Advancing drug safety and mitigating health concerns: High-resolution mass spectrometry in the levothyroxine case study.

Levothyroxine is a drug with a narrow therapeutic index. Changing the drug formulation composition or switching between pharmaceutical brands can alter the bioavailability, which can result in major health problems. However, the increased adverse drug reactions have not been fully explained scientifically yet and a thorough investigation of the formulations is needed. In this study, we used a non-targeted analytical approach to examine the various levothyroxine formulations in detail and to reveal possible chemical changes. Ultra-high-performance liquid chromatography coupled with a data-independent acquisition high-resolution mass spectrometry (UHPLC-DIA-HRMS) was employed. UHPLC-DIA-HRMS allowed not only the detection of levothyroxine degradation products, but also the presence of non-expected components in the formulations. Among these, we identified compounds resulting from reactions between mannitol and other excipients, such as citric acid, stearate, and palmitate, or from reactions between an excipient and an active pharmaceutical ingredient, such as levothyroxine-lactose adduct. In addition to these compounds, undeclared phospholipids were also found in three formulations. This non-targeted approach is not common in pharmaceutical quality control analysis. Revealing the presence of unexpected compounds in drug formulations proved that the current control mechanisms do not have to cover the full complexity of pharmaceutical formulations necessarily.

Comparison of patch testing Brazilian (Green) propolis and Chinese (poplar-type) propolis: Clinical epidemiological study using data from the Information Network of Departments of Dermatology (IVDK).

BACKGROUND: Propolis types differ regarding their chemical composition. OBJECTIVES: To compare patch test results based on Brazilian (Green) propolis with data based on Chinese (poplar-type) propolis, and to evaluate the specifications of raw materials used for the PT preparations. METHODS: In the Information Network of Departments of Dermatology (IVDK), 1290 consecutive patients were patch tested with Brazilian (Green) propolis (NH400, SmartPractice Europe). Patch test reactivity was compared with results obtained with Chinese (poplar-type) propolis (NA71, SmartPractice Europe) by calculating frequencies and corresponding 95% confidence intervals. Data on the specifications of raw materials used for NH400 and NA71 were obtained from the manufacturer. RESULTS: Positive reactions to NH400 were found in 303 (23.5%) patients with unclear clinical relevance in most cases. Patients reacting to NH400 were less often sensitised to fragrances and colophony, but more often to nickel sulphate and cobalt chloride than patients reacting to NA71. The NH400 batch used contained high levels of aerobic bacteria, and was not purified by ethanolic extraction. CONCLUSIONS: Pattern of concomitant reactivity along with raw material properties suggests that the high frequency of positive reactions to NH400 may primarily result from bacterial contamination or impurities in the PT preparation rather than from propolis constituents.

Robustness evaluation of weak anion exchange chromatography method for the purity analysis of therapeutic oligonucleotides.

Therapeutic oligonucleotides are becoming an important class of therapeutics. Their manufacturing processes can result in the formation of impurities, particularly truncated species. To ensure the quality and safety of the product, it is crucial to evaluate the presence of these species. Liquid chromatography analysis enables such purity determination. In this context, a recently described weak anion exchange chromatography method was optimized to allow the effective separation of different impurities. The optimization addressed the complexity and instability of the mobile phases, which contained salts and organic compounds. Adjustments were made to the mobile phase composition and gradient to meet the requirements of QC laboratories. Additionally, to ensure the method's reliability, a robustness study was conducted based on a risk assessment. Five factors were considered potential risks and were assessed experimentally on different chromatographic outputs. This led to the definition of a robust space, ensuring the method's reliability for the purity determination of oligonucleotides.

Characterization of host cell proteins in the downstream process of plant-Based biologics using LC-MS profiling.

Host cell proteins (HCPs) are process-related impurities found in biopharmaceutical products that can impair their safety and efficacy. While ELISA has traditionally been employed to quantify HCPs, LC-MS emerges as a powerful alternative for precise identification of individual HCPs. In this study, we used LC-MS for profiling HCPs from Nicotiana benthamiana-derived biopharmaceuticals. Our approach involved rigorous false discovery rate control to ensure data integrity and reliability. Comprehensive analysis revealed a systematic reduction of HCPs following purification, demonstrating the efficiency of purification processes in removing non-essential proteins. Furthermore, LC-MS enabled the identification of potential contaminants, refining purification strategies and improving product purity and integrity. Our findings highlight the potential of LC-MS as an analytical tool for HCPs analysis in biopharmaceutical development and manufacturing. By providing detailed insights into HCPs profiles and contaminants, LC-MS facilitates informed decision-making in downstream processing steps, benefiting product quality, patient safety, and the biopharmaceutical sector.

