-indicating HPLC method for the determination of related substances in a sustained-release tablet formulation of an antidepressant drug, adhering to ICH Q3B(R2) and Q2(R1) guidelines.

Methodology:

Drug Product Considerations: The sustained-release matrix and various excipients (e.g., cellulose derivatives, lubricants, binders) presented potential interference.
Sample Preparation: Tablets were accurately weighed, finely powdered, and extracted with a mixture of mobile phase and sonication to ensure complete dissolution of the API and impurities from the matrix. Centrifugation and filtration were performed to remove insoluble excipients. A placebo (excipient-only) solution was run to confirm no interference from the matrix.
Chromatographic Conditions: A short C18 column (100 mm x 4.6 mm, 3.5 µm) was used to achieve faster run times compatible with routine quality control. A gradient of phosphate buffer (pH 6.5) and methanol was optimized. Detection was at 230 nm.
Method Validation (as per ICH Q2(R1)⁶):

○ Specificity: Demonstrated by:

■ Chromatographic separation of the main drug peak from all known impurities and degradation products (generated from forced degradation of the drug product).

■ Absence of interfering peaks from excipients in the placebo chromatogram.

■ Peak purity assessment using DAD for the main peak and all impurity peaks.

○ Linearity: Established by analyzing five concentration levels (e.g., LOQ, 50%, 100%, 120%, 150% of the specification limit) for each known impurity. The linear regression equation y=mx+c was determined, and the correlation coefficient (R2) values were all greater than 0.999, indicating excellent linearity across the range.

○ Accuracy: Evaluated by spiking known amounts of impurities (e.g., 0.1%, 0.5%, 1.0% of the nominal drug concentration) into a placebo matrix and analyzing the recovery. Recovery percentages for all impurities ranged from 98.5% to 101.2%, well within the acceptance criterion of 95-105%¹⁷.

○ Precision:

■ Repeatability (Intra-day): Six replicate injections of a sample containing impurities at the specification limit. The RSD for peak areas of all impurities was found to be \<1.5.

■ Intermediate Precision (Inter-day, Inter-analyst): Performed by a different analyst on a different day using a different instrument. RSD values were again \<2.0.

○ Limit of Detection (LOD) and Limit of Quantification (LOQ):

■ LOD (smallest amount detectable): Signal-to-noise ratio (S/N) of 3:1.

■ LOQ (smallest amount quantifiable with acceptable accuracy/precision): S/N of 10:1. The LOQ for all impurities was determined to be 0.05%, below the ICH Q3B reporting threshold for this drug's MDD².

○ Robustness: Small, deliberate variations were introduced to parameters like mobile phase pH (pm0.2), column temperature (pm2circC), and flow rate (pm0.1 mL/min). The resolution between critical pairs and impurity percentages remained within acceptable limits, confirming the method's robustness.

CHALLENGES AND SOLUTIONS IN ANALYTICAL METHOD IMPLEMENTATION

Despite advancements, developing and implementing impurity methods present significant challenges, demanding innovative solutions and a deep understanding of analytical chemistry.

Co-elution of Impurities

Challenge: The most critical challenge is the co-elution of an impurity with the main drug peak or with another impurity. This directly impacts the accuracy of quantification and can lead to the erroneous conclusion that impurity is absent, potentially compromising patient safety¹⁸.

Solution:

Extensive Chromatographic Optimization: This involves systematic adjustments of mobile phase composition (e.g., different organic modifiers, buffer concentrations, pH gradients), column chemistry (screening various C18 columns, or switching to C8, Phenyl, or even highly polar end-capped columns), and temperature.
Diode Array Detector (DAD) Peak Purity: The DAD is indispensable. It allows for the acquisition of UV spectra across the entire peak. A "peak purity" algorithm compares spectra across the peak. If the purity angle (or threshold) exceeds a predefined limit, it indicates that multiple components are co-eluting¹².
Multi-dimensional Chromatography (2D-LC): For highly complex mixtures, 2D-LC couples two different separation dimensions orthogonally. For example, a non-polar column followed by a polar column. This can significantly enhance peak capacity and resolve highly challenging co-elutions¹⁹.
LC-MS Detection: Mass spectrometry offers unparalleled selectivity. Even if two compounds co-elute chromatographically, if they have different molecular masses, they can be differentiated and quantified by the MS detector¹³.

Low Levels of Impurities

Challenge: Many impurities are present at very low concentrations (e.g., below 0.1%), making their detection and accurate quantification challenging, especially when approaching the LOD/LOQ limits.

