Genotoxic Impurities Research Articles

Two genotoxic impurities of sulfonate esters in Posaconazole: Synthesis, method validation and mechanism of action

AbstractSulfonate esters are a class of organic impurities that includes highly potent mutagenic substances that typically need to be controlled down to a low ppm level in pharmaceutical development. In this work, two genotoxic impurities of sulfonate esters in Posaconazole, for example, ((3S,5R)‐5‐((1H‐1,2,4‐triazol‐1‐yl)methyl)‐5‐(2,4‐difluorophenyl)tetrahydrofuran‐3‐yl)methyl‐4‐methylbenzenesulfonate and (2S,3S)‐3‐ (4‐(4‐(4‐(4‐(((3R,5R)‐5‐((1H‐1,2,4‐triazol‐1‐yl)methyl)‐5‐(2,4‐difluorophenyl)tetrahydrofuran‐3‐yl)methoxy)phenyl)piperazin‐1‐yl)phenyl)‐5‐oxo‐4,5‐dihydro‐1H‐1,2,4‐triazol‐1‐yl)pentan‐2‐yl‐4‐methylbenzenesulfonate, were synthesized and characterized. The genotoxicity which was evaluated by two (quantitative) structure‐activity relationships prediction methodologies showed positive. Furthermore, a novel high‐performance liquid chromatography‐tandem mass spectrometry limit test method for detecting these two impurities in Posaconazole has been established and validated. The chromatographic separation of analytes was conducted on an ACQUITY BEH C18 column, and performed with 0.05% methanoic acid‐water and 0.05% methanoic acid‐acetonitrile as mobile phases A and B, respectively. Six batches of commercial‐scale Posaconazole samples were detected by the validated method and the results were in accordance with the Food and Drug Administration acceptance criteria for the genotoxic impurities in drug substances. Finally, a tentative mechanism for these two impurities was proposed, they are generally accepted to undergo an SN2‐type reaction, resulting in 7‐methylguanine as the predominant adduct in double‐stranded DNA.

Impurity study of tecovirimat

This article delineates the systematic identification, synthesis, and impurity control methods used during the manufacturing process development of tecovirimat, an antiviral drug that treats monkeypox. Critical impurities were synthesized, and their chemical structure was confirmed through NMR analysis, GC, and HPLC mass spectrometry. The results established a thorough approach to identify, address, and control impurities to produce high-quality tecovirimat drug substance in accordance with International Conference on Harmonization (ICH)-compliant standards. This study is the first of its kind to evaluate both process and genotoxic impurities in tecovirimat, demonstrating effective control measures during commercial sample investigations and scaling up to a 60-kg batch size. The findings highlight the importance of critical impurity characterization and control in pharmaceutical development and production to ensure the safety and efficacy of the final product.

Preparation of modified graphyne to detect genotoxic impurities in gefitinib: Effects of ionic liquid structures and carbon nanotube composite modes

Seven kinds of ionic liquids (ILs) modified oxidized hydrogen-substituted graphyne (O-HsGY) were prepared and combined with carbon nanotubes (CNTs) by covalent and non-covalent modes. The effects of the structure of ILs and two different CNTs composite modes on the structure and properties of the materials were investigated. A series of characterization methods were used to systematically study the physical and chemical properties of the materials. The genotoxic impurity 2-chloro-1-fluoro-4-nitrobenzene was extracted from gefitinib by the prepared adsorbent and quantitatively analyzed by high performance liquid chromatography. The experimental results showed that the longer the alkyl chain of the ILs, the greater the adsorption capacity of the modified material to the target. The composite mode of carbon nanotubes determined its distribution state, which in turn determined the strength of the interaction between the composite material and the target. Under the optimized conditions, 1-hexyl-3-methylimidazole hexafluorophosphate modified graphyne interacted more strongly with the target, with average recovery of 97.2–100.4% of the target substance. Because surface CNTs impeded ILs adsorption, the average recoveries of covalent and non-covalent composites fell to 96.1–99.5% and 95.9–101.6%, respectively. The above results showed that 1-hexyl-3-methylimidazolium hexafluorophosphate modified graphene had the strongest adsorption performance for the target, while the adsorption properties of the material decreased after composite CNTs.