High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method Development and Validation for Simultaneous Determination of Seven Nitrosamine and Azidomethyl-Biphenyl-Tetrazole Impurities in Losartan.

Nitrosamine-related impurities (N-nitrosomethylamino butyric acid [NMBA], N-nitrosodiethylamine [NDEA], N-nitrosodiisopropylamine [NDIPA], N-nitrosomethylphenylamine [NMPA], N-nitrosodibutylamine [NDBA], N-nitrosodimethylamine [NDMA], and N-nitrosoethylisopropylamine [NEIPA]) and 5-[4'-(azidomethyl)-[1,1'-biphenyl]-2-yl]-2H-tetrazole (AZBT) formed during the manufacture of sartan medicines have been classified into human mutagens and carcinogens after long-term treatment. The study developed a simple, economical but highly sensitive procedure for the simultaneous quantification of seven nitrosamines and AZBT impurities in sartan pharmaceuticals. After extraction with methanol (MeOH) 50%, the compounds were analyzed with a reversed-phase liquid chromatography-tandem mass spectroscopy with atmospheric-pressure chemical ionization (APCI) mode (APCI[+] for nitrosamines and APCI[-] for AZBT), selected reaction monitoring, C18 column, gradient elution with 0.1% formic acid in water and in MeOH, respectively. The validated procedure obtained high extraction efficiency (>90%), wide linear range (0.2-50.0 ng/mL NMBA, NDEA, NDIPA, NMPA, and NDBA; 0.5-50.0 ng/mL NDMA and NEIPA; 2.0-100 ng/mL AZBT), limit of quantification < 10% of the acceptance level, recovery range of 85%-115% with relative standard deviation < 15% and minimum matrix effects for all impurities. The procedure was applied to test 16 commercial losartan samples. As a result, eight samples contained AZBT within the current regulatory limits, but no nitrosamine impurities were detected in all samples.

Establishing a multi-method approach for unprecedented detection and quantification of 13 genotoxic impurities (GTIs) in Apixaban drug substance through ultra-performance liquid chromatography (UPLC).

This groundbreaking study introduces a pioneering development of multi-method approach for the first-ever detection and quantification of 13 genotoxic impurities (GTIs) in Apixaban (Apx) drug substance using ultra-performance liquid chromatography (UPLC) with ultraviolet (UV) detector. In this novel endeavor, two distinct UPLC-UV methods, Method A (for impurities A to G) and Method B (for impurities H to M), were meticulously developed and validated as per International Council for Harmonization (ICH) guidelines to address the challenge of identification and control of 13 GTIs in Apx drug substance. The validation process included rigorous assessment of linearity, accuracy, specificity, precision, limit of quantification (LOQ), and limit of detection (LOD) for each impurity in each method which marks a significant advancement in pharmaceutical analysis. The developed methods address the regulatory requirements set forth by ICH M7(R2) guidelines by providing a reliable approach for quantifying GTIs in Apx drug substance at trace levels to minimize the potential carcinogenic risk to the patients.

Selection of Solvent and Positive Control Concentration for Enhanced Ames Test Conditions for N-Nitrosamine Compounds.

The Ames test is a widely used bacterial mutagenicity assay to evaluate the potential of chemical compounds to induce mutations. In recent years, there has been growing concern regarding the presence of N-nitrosamines in pharmaceuticals, food, and other consumer products. N-Nitrosamines are probable mutagens and carcinogens. To address the reduced sensitivity of the standard Ames test for N-nitrosamines, particularly N-nitrosodimethylamine, the European Medicines Agency (EMA) and United States Food and Drug Administration (FDA) have recently published recommendations for enhanced Ames test (EAT) conditions. However, there is a lack of clear guidance on the selection of N-nitrosamine positive control concentrations, particularly for 1-cyclopentyl-4-nitrosopiperazine, and the amount of solvent to be used in the EAT. This study aims to address the current gap in concentration and volume specifications by providing a comprehensive guide to set up enhanced Ames test conditions specifically for N-nitrosamine compounds using appropriate amounts of solvent, new solvents, and strain-specific positive control concentrations.