Solution:

Increased Sample Load: Injecting a higher concentration of the sample or a larger injection volume can increase the signal for low-level impurities, provided it doesn't overload the column or compromise peak shape.
More Sensitive Detectors: Employing more sensitive detectors such as Mass Spectrometry (MS) or Charged Aerosol Detectors (CAD) can significantly lower detection limits compared to standard UV-Vis detectors.
Sample Pre-concentration: Techniques such as solid-phase extraction (SPE) can be used to selectively extract and concentrate impurities from a large sample volume before injection.

Identification of Unknown Impurities

Challenge: When a new impurity peak appears in the chromatogram (e.g., from a new manufacturing batch or during stability testing) for which no reference standard is available, its identification is crucial, especially if it approaches or exceeds the identification threshold.

Solution:

LC-MS/MS (Tandem Mass Spectrometry): This is the gold standard for impurity identification. The first MS stage provides the precise molecular weight of the impurity, while the second MS stage fragments the impurity, providing structural information that can be used to deduce its chemical structure¹³,²⁰. High-resolution accurate mass (HRAM) MS (e.g., Orbitrap, TOF) can provide elemental composition, significantly aiding identification.
NMR (Nuclear Magnetic Resonance) Spectroscopy: If sufficient quantity of the isolated impurity is available, NMR (proton NMR, carbon NMR, 2D NMR) can provide definitive structural elucidation. This often requires isolation of the impurity via preparative HPLC²¹.
Data Archiving and Spectral Libraries: Building in-house spectral libraries (UV, MS) of known impurities and degradation products is invaluable for rapid identification.

Matrix Effects in Drug Products

Challenge: In drug product analysis, the excipients can interfere with the analysis by co-eluting, suppressing the detector signal, or degrading into compounds that mimic impurities.

Solution:

Thorough Placebo Analysis: Always run a placebo solution (containing all excipients but no API) to identify any excipient-related peaks or interferences.
Optimized Sample Preparation: Develop robust extraction procedures to selectively extract the API and impurities from the complex excipient matrix.
Orthogonal Methods: If direct interference cannot be overcome by HPLC, consider alternative techniques for specific impurities, or use a second HPLC method with different selectivity (e.g., HILIC instead of RP-HPLC).

Ensuring Method Robustness and Transferability

Challenge: A validated method must perform consistently across different instruments, laboratories, and analysts. Lack of robustness can lead to out-of-specification results during routine testing or method transfer.

Solution:

Robustness Study (during validation): Systematically vary key method parameters (e.g., mobile phase pH, column temperature, flow rate, organic modifier percentage) within small, deliberate ranges and observe their impact on critical method performance parameters (e.g., resolution, retention time, peak area).
Clear Method Documentation: Provide explicit details in the method standard operating procedure (SOP) regarding instrument setup, reagent preparation, and analytical procedure.
Analyst Training and Qualification: Ensure analysts are thoroughly trained and qualified on the method.

FUTURE PERSPECTIVES AND EMERGING TRENDS IN IMPURITY ANALYSIS

The landscape of impurity analysis is undergoing continuous evolution, driven by technological advancements, increasing regulatory expectations, and a desire for greater efficiency and understanding.

Ultra-High-Performance Liquid Chromatography (UHPLC)

UHPLC has become a mainstream technology for impurity analysis²². By utilizing columns packed with sub-2 µm particles, UHPLC offers several significant advantages:

Enhanced Resolution: The smaller particle size leads to a more efficient column, resulting in narrower peaks and improved separation of closely eluting impurities. This is particularly beneficial for complex impurity profiles.
Reduced Analysis Time: Higher flow rates and shorter columns, combined with smaller particles, drastically reduce run times (e.g., from 30 minutes to 5 minutes), leading to increased sample throughput and reduced cost per analysis.
Lower Solvent Consumption: Faster run times translate directly to less mobile phase consumption, aligning with green chemistry principles.

The transition from HPLC to UHPLC for impurity analysis is a significant trend, allowing for faster development and routine testing.

Advanced Mass Spectrometry and Hyphenated Techniques

The integration of Mass Spectrometry (MS) with HPLC (LC-MS) continues to be the workhorse for impurity identification and quantification¹³. Future trends include:

High-Resolution Accurate Mass (HRAM) MS: Technologies like Orbitrap and Quadrupole-Time-of-Flight (Q-TOF) provide exceptionally precise mass measurements (e.g., to 5 decimal places), enabling elemental composition determination without prior knowledge of the impurity's formula. This significantly streamlines the identification process²³.
Ion Mobility Mass Spectrometry (IM-MS): This emerging technique adds another dimension of separation based on the molecule's size, shape, and charge, further aiding in the separation and identification of isomers or isobaric compounds that are difficult to resolve by LC or conventional MS²⁴.
ICP-MS (Inductively Coupled Plasma Mass Spectrometry): Used for the determination of elemental impurities, which are also a critical aspect of drug quality (ICH Q3D) but distinct from organic impurities²⁵.