Forced Degradation Products of Olmesartan Medoxomil and their In Vitro Genotoxicity Assessment by Comet Assay using HEK Cells

AbstractThis study designed to characterize major forced degradation products of Olmesartan Medoxomil (OM) and to perform genotoxicity profiling using acid, base, and hydrogen peroxide‐induced stress degradation studies. The isolation of degradation products was conducted on flash chromatography using FlashPure ECOFLEX C18 4 g column with gradient elution and diode array detection‐based fraction collection, and characterized by mass spectrometry. Three impurities (impurity‐1, 2, and 3) were identified with respective m/z values of 447.213, 461.229, and 463.208. The genotoxic potential of impurities was assessed by in silico prediction, and in vitro comet assay using HEK cells. The extent of DNA damage was analyzed using Comet Assay IV imaging software. For impurity‐1, head and tail lengths and their respective total intensities were significantly (p<0.001) larger than those of dimethyl sulfoxide (solvent) control and comparable to positive control. Other parameters such as mean grey level and total area of comet were statistically significant as compared with the solvent and positive controls. Overall, impurity‐1 was a possible genotoxic impurity and a known major degradation product in OM tablet dosage form. In silico prediction also revealed impurity‐1 as toxicity class 3, whilst impurities 2 and 3 were of toxicity class 4.

Trace level Quantification of N-Nitroso Safinamide: A Genotoxic Impurity in Safinamide Mesylate Using ESI-MS/MS

This study presents a novel, sensitive and highly reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology for the determination of N-Nitroso Safinamide, a genotoxic impurity in Safinamide mesylate. The optimized chromatographic conditions utilized Poroshell HPH-C18 (150 x 4.6 mm, 2.7 µm) and Ghost Buster columns with a flow rate of 0.8 ml/min and a column temperature of 40 °C. The mobile phase consisted of formic acid and methanol. Mass spectrophotometric analysis employed the Agilent Jet Stream Electrospray Ionization (AJS ESI) mode with a capillary voltage of 3500 V and a nitrogen gas flow rate of 12 L/min at 350 °C, while Multiple Reaction Monitoring (MRM) mode was used for scanning. Rigorous validation demonstrated the method's reliability and robustness. Linearity experiments showed a strong correlation coefficient of 0.999 between analyte concentration and peak area response. The method exhibited low limits of detection (LOD) and quantification (LOQ) of 0.00593 ppm and 0.01797 ppm, respectively, enabling accurate detection and quantification of N-Nitroso Safinamide at low concentrations. Precision studies demonstrated good repeatability and reproducibility, with % RSD values falling within acceptable limits. Specificity evaluations confirmed the absence of interferences in blank, standard, sample, and spiked sample solutions. The method's accuracy was established through satisfactory recovery values, ranging between 70 % and 130 % in spiked samples. The developed LC-MS/MS methodology offers a highly reliable and precise approach for quantifying N-Nitroso Safinamide in Safinamide mesylate.

Five impurities in posaconazole: detection, genotoxicity prediction and action-mechanism analysis

Genotoxic impurities are a class of highly potent mutagenic substances that typically needs to be controlled down to low ppm level in pharmaceuticals. A novel LC-MS/MS limit test method (single impurity ≤16.7 ppm) for detecting five impurities in Posaconazole has been established and partially validated. The chromatographic separation of analytes was conducted on an YMC-Triart C18 column and gradient elution was performed with 0.1% formic acid aqueous solution and acetonitrile as mobile phase A and B, respectively. Five impurities were carried out in ESI+ and MRM with the following mass transition pairs: m/z 390.2→296.2 for BSKZ029, m/z 300.4→136.2 for BSKZ117, m/z 207.0→162.2 for BSKZ135, m/z 209.0→91.0 for BSKZ155 and m/z 499.1→258.1 for BSKZ160. The established method exhibited good specificity, well robustness and high sensitivity with the LOD and LOQ was 1.34-1.56 ng/mL and 4.01-4.69 ng/mL, respectively. The genotoxicity of these impurities were evaluated by two (Q)SAR prediction methodologies, and results showed that BSKZ135 and BSKZ029 exhibited no genotoxicity, while BSKZ155, BSKZ117 and BSKZ160 possessed potent mutagenicity risk. Furthermore, the possible action-mechanisms of which were analyzed and proposed. This study has instructive significance to the analysis and genotoxicity research of genotoxic impurities, and also provides technical support for the supervision of the drug administration.

