Advances in Impurity Profiling of Pharmaceutical Formulations Volume 60- Issue 1

Ramesh Chaughule¹* and Anushree Lokur²

• ¹Department of Chemistry, Ramnarain Ruia Autonomous College, Matunga, Mumbai, 400019, India
• ²Department of Microbiology, Ramnarain Ruia Autonomous College, Matunga, Mumbai, 400019, India

Received: December 14, 2024; Published: December 26, 2024

*Corresponding author: Ramesh Chaughule, Department of Chemistry, Ramnarain Ruia Autonomous College, Matunga, Mumbai, 400019, India

DOI: 10.26717/BJSTR.2024.60.009393

Abstract PDF

Impurity profiling has become a critical component of pharmaceutical development, quality control, and regulatory compliance. During drug manufacturing, impurities—introduced through synthesis processes, excipients, residual solvents, or degradation products—pose significant challenges to the safety, efficacy, and stability of pharmaceuticals. Impurity profiling, a systematic approach to identifying, characterizing, and quantifying these impurities, is essential for ensuring that pharmaceutical products meet stringent safety and quality standards. This article explores recent trends in impurity profiling, focusing on advanced analytical techniques, including chromatographic methods, spectroscopic methods, and hyphenated techniques like LC-MS and GC-MS. These techniques have significantly enhanced the detection and characterization of impurities at trace levels, contributing to the development of safer and more effective medications. Complexities in the analysis of biosimilars against innovators is also briefly discussed since the biosimilars play a pivotal role in making biologic therapies more accessible and affordable for patients. Additionally, the regulatory landscape governing impurity profiling is discussed, highlighting the importance of adhering to international guidelines to ensure public health and safety.

Keywords: Drug Impurities; Analytical Techniques; Spectrometric Techniques; Data Analysis; Impurity Profiling

Abbreviations: APIs: Active Pharmaceutical Ingredients; FDA: Food and Drug Administration; EMA: European Medicines Agency; HPTLC: High-Performance Thin-Layer Chromatography; HPLC: High-Performance Liquid Chromatography; GC: Gas Chromatography; LC: Liquid Chromatography; MS: Mass Spectrometry; LC-MS: Liquid Chromatography-Mass Spectrometry; PTMs: Post-Translational Modifications; FT-IR: Fourier Transform Infrared Spectroscopy; GC-MS: Gas Chromatography-Mass Spectrometry; CQAs: Critical Quality Attributes

During the manufacturing of drugs, the Active Pharmaceutical Ingredients (APIs) and excipients are rarely 100% pure. This lack of purity is due to the presence of various components introduced from sources such as the synthesis process, excipients themselves, residual solvents, or degradation products. These unintended and undesirable substances are collectively known as impurities. In any pharmaceutical product or drug substance, when impurities are expected to be present, it is essential to identify and characterize them using appropriate analytical methods—a process known as impurity profiling. Impurity profiling is a systematic approach designed to identify, isolate, and elucidate the structure of unknown impurities. This process is crucial for accurately determining and quantifying the impurities within a medicinal substance. Impurity profiling is a critical aspect of pharmaceutical quality control and drug development, ensuring that medications are safe, effective, and of high quality. Impurities in pharmaceutical formulations can arise from various sources during the manufacturing process, including raw materials, intermediates, manufacturing processes, degradation, and packaging. These impurities, even in trace amounts, can significantly impact the safety, efficacy, and stability of the final drug product. It ensures that these impurities remain within acceptable limits, preventing any toxicological effects on the human body. Therefore, producing high-quality and efficacious medicinal products requires stringent impurity reporting.

Impurity profiling necessitates the use of highly sensitive, selective, and efficient analytical techniques to detect trace amounts of impurities. The current demands for impurity profiling underscore the need for techniques that offer high accuracy, precision, and sensitivity, as impurities may exist in minute quantities within the drug substance [1]. Even trace amounts of impurities can pose significant risks, making impurity profiling essential in the pharmaceutical industry.

Impurity Profiling

It is a crucial aspect of pharmaceutical development and quality control. It involves identifying, quantifying, and controlling the presence of impurities in drug substances and products to ensure their safety, efficacy, and quality.

Safety Concerns

Some impurities can be toxic, mutagenic, or carcinogenic, posing serious health risks to patients. For instance, certain degradation products might form during the shelf life of a drug, potentially interacting with biological systems in unpredictable and harmful ways. Identifying and controlling these impurities is crucial to prevent adverse health outcomes.