NDSRIs Crisis in Pharmaceuticals; Insights on Formation Pathways, Root Causes, Risk Management, and Novel Analytical Techniques.

The discovery of a new class of nitrosamine impurities called N-nitroso drug substance related impurities (NDSRIs) in pharmaceuticals has emerged as a significant challenge for the pharmaceutical sector due to their significant genotoxic and mutagenic effects. Regulatory bodies globally in active collaboration with all the concerned stake holders, are taking effective measures to prevent and control NDSRIs. This comprehensive review on NDSRIs discusses formation pathways, root cause analysis, acceptable intake limits, case studies, control strategies and regulatory responses pertaining to recent NDSRI incidents. This review discusses the novel liquid chromatographic techniques (LC- MS/MS, GC-MS/MS) used to identify and quantify of NDSRIs. This review would aid pharmaceutical professionals, R&D analytical and formulation scientists, and regulatory bodies in gaining deeper insights into the NDSRIs crisis, facilitating formulation of NDSRI-free drug products, and ensuring their sensitive detection with accurate risk evaluation.

Identification and quantitative analysis of genotoxic impurities in rifampicin: Development and validation of a targeted LC-MS/MS method for 1-amino-4-methylpiperazine.

Rifampicin, essential for long-term tuberculosis treatment, requires rigorous control of non-therapeutic impurities due to their potential adverse, including mutagenic effects. Reports on control strategies for genotoxic impurities in rifampicin have been limited. This study introduced an analytical method to identify potential genotoxic impurities from the synthesis of raw materials. The structure of the 25-deacetyl-23-acetyl-rifampicin genotoxic impurity was confirmed using nuclear magnetic resonance, high-resolution mass spectrometry (HRMS), and high-performance liquid chromatography (HPLC). An HPLC-HRMS method was established and validated for detecting another genotoxic impurity, 1-amino-4-methylpiperazine, adhering to the International Council on Harmonization guidelines, which include specificity, linearity, detection and quantification limits, accuracy, precision, and robustness. These developments improve the quality control strategy for genotoxic impurities in rifampicin, ensuring product safety.

Determination of volatile impurities and ethanol content in ethanol-based hand sanitizers: Compliance and toxicity.

This study aimed to assess volatile impurities and ethanol content in ethanol-based hand sanitizers. A total of 31 different brands of hand sanitizers were analyzed using headspace gas chromatography-mass spectrometry to detect impurities and determine alcohol content for compliance. Volatile impurities were identified through Mass Spectrometry database analysis, and regression analysis was employed to ascertain ethanol percentage. Furthermore, a simulated toxicological analysis was conducted to evaluate the potential toxic effects associated with hand sanitizer usage. The detected impurities primarily included ethyl acetate, benzene, acetone, and acetal, along with contaminations such as isobutanol and non-recommended alcohols. In addition, 71 % of samples contained less than the recommended 60 % v/v alcohol concentration, failing to comply with guidelines from the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO). Additionally, the simulation study underscored acute and chronic toxicities primarily linked to benzene contamination. Given that some of the studied products are imported while others are locally produced, it is imperative for consumers worldwide to be informed that certain hand sanitizers may not only be ineffective but also contain harmful residues.

Aspects of complexity in quality and safety assessment of peptide therapeutics and peptide-related impurities. A regulatory perspective.

In recent years, a number of therapeutic peptides have been authorized in the EU market, and several others are in the clinical development phase or under assessment for full dossier or generic applications. Quality and safety guidelines specific to peptides are limited, and some aspects have to be considered. In particular, concerns relate to the analytical investigation for impurities and the toxicological assessment of these substances. The guidelines and the compendial pharmacopoeias provide certain references but that may be questionable if interpreted according to whether therapeutic peptides are considered chemical or biological entities, large or small. The characterization of peptide-related impurities cannot follow the small molecule approach but should consider aspects closely linked to the complex mechanisms of action that these large molecules can exert in the human body. Although direct genotoxic mechanisms cannot be excluded, hazardous interactions on biological systems cannot be ruled out, as in the case of natural peptide toxins and their specific interactions with cellular or membrane targets. From a regulatory perspective, only after specific risk identification and characterization should an equally specific safety threshold in relation to potential toxicity be defined.