A novel LC-QTOF-MS/MS method for trace level identification and quantification of potential genotoxic impurity, 5-Nitro-2-(propylthio)pyrimidine-4,6-diol in ticagrelor drug substance

An important objective of the present work is to develop and validate a novel LC-QTOF-MS/MS method to identify and quantify 5-Nitro-2-(propylthio) pyrimidine-4,6-diol genotoxic impurity at trace level (∼8.33 ppm) in ticagrelor drug substance, is an antiplatelet medicine used in the treatment of heart attacks and strokes. The potential genotoxic impurities must be controlled below acceptance limit during process of ticagrelor synthesis and release. In reversed-phase mode, the ACQUITY UPLC CSH C18 (2.1 mm x 100 mm, 1.7 μm) with ambient column temperature was used. 0.1% formic acid in Milli-Q water and methanol in the ratio of 30:70 v/v used as mobile phase A and mobile phase B with isocratic elution mode and the run time was 8.0 min. The flow rate was maintained at 0.25 mL/min, autosampler temperature was 5°C, Injection volume was 20 μL. The Retention time of 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was found at 1.345 minutes. Limit of detection (LOD) and limit of quantification (LOQ) were found at 0.56 ppm and 1.67 ppm, respectively. The 5-Nitro-2-(propylthio)pyrimidine-4,6-diol was linear (R2=0.9986) over the concentration range 1.666 to 12.495 ppm. The method was accurate and precise, it was demonstrated by calculating the %mean recovery values ranging from 98.0-106.9%.

Assessment and Control of Organic Impurities in Medicinal Products: A Review

INTRODUCTION. The determination of impurities is a key requirement for the quality assessment of medicines because impurities can significantly impact the quality and therapeutic effectiveness. Pharmacopoeias are the most important scientific and methodological guidelines for manufacturers developing medicinal product specifications and regulators assessing these specifications as part of registration dossiers. Therefore, it is essential to harmonise national and international approaches to impurities.AIM. This study aimed to analyse and summarise pharmacopoeial requirements for and methodological approaches to the control, evaluation, and identification of organic impurities in medicines.DISCUSSION. The authors compared requirements for the control of organic impurities in small-molecule medicines set forth in national and international pharmacopoeias and guidelines of the International Council for Harmonisation of Technical Requirements for Medicinal Products for Medical Use (ICH) and the Eurasian Economic Union (EAEU). This comparison highlighted the differences in current approaches that require further harmonisation of the existing regulatory documentation. Additionally, this study analysed the popularity, advantages, and disadvantages of different options for determining impurities in two-component combination products (i.e. identified and/or unidentified impurities in one or each of the active substances). The analysis demonstrated the need to control nitrosamines and genotoxic impurities and to use selective and highly sensitive chromatographic methods.CONCLUSIONS. When drafting pharmacopoeial monographs for medicinal products, experts should consider the general approach set forth in the EAEU pharmacopoeia and regulations and in the ICH Q3B guideline. This approach recommends that process-related impurities of active substances should not be controlled at the medicinal product level. Therefore, pharmacopoeial monographs for active substances should distinguish degradation products from process-related impurities. Impurities should be determined for each active substance to ensure the quality and safety of fixed combination medicinal products. Priorities for improving the methodological approach to the control of organic impurities include using reference standards for impurities and acknowledging the necessity of impurity quantification.