Quality by Design (QbD) in Analytical Method Development

The pharmaceutical industry is increasingly embracing Quality by Design (QbD), a systematic approach to development that begins with predefined objectives and emphasizes understanding the product and process based on sound science and quality risk management²⁶. For analytical method development, QbD entails:

Analytical Target Profile (ATP): Defining the desired performance characteristics of the method (e.g., required resolution, accuracy, precision, quantitation limits).
Risk Assessment: Identifying critical method parameters (CMPs) that could impact method performance (e.g., pH, temperature, gradient slope).
Method Design Space: Experimentally determining the range of CMPs within which the method consistently meets the ATP. This often involves Design of Experiments (DoE), a statistical approach to efficiently explore the parameter space²⁷.
Control Strategy: Establishing ongoing monitoring and control measures to ensure the method remains within the defined design space throughout its lifecycle. QbD leads to more robust, reproducible, and transferable analytical methods compared to traditional empirical approaches

Chemometrics and Data Analytics

The vast amount of data generated by modern analytical instruments necessitates advanced data analysis tools. Chemometrics applies mathematical and statistical methods to chemical data [28].

Multivariate Data Analysis: Used to identify patterns, relationships, and anomalies in complex chromatographic data, aiding in method optimization and troubleshooting.
Spectroscopic Data Analysis: Chemometric tools are used to interpret complex UV or MS spectra for identification purposes.
Process Analytical Technology (PAT): Integrates analytical measurements into the manufacturing process, allowing for real-time monitoring and control of impurity formation, thereby enhancing process understanding and product quality²⁹,³⁰.

Green Chemistry in Analytical Methods

There is a growing emphasis on "green" analytical methods that minimize environmental impact. This includes:

Reduced Solvent Consumption: Achieved through UHPLC, miniaturized columns, and micro-HPLC.
Use of Safer Solvents: Replacing toxic or hazardous solvents (e.g., chloroform) with more environmentally benign alternatives (e.g., ethanol, water).
Waste Reduction: Optimizing methods to generate less hazardous waste.

Conclusion

The analytical method development for related substances (organic impurities) is a multifaceted and critical endeavour, inextricably linked to the safety and quality of pharmaceutical products. Adherence to stringent regulatory guidelines, notably ICH Q3A, Q3B, and Q6A, along with pharmacopoeial standards such as the USP, forms the bedrock of a successful development programme. The systematic approach to method creation, with High-Performance Liquid Chromatography (HPLC) as the central technique, coupled with rigorous forced degradation studies and comprehensive method validation, is indispensable for generating stability-indicating methods that accurately characterize the impurity profile of both drug substances and drug products.

While the pursuit of a perfectly resolved and quantified impurity profile presents inherent challenges such as co-elution, the complexities of low-level impurities, and the daunting task of identifying unknown components, continuous innovation in analytical science offers compelling solutions. The widespread adoption of UHPLC for enhanced speed and resolution, the indispensable power of high-resolution mass spectrometry for structural elucidation, and the strategic implementation of Quality by Design (QbD) principles are transforming the landscape of impurity analysis. These advancements not only facilitate more efficient and robust method development but also significantly elevate the overall quality assurance framework within the pharmaceutical industry. Ultimately, the relentless drive to refine and advance these analytical methodologies underscores the industry's unwavering commitment to delivering safe, effective, and high-quality medicines to patients worldwide.

ACKNOWLEDGEMENT

The authors express thanks to Suven Pharmaceuticals Limited, Hyderabad for support and providing the research facility to carry out this work.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

References

1. International Council for Harmonisation. (1995). ICH Q3A(R2): Impurities in New Drug Substances.

2. International Council for Harmonisation. (2006). ICH Q3B(R2): Impurities in New Drug Products.

3. International Council for Harmonisation. (2003). ICH Q6A: Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances.

4. United States Pharmacopeia. (2020). USP General Chapter <1086> Impurities in Drug Substances and Drug Products.

5. Roy, A., & Singh, R. (2012). Impurity profiling in pharmaceuticals: A review. Journal of Pharmaceutical Analysis, 2(1), 21-27. [https://doi.org/10.1016/j.jpha.2011.09.004]

6. International Council for Harmonisation. (2005). ICH Q2(R1): Validation of Analytical Procedures: Text and Methodology.