A practical HPLC-MS method for the analysis of nitrosamine drug substance related impurities using an inexpensive single quadrupole mass spectrometer.

Nitrosamine drug substance related impurities (NDSRIs) are often analyzed using high performance liquid chromatography (HPLC) with mass spectrometry (MS) detection. Due to high sensitivity requirements, high resolution MS or MS/MS is commonly used. However, it is difficult to implement this type of method for routine analysis at a supply site. Herein, we report a systematic approach to develop and validate a practical, robust, and user-friendly method for the analysis of NDSRIs using an inexpensive single quadrupole MS instrument such as QDa. We used 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro- [1,2,4] triazolo [4,3-a] pyrazine (NTTP) as an example to demonstrate the method development process. By optimizing the HPLC and MS parameters, we were able to develop a simple HPLC-MS method that provides the desired specificity and sensitivity for the analysis of NTTP and can be easily implemented in an analytical lab. The limit of quantitation is 0.5 ng/mL, corresponding to 0.1 ppm with respect to 5 mg/mL sitagliptin. The method has been successfully validated per ICH guidelines.

The Effect of Electrolyte pH and Impurities on the Stability of Electrolytic Bicarbonate Conversion.

Electrolytic bicarbonate conversion holds the promise to integrate carbon capture directly with electrochemical conversion. Most research has focused on improving the faradaic efficiencies of the system, however, the stability of the system has not been thoroughly addressed. Here, we find that the bulk electrolyte pH has a large effect on the selectivity, where a higher pH results in a lower selectivity. However, the bulk electrolyte pH has no effect on the stability of the system. A decrease in CO selectivity of 30% was observed within the first three hours of operation in an optimized system with 3 M KHCO3 and gap between the membrane and electrode. Single-pass electrolyte experiments at various constant pH values (8.5, 9.0, 9.5, and 10.0), show that only at a pH of 10 the CO selectivity was stable during three hours, reaching a faradaic efficiency toward CO of only 18% as compared to an initial 55% at pH 8.5. Trace metal impurities present in the electrolyte were found to be the cause of the decrease in stability as these deposit on the electrode surface. By complexing the trace metal ions with ethylenediaminetetraacetic acid (EDTA), the metal deposition was avoided and a stable CO selectivity was obtained.

A comprehensive approach for N-nitrosamine determination in pharmaceuticals using a novel HILIC-based solid phase extraction and LC-HRMS.

N-nitrosamines (NAs) are potentially highly carcinogenic compounds that have recently been detected in traces in various drug products (DPs). Due to the different physicochemical properties of NAs and active pharmaceutical ingredients (APIs), there is a lack of appropriate analytical methods for simultaneously determining multiple NAs in various DPs. To overcome these limitations, a versatile and innovative analytical approach was developed using a unique sample clean-up procedure by solid phase extraction based on hydrophilic interaction chromatography, which retains high amounts of APIs and polar excipients while allowing NAs of interest to pass through. The samples were analyzed by liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry. The proposed highly sensitive, selective, and robust method was successfully validated, resulting in excellent linearity (R2 > 0.999), accuracy (85-115 %), and precision (RSD <10 %) with adequate recoveries (>80 %), achieving limits of quantitation of at least 42.5 % of regulatory limits. Furthermore, robustness was confirmed for ten DPs (recoveries >80 % and RSD <15 % for all NAs), including those containing up to three APIs. The analytical approach was utilized to examine 26 commercially available and expired DPs. Three NAs (N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, and N-nitroso-di-n-butylamine) were detected, only NDMA exceeded the limits in expired DPs by up to 32-fold. It was found that special care should be taken when handling samples as NDMA content can be decreased by almost 50 % if samples are not prepared immediately. The approach was tested on 59 different APIs and was confirmed as reliable tool for routine monitoring of 15 NAs in various DPs. Due to its flexibility, the method can be further adapted to the specific API of interest or extended to the newly emerging NA drug substance-related impurities to ensure the safety of DPs and thereby mitigate potential health risks.