Determination of genotoxic impurities of aromatic aldehydes in pharmaceutical preparations by high performance liquid chromatography after derivatization with N-Cyclohexyl-4-hydrazino-1,8-naphthalenediimide

The detection of aromatic aldehydes, considered potential genotoxic impurities, holds significant importance during drug development and production. Current analytical methods necessitate complex pre-treatment processes and exhibit insufficient specificity and sensitivity. This study presents the utilization of naphthalenediimide as a pre-column derivatisation reagent to detect aromatic aldehyde impurities in pharmaceuticals via high-performance liquid chromatography (HPLC). We screened a series of derivatisation reagents through density functional theory (DFT) and investigated the phenomenon of photoinduced electron transfer (PET) for both the derivatisation reagents and the resulting products. Optimal experimental conditions for derivatisation were achieved at 40 °C for 60 min. This approach has been successfully applied to detect residual aromatic aldehyde genotoxic impurities in various pharmaceutical preparations, including 4-Nitrobenzaldehyde, 2-Nitrobenzaldehyde, 1,4-Benzodioxane-6-aldehyde, and 5-Hydroxymethylfurfural. The pre-column derivatisation method significantly enhanced detection sensitivity and reduced the limit of detection (LOD), which ranged from 0.002 to 0.008 μg/ml for the analytes, with relative standard deviations < 3 %. The correlation coefficient (R2) >0.998 demonstrated high quality. In chloramphenicol eye drops, the concentration of 4-Nitrobenzaldehyde was measured to be 8.6 µg/mL below the specified concentration, with recoveries ranging from 90.0 % to 119.2 %. In comparison to existing methods, our work simplifies the pretreatment process, enhances the sensitivity and specificity of the analysis, and offers comprehensive insights into impurity detection in pharmaceutical preparations.

Does Price Really Matter for Generic Alternatives? Supervised learning Approaches in Deciding the Right Price for Acceptable Quality Attributes of Amlodipine Besylate among Generic Alternatives.

The price and safety of finished pharmaceutical preparations are two major concerns while prescribing medicine. In this work, machine learning-based classification models were developed with respect to the quality attributes of 258 samples covering 9 marketed amlodipine (AMLO) formulations. The quantitation of AMLO and its three sulfonate ester genotoxic impurities of besylate counter ion was settled using a validated high-performance liquid chromatography-diode-array detection method. The classification of correlation between dependent and independent variables was exercised using linear discriminant analysis models. The linear dispersion of acceptable quality attributes was significantly different for AMLO besylate formulation with unit price per tablet "<1 Rs." Although the correlations between price and quality are well-understood associations group centroid distance for price group "2-3 Rs." and "1-2 Rs." reveal that acceptable quality dispersion was similar for both groups. Nonetheless, a higher price could allow storage of the finished formulation to be kept on the shelf for a longer period.

TANDEM MASS SPECTROMETRIC METHOD FOR THE TRACE LEVEL DETERMINATION OF 2-AMINOPYRIDINE: A POTENTIAL GENOTOXIC IMPURITY IN TENOXICAM API

Objective: This study aimed to develop a highly sensitive method for the determination of the genotoxic impurity 2-amino pyridine in Tenoxicam, employing hyphenated techniques. Methods: The determination of 2-amino pyridine was carried out using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in Selected Ion Monitoring mode (SIM). A LiChrospher RP-18 (100×4.6 mm) 5.0 µm column was utilized for the separation. A gradient elution technique was employed with acetonitrile (mobile phase A) and 0.01M ammonium acetate buffer (mobile phase B) in varying ratios. The gradient program (T/%B) was set as 0/5, 2.50/15, 5.00/30, 10.00/50, 15.00/95, 20.00/95. The developed method was validated according to the International Conference on Harmonization guidelines. Results: The limits of detection (LOD) and quantification (LOQ) for 2-amino pyridine were found to be 0.09 ppm and 0.3 ppm, respectively. The method demonstrated accuracy within the range of 89.1% to 106.6% for the analyte. The method's linearity was confirmed through a six-point calibration graph spanning 6 ppm to 75 ppm, corresponding to a concentration of 20 mg/ml of Tenoxicam. Conclusion: Developed hyphenated LC-MS/MS method presented in this study offers a highly sensitive and accurate means for the determination of the genotoxic impurity 2-amino pyridine in Tenoxicam. With validated LOD and LOQ values, as well as demonstrated accuracy, this method proves to be a robust quality control tool suitable for the quantitation of 2-amino pyridine at very low concentrations in the pharmaceutical compound Tenoxicam.