Efficacy Issues

Impurities can interfere with the therapeutic activity of the drug’s API, leading to reduced efficacy. This is particularly concerning in drugs with a narrow therapeutic index, where even small variations in drug concentration can lead to suboptimal treatment outcomes or adverse effects.

Stability Considerations

The presence of impurities can accelerate the degradation of drug products, thereby reducing their shelf life and stability. Stability studies are conducted to understand how impurities develop over time and under various storage conditions, which is essential for establishing appropriate storage guidelines and expiration dates.

Regulatory bodies across the globe have established comprehensive guidelines to ensure that impurity profiling is rigorously conducted during the development and manufacturing of pharmaceuticals. These guidelines are designed to protect public health by setting acceptable limits for impurities and mandating robust analytical methods for their detection and quantification. Thus impurity profiling is a cornerstone of pharmaceutical development, ensuring that drug products are safe, effective, and of high quality. Regulatory guidelines such as FDA (Food and Drug Administration, USA), EMA (European Medicines Agency), Pharmacopoeias etc., provide a framework for controlling impurities, safeguarding public health by minimizing the risks associated with their presence in pharmaceuticals.

Impurities in pharmaceutical products can arise from various sources during the manufacturing process, storage, or degradation. These impurities must be identified, quantified, and controlled to ensure the safety, efficacy, and quality of the final product. Below is a detailed overview of the different types of impurities:

Organic Impurities

Organic impurities are carbon-based compounds that can be present in the final product due to the manufacturing process or chemical degradation. These impurities can be categorized into two main types:

Inorganic Impurities

Inorganic impurities are non-carbon-based substances that may be introduced during the production process. These impurities are typically residues from reagents, catalysts, or other materials used in the synthesis or formulation of the product.

Residual Solvents

Residual solvents are organic volatile chemicals used during the production of the API or during formulation, which may remain in trace amounts in the final product. These solvents must be controlled to acceptable levels due to their potential toxicity.

Impurity profiling is a critical aspect of forensic science, particularly when dealing with trace evidence or contaminants. The accuracy and reliability of impurity profiling depend on the analytical techniques used. As drug products become more complex, the importance of thorough impurity profiling continues to grow, driving advancements in analytical techniques and regulatory science. Below is an overview of the main techniques used for impurity profiling, categorized into chromatographic techniques, spectroscopic methods, and hyphenated techniques.

Chromatographic Techniques

Chromatographic techniques are essential for separating complex mixtures into individual components. They are widely used in impurity profiling to isolate and quantify impurities in samples. In pharmaceutical analysis, the identification and quantification of these impurities are crucial for ensuring that the product meets regulatory standards and is safe for consumption. Advanced analytical techniques such as chromatography, mass spectrometry, and spectroscopy are often employed to detect and control these impurities at trace levels.

Thin Layer Chromatography (TLC): TLC is a technique used to separate and identify organic and inorganic substances based on their affinity to a stationary phase and a mobile phase. It requires minimal sample clean-up, wide choice of mobile phases, flexibility in sample distinction, high sample loading capacity. This old and inexpensive technique finds a lot of applications in the field of pharmaceutical analysis. The adsorbent (a solid state) is coated onto a solid support as a thin layer usually on a glass, plastic, or aluminium support. Several factors determine the efficiency of this type of chromatographic separation. TLC plays a crucial role in the early stage of drug development when information about the impurities and degradation products in drug substance and drug product is inadequate [2,3]. TLC can be applied in preliminary screening of pharmaceutical impurities in drugs like aspirin. It offers a fast, cost-effective method for separating known degradation products, especially during stability testing [4].

High Performance Thin Layer Chromatography (HPTLC):

With the advancement of techniques, High-Performance Thin-Layer Chromatography (HPTLC) has emerged as a significant tool in drug analysis. HPTLC is a rapid separation technique that is versatile enough to analyse a wide range of samples. This method offers several advantages, including ease of use and short analysis times, making it suitable for analysing complex or crude sample preparations. HPTLC evaluates the entire chromatogram using various parameters without time constraints. Additionally, it allows for the simultaneous yet independent development of multiple samples and standards on each plate, enhancing the reliability of the results. HPTLC has been used to quantify drugs such as ethinyl estradiol and cyproterone [5], alfuzosin [6],pentazocine and tramadol [7]. The technique’s ability to handle multiple samples simultaneously makes it ideal for large-scale drug screening.