N-Nitrosamine Drug Substance Related Impurities (NDSRIs) - A proposal for the addition of subcategories to Carcinogenic Potency Categorisation Approach categories 1 and 2 for NDSRIs with a molecular weight > 200 Da.

The carcinogenicity potency categorization approach (CPCA) derived and harmonized by Health Authorities was a significant milestone, as it defined molecular properties that allowed for the rapid evaluation of the chemical structures of N-nitrosamine drug substance related impurities (NDSRIs) and the assignment of associated lifetime Acceptable Intake limits to inform on appropriate impurity control strategies in certain drug products. Nonetheless, it is important to continue to refine and improve on the CPCA based upon data-derived evidence. Herein, we focus on the default CPCA AI for NDSRIs, which is largely based on the small molecule N-nitrosamines (NAs). Considering the carcinogenic potency of NAs with a molecular weight > 200 Da (NDSRIs molecular weight is typically 200-600 Da), we propose that in the absence of any compound specific data, the lowest lifetime Acceptable Intake for NAs, such as NDSRIs, should be 10x less (i.e., 150 ng/day) than the ICH M7 Threshold of Toxicological Concern of 1500 ng/day, (even for NDSRIs that are considered CPCA Category 1 and 2) which would conservatively result in a theoretical cancer risk of <1 in 100,000.

Research on wheat impurity identification method based on terahertz imaging technology.

The traditional detection of impurities in wheat has difficulties such as low precision, time-consuming, and cumbersome, therefore, it is important to study the method of rapid and accurate detection of impurities in wheat for correctly assessing the quality grade of wheat. Terahertz (THz) technology has many superior properties such as transient, broadband, low-energy, and penetrating, which can realize rapid and nondestructive detection of wheat quality. In this study, a classification and recognition algorithm AHA-RetinaNet-X for wheat impurity terahertz images based on RetinaNet and Artificial hummingbird algorithm (AHA) is proposed.A THz three-dimensional tomography imaging system is used to image wheat and its impurities, and two THz image datasets, respectively the wheat and impurity dataset for verifying the classification effect of wheat and impurities and the impurity dataset for verifying the classification effect of impurities. The experimental results show that the AHA-RetinaNet-X model outperforms other detection and classification models in terms of accuracy, F1-score, precision, recall, and specificity, and is able to achieve 96.1%, 94.9%, 95.2%, 95.8%, 95.5%, 95.3%, and 93.3% for the wheat and impurity dataset and the impurity dataset, respectively, 95.6%, 96.3%, and 95.2%, and the mAP value of AHA-RetinaNet-X is also higher than the other models and can reach 92.1%. The combination of THz imaging technology and AHA-RetinaNet-X can realize the classification and identification of wheat and impurities, which provides a new method for the non-contact rapid nondestructive detection and identification of wheat and impurities, and also provides a reference for the research of the identification and detection methods of other substances.

Elemental impurities (heavy metals) in kratom products: an assessment of published individual product analyses.

INTRODUCTION: Kratom is commonly used by consumers, and the elemental impurity exposure that consumers would have at different kratom ingestion doses has been determined. METHODS: This assessment used original data from independent third-party laboratory testing of kratom products to identify the percentage of products that exceeded permissible daily exposure limits for lead (5 µg/day), nickel (200 µg/day), arsenic (15 µg/day), and cadmium (5 µg/day), the interim reference level for lead in adults (12.5 µg/day), and the tolerable upper intake level for manganese (11 mg/day) and nickel (1 mg/day). We assessed all products regardless of type and then evaluated non-extract products, extract products, and a soda preparation separately for elemental impurities. RESULTS: Three assessments of elemental impurities in kratom products have been published, totaling 68 products. Assessing all products and assuming a 3 g daily dose of kratom, 7.4% would exceed the permissible daily exposure limits for lead, 0% for nickel, 3.1% for arsenic, and 0% for cadmium. At a kratom dose of 25 g daily, 70.6% would exceed the permissible daily exposure limits for lead, 20.6% for nickel, 9.4% for arsenic, and 0% for cadmium. The interim reference level for lead would be exceeded by 1.5% of products at a kratom daily dose of 3 g and 33.8% of products at 25 g. The tolerable upper intake level for manganese would be exceeded by 12.5% of products at a kratom daily dose of 3 g and 41.7% of products at 25 g. Non-extract products generally contain greater concentrations of elemental impurities than extract products or the soda preparation. DISCUSSION: Apart from their concentrations in a gram of product, assessing the amount of exposure to elemental impurities at different kratom ingestion doses is also important. Elemental impurities exceeding regulatory permissible concentrations for many products, especially with greater daily kratom ingestion doses, may impact human health. CONCLUSIONS: Some kratom products contain excessive concentrations of elemental impurities of toxicological concern, such as lead and arsenic. Non-extract products (powders, capsules, tablets) generally contain greater concentrations of elemental impurities than extract products or the soda preparation. Daily use of these products can result in exposures exceeding regulatory thresholds and adverse health effects.