Trace Level Quantification of Genotoxic Impurities through Atmospheric Pressure Chemical Ionization (APCI) Coupled with Triple Quardrapole Analyzer in Combination Diabetic Drug Product

N-Nitrosamine impurities have been found in several drug products, showing a concern for regulatory aspects. The study presents the development and validation of a sensitive LC-MS/MS method for detecting eight nitrosamines in vildagliptin and metformin drug products. The developed method was specific and linearity was ranged 3.53-55.92 ppb for NDMA, 3.18-50.37 ppb for NMBA, 0.97-15.41 ppb for NDEA, 0.98-15.52 ppb for NEIPA, 1.00-15.86 ppb for NDIPA, 0.98-15.46 ppb for NDBA, 0.98-15.58 ppb for NMPA and 0.7-15.32 ppb for NDPA with correlation coefficient (r) was more than 0.98 and square of correlation coefficient (r2) was found to be greater than 0.96. The LOQ were obtained in the range of 0.97-3.53 ppb with %RSD in the range of 2.5-10.9% for eight nitrosamines showed good sensitivity. Accuracy was found in the range of 98.63 ± 5.77-125.17 ± 3.68% at LOQ level, 74.77 ± 0.83-88.17 ± 0.15% at 50% level, 79.93 ± 8.47-89.00 ± 0.61% at 100% level and 74.67 ± 2.31-84.03 ± 0.93% at 150% level for the eight nitrosamine impurities that were well within acceptance criteria of not less than 70% and not more than 130%. The method validation results demonstrated that the method is precise, accurate and linear, can be applied to quantify the nitrosamines in vildagliptin and metformin drug products.

Deep eutectic solvents as green and sustainable diluents in headspace gas chromatography for the determination of trace level genotoxic impurities in pharmaceuticals

Genotoxic impurities (GTIs) are potential carcinogens that need to be controlled down to ppm or lower concentration levels in pharmaceuticals under strict regulations. The static headspace gas chromatography (HS-GC) coupled with electron capture detection (ECD) is an effective approach to monitor halogenated and nitroaromatic genotoxins. Deep eutectic solvents (DESs) possess tunable physico-chemical properties and low vapor pressure for HS-GC methods. In this study, zwitterionic and non-ionic DESs have been used for the first time to develop and validate a sensitive analytical method for the analysis of 24 genotoxins at sub-ppm concentrations. Compared to non-ionic diluents, zwitterionic DESs produced exceptional analytical performance and the betaine : 7 (1,4- butane diol) DES outperformed the betaine : 5 (1,4-butane diol) DES. Limits of detection (LOD) down to the 5-ppb concentration level were achieved in DESs. Wide linear ranges spanning over 5 orders of magnitude (0.005–100 µg g−1) were obtained for most analytes with exceptional sensitivities and high precision. The method accuracy and precision were validated using 3 commercially available drug substances and excellent recoveries were obtained. This study broadens the applicability of HS-GC in the determination of less volatile GTIs by establishing DESs as viable diluent substitutes for organic solvents in routine pharmaceutical analysis.

Analyzing Alkyl Bromide Genotoxic Impurities in Febuxostat Based on Static Headspace Sampling and GC-ECD.

Herein, a sensitive and selective gas chromatography-electron capture detector (GC-ECD) method was developed and validated for the quantification of trace levels of five bromo-containing genotoxic impurities in Febuxostat active pharmaceutical ingredient (API) after headspace sampling (HS). Multivariate experimental designs for the optimization of static headspace parameters were conducted in two stages using fractional factorial design (FFD) and central composite design (CCD). The optimum headspace conditions were 5 min of extraction time and a 120 °C extraction temperature. Baseline separation on the analytes against halogenated solvents was carried out using an Agilent DB-624 (30 m × 0.32 mm I.D., 1.8 μm film thickness) stationary phase under isothermal conditions. The method was validated according to ICH guidelines in terms of specificity, linearity, the limits of detection and quantification, precision and accuracy. The linearity was assessed in the range of 5-150% with respect to the specification limit. The achieved LOD and LOQ values ranged between 0.003 and 0.009 and 0.01 and 0.03 μg mL-1, respectively. The accuracy of the method (expressed as relative recovery) was in the range of 81.5-118.2%, while the precision (repeatability, inter-day) was less than 9.9% in all cases. The validated analytical protocol has been successfully applied to the determination of the impurities in various Febuxostat API batch samples.