High-Performance Liquid Chromatography (HPLC): High-Performance Liquid Chromatography (HPLC) is an advanced technique used to separate complex mixtures of molecules in chemical and biological systems. It is a powerful tool for drug impurity characterization, providing precise identification, quantification, and profiling of impurities, thereby ensuring the safety and efficacy of pharmaceutical products. HPLC separates components based on their interactions with the stationary phase and their solubility in the mobile phase, making it especially useful for analyzing non-volatile and thermally unstable compounds. By adjusting parameters such as mobile phase composition, flow rate, and column type, HPLC can be tailored to resolve specific impurities. HPLC is widely applied in detecting and quantifying impurities in pharmaceuticals, environmental samples, and biological fluids. It plays a key role in studying drug stability by identifying and quantifying degradation products formed under various stress conditions, such as heat, light, and pH. The high resolution, sensitivity, and versatility of HPLC in analyzing a wide range of compounds are significant advantages. HPLC’s ability to generate detailed impurity profiles is crucial for regulatory compliance, as it ensures that pharmaceutical products meet strict quality standards. It is particularly valuable in stability testing, helping monitor the formation of degradation products in antibiotics like penicillin, where the drug’s stability is critical for efficacy.

Other classes of antibiotics consist of macrolides, amidoglycosides, sulfonamides and tetracyclines. Antibiotics are the chemotherapeutic agents that kill or inhibit the growth of microorganisms. These chemical agents are used to treat disease by destroying pathogenic microorganisms or inhibiting their growth at concentration low enough to avoid undesirable damage to the host [8]. HPLC is also compatible with various detection methods, including UV-Vis, fluorescence, and mass spectrometry, further enhancing its versatility in impurity profiling.

Gas Chromatography (GC): Gas Chromatography (GC) is an advanced technique widely utilized for the detection and characterization of volatile and semi-volatile impurities in pharmaceuticals, playing a vital role in safeguarding drug quality, efficacy, and regulatory adherence. By separating analytes based on their boiling points and interactions with a stationary phase, GC is particularly well-suited for volatile organic compounds, environmental pollutants, and pharmaceutical impurities. A typical application includes the quantification of residual organic solvents, such as ethanol and methanol, which are frequently employed during Active pharmaceutical Ingredient (API) synthesis for roles like reaction media, purification, yield enhancement, and crystallization control. The technique’s high sensitivity and separation efficiency make it suitable for accurately quantifying complex mixtures, including trace-level compounds down to parts per trillion in some cases. Although GC is highly effective for volatile and semi-volatile substances, it encounters limitations with high-molecular-weight compounds like polypeptides or thermally unstable antibiotics, where derivatization may be required due to their non-volatility. Despite these limitations, GC is widely applied in pharmaceutical analysis for tasks such as batch consistency verification, process control, residual solvent determination, and the assay of drugs like isotretinoin, cocaine, and betamethasone valerate. It is also instrumental in detecting process-related impurities, using a range of detectors to ensure compliance with standards set by bodies such as the International Conference on Harmonization [9-11].

Liquid Chromatography-Mass Spectrometry (LC-MS): Liquid Chromatography-Mass Spectrometry (LC-MS) is a highly effective analytical method frequently employed in the characterization of drug impurities. By integrating the separation power of Liquid Chromatography (LC) with the detection capabilities of Mass Spectrometry (MS), LC-MS serves as a crucial technique for both identifying and quantifying impurities in pharmaceuticals. The liquid chromatography component efficiently separates impurities based on characteristics such as polarity and solubility, while the mass spectrometer provides molecular identification through mass-to-charge ratio analysis [12]. This combination enables LC-MS to detect impurities at extremely low concentrations, making it ideal for tracing degradation products, residual solvents, and other contaminants that may compromise drug safety and efficacy. Furthermore, LC-MS can reveal the molecular structure of unknown impurities, aiding in understanding their impact on drug performance and stability. This technique is vital in pharmaceutical processes such as stability studies, method development, and meeting stringent quality standards. It is especially effective in the detection of trace degradation products in biologic drugs, including monoclonal antibodies, and in identifying Post-Translational Modifications (PTMs) that may influence drug efficacy [13].