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test.

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity. In addition, preincubations were conducted for both 60 min and 30 min. These test variables were evaluated by testing 12 small-molecule N-nitrosamines and 17 NDSRIs for mutagenicity in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA (pKM101). Eighteen of the 29 N-nitrosamine test substances tested positive under one or more of the testing conditions and all 18 positives could be detected by using tester strains TA1535 and WP2 uvrA (pKM101), preincubations of 30 min, and S9 mixes containing 30% hamster liver S9. In general, the conditions under which NDSRIs were mutagenic were similar to those found for small-molecule N-nitrosamines.

Green Hydrogen Production From Non-Traditional Water Sources: A Sustainable Energy Solution With Hydrogen Storage and Distribution.

Green hydrogen development plays an essential role in creating a sustainable and environmentally conscious society while reducing reliance on traditional fossil fuels. Proton Exchange Membrane Water Electrolysers (PEMWEs), are sensitive to water quality, with various impurities impacting their efficiency, the quality of the hydrogen produced, and the device's lifespan. High-purity water is required for PEM electrolyzers; Type II water, which is required for commercial electrolyzers, must have a resistivity greater than 1 MΩ cm, sodium, and chloride concentrations less than 5 µg/L, and total organic carbon (TOC) content less than 50 parts per billion. The majority of electrolyzers operate on freshwater, or total dissolved solids (TDS) <0.5 g/kg, whereas brackish, rainwater, wastewater, and seawater have TDSs of 1-35 g/kg, 0.01-0.15 g/kg, 0.5-2 g/kg, and 35-45 g/kg, respectively. This critical review offers, for the first time, a comprehensive overview of relevant impurities in operating electrolyzers and their impact. The findings of this study indicate that electrolysis-based H2 processes are promising options that contribute to the H2 production capacity but require improvements to produce larger competitive volumes. In addition, the main challenges and opportunities for generating, storing, transporting, and distributing hydrogen, as well as large-scale adoption are discussed.

Identification and characterization of two new oxidation degradation impurities in cinnarizine through LC-HRMS/MS and 1H NMR, along with in silico toxicity predictions of its degradation products.

Cinnarizine (CIN) drug substance is a US FDA and EMA approved antihistaminic drug, There is no report available on CIN for the identification of degradation products and their degradation pathway. Herein, we report a stability-indicating assay method for CIN, the formation and characterization of its major degradation products using LC-HRMS/MS and 1H-NMR techniques. CIN was subjected to oxidation, acid, base, thermal and photolytic degradation conditions. Two unknown degradation products (DP-1 and DP-2) of CIN were formed under oxidative conditions. We successfully separated these degradants using gradient elution on an Inertsil ODS 3 V column (150 × 4.6 mm, 5 µm) using mobile phase A consisting of 0.1% formic acid and the mobile phase B consisting of 0.1% formic acid/acetonitrile (20/80, v/v). CIN was labile to oxidative conditions and stable to acidic, alkaline hydrolytic, photolytic and thermal conditions. The degradation pathways were derived from the nature of the product formed under oxidative degradation conditions and available reports for confirmation of the mechanism. Since the stability-indicating assay method can be utilized for stability studies and routine quality control of CIN in both the pharmaceutical industry and research laboratories. This method has been validated in compliance with the guidelines set forth by the ICH.