A Rapid, Selective and Sensitive Electrospray Ionization Assisted LC-MS Method for Ranolazine and Identification of Its Two Potential Genotoxic Impurities

A Rapid, Selective and Sensitive Electrospray Ionization Assisted LC-MS Method for Ranolazine and Identification of Its Two Potential Genotoxic Impurities

DNA adductomics aided rapid screening of genotoxic impurities using nucleosides and 3D bioprinted human liver organoids

Current genotoxicity assessment methods are mainly employed to verify the genotoxic safety of drugs, but do not allow for rapid screening of specific genotoxic impurities (GTIs). In this study, a new approach for the recognition of GTIs has been proposed. It is to expose the complex samples to an in vitro nucleoside incubation model, and then draw complete DNA adduct profiles to infer the structures of potential genotoxic impurities (PGIs). Subsequently, the genotoxicity is confirmed in human by 3D bioprinted human liver organoids. To verify the feasibility of the approach, lansoprazole chloride compound (Lanchlor), a PGI during the synthesis of lansoprazole, was selected as the model drug. After confirming genotoxicity by Comet assay, it was exposed to different models to map and compare the DNA adduct profiles by LC-MS/MS. The results showed Lanchlor could generate diverse DNA adducts, revealing firstly its genotoxicity at molecular mechanism of action. Furthermore, the largest variety and content of DNA adducts were observed in the nucleoside incubation model, while the human liver organoids exhibited similar results with rats. The results showed that the combination of DNA adductomics and 3D bioprinted organoids were useful for the rapid screening of GTIs.

Development and Validation of Simple and Stable LCMS Method for the Quantification of Potential Genotoxic Impurities in Ozenoxacin Pure Drug and its Commercial Preparations

Ozenoxacin is an antibiotic drug prescribed to treat various skin infections caused by various bacteria. Various chemical mechanisms such as stille coupling, Buchwald–Hartwig coupling, cyclization and saponification are involved during the process of synthesis of Ozenoxacin. In the process of synthesis, there is a possibility of the formation of related impurities and among them, some are genotoxic impurities. To date, in literature, there is no method reported for analysing Potential Genotoxic Impurities (PGIs) in Ozenoxacin and hence this study was initiated to develop an LCMS method for quantification of two genotoxic impurities of Ozenoxacin viz., nitroso impurity, ester impurity. The analytes were resolved on Alltima C18 column (150×4.6mm; 5 μm particle size) using 0.01 mM ammonium acetate at pH 4.8 and methanol in 80:20 (v/v) at 0.5 mL/min flow rate and 10 μ sample injection volume. The multiple reaction monitoring of the mass fragments confirms the parent ion at m/z of 364, 393 and 496 for Ozenoxacin, Nitroso and Ester impurity respectively with characteristic product ion at m/z 196. The method has a linearity range of 0.05 μg/mL to 1.0 μg/mL for three analytes with detection limits of 0.015, 0.011 and 0.015 μg/mL for Ester impurity, Ozenoxacin and Nitroso impurity respectively. The method was validated and produces acceptable results and can successfully separate the potential genotoxic impurities in spiked commercial samples. Based on the findings, it was concluded that this method can be practically useful for the identification and quantification of potential genotoxic impurities and may apply to the safe use of Ozenoxacin in clinical treatment.