7. Singh, R., & Singh, S. (2009). Forced degradation studies to develop stability indicating methods--a review. Journal of Pharmaceutical and Biomedical Analysis, 49(4), 1010–1023. [https://doi.org/10.1016/j.jpba.2009.01.032]

8. U.S. Food and Drug Administration. (2018). Guidance for Industry: ANDAs: Impurities in Drug Substances.

9. Ramesh, M., & Rao, A. J. (2018). Impurity profiling of pharmaceuticals: An overview. Journal of Applied Pharmaceutical Science, 8(08), 159-166. [https://doi.org/10.7324/JAPS.2018.8825]

10. Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2010). Introduction to Modern Liquid Chromatography. John Wiley & Sons.

11. D'Orazio, G. (2013). High performance liquid chromatography in pharmaceutical analysis. Journal of Pharmaceutical and Biomedical Analysis, 87, 240-256.

[https://doi.org/10.1016/j.jpba.2013.04.020]

12. Ermer, J. (2001). Validation of HPLC assays: peak purity and impurity profiling. Journal of Pharmaceutical and Biomedical Analysis, 25(3-4), 369-383. [https://doi.org/10.1016/S0731-7085(00)00516-4]

13. Klick, S., O'Connor, S., & Schug, K. A. (2018). Liquid Chromatography-Mass Spectrometry (LC-MS) in pharmaceutical analysis. In Separation Science and Technology (pp. 53-83). Springer.

14. Bakshi, M., & Singh, S. (2002). Development of validated stability-indicating assay methods—critical review. Journal of Pharmaceutical and Biomedical Analysis, 28(6), 1011-1040. [https://doi.org/10.1016/S0731-7085(02)00032-X]

15. Dolan, J. W., & Snyder, L. R. (2006). Troubleshooting HPLC Systems: A Guide for the Experienced Chromatographer. Humana Press.

16. Vashist, M., Sharma, M., & Kumar, D. (2015). Relative response factor: An overview. Journal of Applied Pharmaceutical Science, 5(03), 162-167. [https://doi.org/10.7324/JAPS.2015.50328]

17. Berridge, J. C. (2004). The role of experimental design in HPLC method development. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 800(1-2), 161-171. [https://doi.org/10.1016/j.jchromb.2003.09.006]

18. Ermer, J., & Miller, J. M. (Eds.). (2005). Method Validation in Pharmaceutical Analysis: A Guide to Best Practice. Wiley-VCH.

19. Gfrörer, P., & Schepers, R. (2014). Two-dimensional liquid chromatography for separation and isolation of impurities from pharmaceutical preparations. Journal of Separation Science, 37(13), 1605-1613. [https://doi.org/10.1002/jssc.201400166]

20. Mazzeo, J. R., et al. (2013). Impurity identification and quantification using LC-MS in pharmaceutical analysis. Journal of Pharmaceutical and Biomedical Analysis, 86, 201-210. [https://doi.org/10.1016/j.jpba.2013.07.016]

21. Holzgrabe, U., Wawer, I., & Diehl, B. (Eds.). (2010). NMR Spectroscopy in Pharmaceutical Analysis. Wiley-VCH.

22. Fekete, S., et al. (2014). Ultra-high-pressure liquid chromatography (UHPLC): An emerging tool for pharmaceutical analysis. TrAC Trends in Analytical Chemistry, 63, 114-124. [https://doi.org/10.1016/j.trac.2014.07.009]

23. Rogers, L. C., & Korfmacher, W. A. (2016). High-resolution mass spectrometry: From pharmaceutical discovery to clinical applications. Mass Spectrometry Reviews, 35(1), 121-140. [https://doi.org/10.1002/mas.21447]

24. Zhang, M., et al. (2020). Ion mobility-mass spectrometry: An emerging tool in pharmaceutical analysis. Journal of Pharmaceutical and Biomedical Analysis, 184, 113175. [https://doi.org/10.1016/j.jpba.2020.113175]

25. International Council for Harmonisation. (2014). ICH Q3D: Guideline for Elemental Impurities.

26. Borman, P., & Elder, D. (2017). QbD for Analytical Methods. Springer.

27. Gemperli, A., et al. (2015). Design of experiments (DoE) in pharmaceutical development. Journal of Pharmaceutical Sciences, 104(2), 522-536. [https://doi.org/10.1002/jps.24278]

28. Brereton, R. G. (2003). Chemometrics: Data Analysis for the Laboratory and Chemical Plant. John Wiley & Sons.

29. Read, E. K., et al. (2013). Process Analytical Technology (PAT) in pharmaceutical development. Biotechnology Progress, 29(1), 1-13. [https://doi.org/10.1002/btpr.1683]

30. Chawla, R. K., et al. (2021). Development and validation of an inductively coupled plasma mass spectrometry method for estimation of elemental impurities in calcium acetate active pharmaceutical ingredient. Indian Journal of Pharmaceutical Sciences, 83(4), 830-837.