Overall, LC-MS is indispensable for drug impurity analysis, offering exceptional sensitivity, specificity, and versatility.

It plays a key role in ensuring the safety, efficacy, and regulatory compliance of pharmaceutical products by providing detailed information about the identity, quantity, and structure of impurities.

Spectrometric Techniques

Spectroscopic methods involve the interaction of light with matter, providing detailed information about molecular structure, functional groups, and impurity composition.

UV-Visible Spectroscopy : UV-Visible spectroscopy is a crucial tool in the pharmaceutical industry for detecting and quantifying drug impurities. It combines simplicity, cost-effectiveness, and accuracy, making it an ideal choice for routine quality control and drug purity monitoring. By measuring the absorbance or transmittance of UV or visible light through a sample, this technique provides valuable insights into the molecular composition and concentration of impurities in a drug substance. Since different impurities exhibit unique absorption spectra, they can be identified based on the specific wavelengths at which they absorb light. The concentration of these impurities can be quantified by measuring absorbance at precise wavelengths, using calibration curves or comparing the results with known standards. UV-Visible spectroscopy is also effective in monitoring drug purity during production and quality control, as it can detect any deviations from the expected absorption profile. Additionally, as drugs degrade over time, leading to the formation of impurities, UV-Visible spectroscopy can identify these degradation products by observing new peaks or shifts in the absorption spectrum [14].

Fourier Transform Infrared (FT-IR) Spectroscopy : Fourier Transform Infrared Spectroscopy (FT-IR) is a fast and cost-effective analytical technique; however, it is not suitable for directly separating and determining impurities in complex mixtures. To address this limitation, chemo metric techniques, particularly multivariate regression, are employed to enhance impurity profiling. Chemo metrics serves as a powerful tool for chemists and researchers, enabling them to extract meaningful information from complex data sets, make informed decisions, and optimize processes. When combined with IR spectroscopy, chemo metrics significantly improves and simplifies quality control processes in drug manufacturing, allowing for more accurate monitoring of impurity levels and ensuring product consistency. In separation methods, the principle often involves distinguishing between components in a mixture based on their unique physical or chemical properties, such as differences in molecular size, polarity, or interaction with a stationary phase. Since FT-IR alone do not separate components, chemo metrics is utilized to analyse and interpret the spectral data obtained, allowing for the identification and quantification of impurities without physical separation [15].

Mass Spectrometry (MS): Mass spectrometry (MS) is a highly sensitive technique known for its high reproducibility and specificity, making it invaluable in the analysis of trace compounds and in elucidating molecular structures. It excels in identifying biomolecules or proteins within biological samples, and with the use of soft ionization techniques, it can effectively study high molecular mass, non-volatile, and thermally sensitive compounds. During the process, the parent molecule is ionized, producing ions or fragments that travel to the analyser compartment, where they are resolved according to their mass-to-charge ratio. The resulting mass spectrum provides detailed data on the molecular composition of the parent compound. Mass spectrometry is often coupled with chromatographic techniques, creating a powerful hyphenated approach for determining the structure of impurities. However, despite its strengths, MS has certain drawbacks. The technique can be complex and costly, requiring highly specialized equipment and expertise. Additionally, the interpretation of mass spectra can be challenging, particularly when dealing with complex mixtures, and soft ionization techniques, while useful, may not always prevent fragmentation, potentially complicating the analysis. These factors can limit the accessibility and ease of use of mass spectrometry in routine applications [16].

Gas Chromatography- Mass Spectrometry (GC-MS): Gas Chromatography- Mass Spectrometry (GC-MS) is an analytical technique that combines the features of Gas Chromatography (GC) and Mass Spectrometry (MS) to identify different substances within a test sample. This powerful tool is widely used in analytical laboratories for applications in environmental analysis (to identify pollutants and volatile organic compounds in air, water, and soil samples), in pharmaceuticals (for the quality control of drugs, determining the purity of compounds, and detecting impurities), food safety (to detect pesticide residues, food additives, and contaminants in food products, and forensic investigations, pharmaceuticals, and food safety testing. GC-MS has been applied to identify and quantify residual solvents in pharmaceutical raw materials and finished products. An example is its use in detecting ethyl acetate as a process-related impurity in the manufacture of certain APIs [17].