In silico Toxicity Assessment and Quantification of Potential Genotoxic Impurity in Olmesartan Medoxomil Drug Substance using GC-MS/MS

This study was designed to assess the in silico toxicity of 4-chloromethyl-5-methyl-1,3-dioxol-2-one (4-CMMD) in olmesartan medoxomil (OLM) using sophisticated advanced analytical GC-MS/MS method. The developed GC-MS/MS method is more sensitive as well as selective, for trace level analysis of genotoxic impurity 4-chloromethyl-5-methyl-1,3-dioxol-2-one (4-CMMD) in olmesartan medoxomil (OLM). In silico genotoxicity of 4-CMMD have been confimed by ICH M7 guidelines and tested to be “POSITIVE” in both knowledge and statistical based approaches. The conditions of gas chromatographic separation and mass spectrometry were optimized on stationary phase DB-35MS, helium carrier gas as at a flow rate of 1.5 mL/min. Quantification was performed in multiple reaction monitoring (MRM) mode. The absence of interference at the retention time of 4-CMMD indicates that the newly developed approach is very specific and selective for accurately measuring trace levels of impurities. Additionally, this method provided linear results which were validated by linearity solutions, with concentrations ranging from 3.74 to 45.12 ppm and an observed coefficient of regression of 0.9981. Sensitivity results shows this method is more sensitive detection limit (DL) achieved at 1.23 ppm and quantification limit (QL) achieved at 3.74 ppm. The developed method is precise and accurate according to the precision results, which show RSD values &lt; 10% and recovery &gt; 90%, both of which are within acceptable standards. The solution stability of the samples was assessed at both room temperature and refrigerated settings, and it was found to be stable for a period of 48 h. As a result, this method has been employed for the intented purpose of quantifying 4-CMMD at the trace levels in testing laboratories, pharmaceutical analytical laboratories, and quality control laboratories.

Identification, trace level quantification, and in silico assessment of potential genotoxic impurity in Famotidine

Potential genotoxic impurities in medications are an increasing concern in the pharmaceutical industry and regulatory bodies because of the risk of human carcinogenesis. To prevent the emergence of these impurities, it is crucial to carefully examine not only the final product but also the intermediates and key starting material (KSM) used in drug synthesis. During the related substances analysis of KSM of Famotidine, an unknown impurity in the range of 0.5–1.0% was found prompting the need for isolation and characterization due to the possibility of its to infiltrate into the final product. In this study, the impurity was isolated and characterized as 5-(2-chloroethyl)-3,3-dimethyl-3,4-dihydro-2H-1,2,4,6-thiatriazine 1,1-dioxide using multiple instrumental analysis, uncovering a structural alert that raises concern. Considering the potential impact of impurity on human health, an in silico genotoxicity assessment was established using Derek and Sarah tool in accordance with ICH M7 guideline. Furthermore, molecular docking and molecular dynamics simulation were performed to evaluate the specific interaction of the impurity with DNA. The findings reveal consistent interaction of the impurity with the dG-rich region of the DNA duplex and binding at the minor groove. Both in silico prediction and molecular dynamic study confirmed the genotoxic character of the impurity. The newly discovered impurity in famotidine has not been reported previously, and there is currently no analytical method available for its identification and control. A highly sensitive HPLC-UV method was developed and validated in accordance with ICH requirements, enabling quantification of the impurity at trace level in famotidine ensuring its safe release.

Analytical determination of ethylenediamine impurity in tripelennamine hydrochloride by gas chromatography-mass spectrometry using phthalaldehyde as the derivatizing agent.

In the pharmaceutical industry, effective risk management and control strategies for potential genotoxic impurities are of paramount importance. The current study utilized GC-MS to evaluate a precise, linear, and accurate analytical method for quantifying ethylenediamine present in tripelennamine hydrochloride using phthalaldehyde as a derivatizing agent. When phthalaldehyde is sonicated for 10 min at room temperature, it reacts with ethylenediamine to form (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine. This approach minimizes matrix interference issues and resolves sample preparation difficulties encountered during ethylenediamine identification in GC-MS. In this method, helium serves as the carrier gas, while methanol acts as the diluent. The stationary phase consists of a DB-5MS column (30 m × 0.25 mm × 0.25 μm) with a flow rate of 1.5 mL/min. The retention time of (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine was determined to be 6.215 min. The method validation demonstrated limits of detection and quantification for (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine at 0.4 and 1.0 ppm, respectively, with a linearity range spanning from 1 to 30 ppm concentration with respect to the specification level. System suitability, precision, linearity, and accuracy of the current method were assessed in accordance with guidelines, yielding results deemed suitable for the intended use.