Nuclear Magnetic Resonance (NMR) Spectrometry: NMR has become an indispensable tool in the analysis of drug impurities, benefiting from advancements that have expanded its theoretical and experimental applications in spin physics. Utilizing the magnetic properties of atomic nuclei, NMR spectroscopy is essential for the structural elucidation of unknown molecules, particularly in pharmaceutical research. Techniques such as ¹H and ¹³C NMR spectroscopy are widely employed for determining molecular structures. NMR is particularly valuable for detecting trace impurities in drug compounds, both before and after chromatographic separation. This technique excels in analyzing the configuration and structure of synthetic and organic molecules, as long as they are available in sufficient purity and quantity, with a molecular mass not exceeding 50 kDa [18].

Biosimilars are biologic medical products that closely resemble an already approved original biologic, known as the innovator or reference product. Although biosimilars are not exact replicas of their reference counterparts, they are engineered to have no clinically meaningful differences in terms of safety, purity, and efficacy. These products are essential in treating a wide range of conditions, including cancers, autoimmune diseases, and chronic inflammatory disorders, where biologic therapies are the standard of care. By offering lower-cost alternatives to expensive biologics, biosimilars significantly enhance access to life-saving treatments. These highly similar versions of approved biologic medicines undergo rigorous evaluation and regulatory approval processes, ensuring they provide the same therapeutic benefits as their reference products at a reduced cost. Consequently, biosimilars play a pivotal role in making biologic therapies more accessible and affordable for patients. The Mass Spectrometry (MS) analysis of biosimilars in comparison to innovator products involves highly complex and detailed procedures. This complexity stems from the necessity to account for structural heterogeneity, Post-Translational Modifications (PTMs), glycosylation patterns, and other subtle molecular differences that could influence the biosimilar’s safety, efficacy, and immunogenicity.

Advanced MS techniques, such as high-resolution mass spectrometry, tandem MS (MS/MS), and isotope labeling, are critical for accurately characterizing these features. Additionally, thorough method validation—including assessments of precision, accuracy, and reproducibility— is essential to ensure that the biosimilar aligns with the innovator product’s Critical Quality Attributes (CQAs). This rigorous analytical approach is crucial for confirming comparability and biosimilarity, thus facilitating regulatory approval and market acceptance.

Prevalence of Impurities in Pharmaceutical Products

Impurities bring down the shelf life of the drugs and cause difficulties during drug formulation. A study analyzing the presence of impurities in various pharmaceutical products revealed that approximately 15% of drug formulations contain impurities above the acceptable limits set by regulatory authorities. The most common sources of these impurities include synthesis by-products (40%), residual solvents (30%), degradation products (20%), and excipient-related impurities (10%). The presence of these impurities affect the efficacy and safety of the drug formulation. (Figure 1) depicts the relationship between percentage impurity levels and percentage drug efficacy. It shows that as impurity levels increase, drug efficacy tends to decrease, illustrating the negative impact of impurities on drug performance. At low levels of impurities, the drug’s efficacy is generally at its maximum. This is because fewer impurities are interfering with the Active Pharmaceutical Ingredient (API), allowing it to function optimally [19].

Figure 1

Comparison of Analytical Techniques

Comparative studies have shown that HPLC and LC-MS offer higher sensitivity and specificity in impurity detection compared to TLC and GC. Specifically, LC-MS can detect impurities at concentrations as low as 0.001%, whereas TLC typically detects impurities down to 0.5%. (Table 1) shows the comparison of analytical techniques for impurity profiling.

Table 1: Comparison of Analytical Techniques.

The evolving landscape of impurity profiling in pharmaceutical formulations underscores its pivotal role in ensuring the safety, efficacy, and quality of drug products. Advances in analytical techniques, such as chromatography, mass spectrometry, and spectroscopy, have made it possible to detect and quantify impurities at increasingly lower levels, thereby enhancing the reliability of impurity profiling. The integration of these techniques into routine pharmaceutical analysis has not only improved the accuracy and precision of impurity detection but also ensured compliance with stringent regulatory standards. Regulatory agencies like the FDA or EMA require strict impurity profiling to ensure that the drug’s efficacy is not compromised by the presence of impurities. The relationship highlighted by the graph helps define the acceptable threshold for impurities, allowing manufacturers to optimize drug purity during production. As pharmaceutical products continue to grow in complexity, the need for robust impurity profiling will remain critical, safeguarding public health by preventing potential toxicological risks associated with impurities. Through continued innovation and adherence to regulatory frameworks, impurity profiling will continue to play a vital role in the development and manufacturing of high-quality pharmaceuticals.

1. Görög S (2006) The importance and the challenges of impurity profiling in modern pharmaceutical analysis. Trends Anal Chem 25(8): 755-757.
2. White D, Varlashkin P, Rusch DN (1992) Detection of Impurities: A Review on Advance in Impurities Detection and Characterization in Pharmaceuticals by Analytical Techniques. J Pharm Sci 81: 1204-1209.
3. Agbaba D, Radovic A, Vladimirov S, Zivanov-Stakic D (1996) Simultaneous TLC determination of co-trimoxazole and impurities of sulfanilamide and sulfanilic acid in pharmaceuticals. J Chromatogr Sci 34: 460-464.
4. Paul A, Paul D, Anindya Bagchi A, Raha A, Mukherjee P et al. (2019) Statistically validated chromatographic study of salicylate and aniline derivatives of NSAID. The Pharma Innovation Journal 8(7): 734-739
5. Pavic K, Cudina O, Agbaba D, Vladimirov S (2003) Quantitative analysis of cyproterone acetate and ethinylestradiol in tablets by use of planar chromatography. J Planar Chromatgr Mod TLC 16(1): 45-47.
6. Fayed AS, Shehata MAA, Hassan NY, El-Weshahy SA (2006) Validated HPLC and HPTLC stability-indicating methods for determination of alfuzosin hydrochloride in bulk powder and pharmaceutical formulations. J Sep Sci 29(18): 2716-2724
7. Desai P, Captain A, Kamdar S (2012) Development and Validation of HPTLC Method for Estimation of Tramadol HCl in Bulk and in Capsule Dosage Form. Int J Pharm Tech Res 4(3): 1261-1265,
8. Peter K, Mlynska MS, Baniszewski B (2020) Determination and Identification of Antibiotic Drugs and Bacterial Strains in Biological Samples. Molecules 25(11): 2556.
9. Reddy BP, Reddy MS (2009) Residual solvents determination by HS-GC with Flame Ionization Detector in Omeprazole Pharmaceutical formulations. Int Pharm Tech Res1(2): 230-234.
10. Hashimoto K, Urakami K, Fujiwara Y, Terada S, Watanabe C (2001) Determination of Residual Solvents in Pharmaceuticals by Thermal Desorption-GC/MS. Anal Sci 17(5): 645-648.
11. Saraji M, Khayamian T, Siahpoosh ZH, Farajmand B (2012) Determination of volatile residual solvents in pharmaceutical products by static and dynamic headspace liquid-phase microextraction combined with gas chromatography-flame ionization detection. Anal Methods (6): 1552-1559.
12. Ermer J (1998) The use of hyphenated LC–MS technique for characterisation of impurity profiles during drug development. J Pharm Biomed Anal 18(4-5): 707-714.
13. Peng W, Pronker MF, Snijder J (2021) Mass spectrometry based De Novo sequencing of monoclonal antibiotics using multiple proteases and a dual fragmentation scheme. J Proteome Res 20(7): 3559-3566
14. Nicolas EC, and Scholz TH (1998) Active drug substance impurity profiling: Part I. LC/UV diode array spectral matching. J Pharm Biomed Anal 16(5): 813-824.
15. Kupcewicz B, Ronowicz J, Balcerowska-Czerniak G, Walasek A, Budzisz E (2013) Evaluation of impurities in simvastatin drug products with the use of FT-IR spectroscopy and selected chemometric techniques. Central Eur. J Chem 11(8): 1320-1329.
16. Rajawat J, Jhingan G (2019) Mass spectroscopy. In Data Processing Handbook for Complex Biological Data Sources p: 1-20.
17. Liu J, Xie B, Mai B, Cai O, He R, et al. (2021) Development of a sensitive and stable GC-MS/MS method for simultaneous determination of four N- nitrosamine genotoxic impurities in sartan substances. J Anal Science and Technology 12(3): 1-8
18. Holzgrabe U (2010) Quantitative NMR spectroscopy in pharmaceutical applications. Prog Nucl Magn Reson Spectrosc 57(2): 229-240.
19. Dhangar KR, Jagtap RB, Surana SJ, Shirkhedkar AA (2017) Impurity Profiling of Drugs towards Safety and Efficacy: Theory and Practice. J Chil Chem Soc 62(2): 3543-3557.