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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. [ABSTRACT FROM AUTHOR]

The objective of the present research work was to demonstrate the application of liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) for the separation, identification, and characterization of degradation products (DPs) of the drug lobeglitazone. The drug was subjected to various stress conditions such as oxidation, hydrolysis, thermal, and photolytic degradation as per the guidelines of the International Conference on Harmonization Q1A (R2). The drug was stable under thermal stress conditions, whereas it was susceptible to degradation in the other stress conditions forming four DPs. DPs were separated using a high‐performance LC system equipped with Zorbax SB C18 column (250 × 4.6 mm, 5 µm particles) using isocratic elution mode. The DPs were identified and characterized using MS and MS/MS. The chemical structure and fragmentation pattern were predicted for each degradant from the data obtained by mass studies. The drug and identified DPs were assessed by in‐silico approaches for predicting absorption, distribution, metabolism, and toxicity behavior. The in‐silico tools used for prediction were the pkCSM web server, ToxTree, and OSIRIS property explorer. [ABSTRACT FROM AUTHOR]

Indacaterol, glycopyrronium, and mometasone furoate triple combination inhalable fixed-dose medicines are effectively used to treat asthma and various chronic pulmonary disorders. The study aimed to develop and validate a simple single-run RP-HPLC impurity quantitation method. The chromatographic separation was accomplished using gradient elution mode of mobile phase A (potassium dihydrogen phosphate buffer pH 2.2) and mobile phase B (mixture of acetonitrile and methanol), with a flow rate of 0.8 mL/min using YMC Triart, C18 (250 × 4.6 mm, 5 µm) HPLC column at 45 °C and the detection wavelength of 210 nm (for indacaterol, glycopyrronium and their impurities) and 248 nm (for mometasone furoate and its impurities). Water and methanol (20:80) were used as a diluent. Quantitation of 10 known and several unknown impurities was successfully performed with the determination of relative response factors for all the known impurities. The developed method was validated as per the ICH Q2(R1) guidelines. The stability indicating the nature of the method was proved by performing stress study on the sample and placebo. The linearity and range of the method were proved by calculating the r2 values (> 0.998). The overall precision was found to be within 1.82 − 7.76% RSD. The recovery for all the actives and known impurities were within 90 − 115% with 0.4 − 12% RSD. The sample solution was stable for 2 days at room temperature. The developed method can be successfully used for the impurity analysis of routine, stability, and commercial samples in a quality control laboratory of the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

The topical lotion of clobetasol propionate was approved for the treatment of psoriasis. Commercially, it is available in the various dosage forms like ointment, cream, foam gel, solution, in various quantities ranges from 0.05 %-0.5 % w/w. The drug is reported in all official pharmacopoeias with their 13 related substances. For the purpose of estimating clobetasol propionate in bulk and formulations using the reversed-phase high performance liquid chromatography technology, an accurate and reliable method was devised. Agilent 1260 Infinity II type high performance liquid chromatography with diode array detector and Phenomenex Luna-C18 column, measuring 250×4.6 mm, 5 µm was utilized in the method. Ammonium acetate buffer, acetonitrile and methanol (60:20:20) made up the mobile phase A combination; acetonitrile and ammonium acetate buffer (20:80) made up the mobile phase B combination. The optimum peak was obtained at retention duration of 15.73 min by maintaining a flow rate of 1.0 ml/min at a wavelength of 240 nm throughout. The percentage error for the instrument, method and intermediate precision was 0.01 %, 0.01 % and 0.02 % respectively. For both method and intermediate precision, the total percentage relative standard deviation was 0.02 %. The procedure was linear and accurate with correlation coefficient of 0.9997 for concentration ranges of 0.05-120 µg/ml with accuracy levels of 0.08 %, 0.02 % and 0.03 % for relative standard deviation of 80 %, 100 % and 120 %. When the drug's stress stability was examined, it was discovered to be unstable under basic conditions, degrading at 99.34 % and under heat conditions, degrading at 14.40 %. Since the established approach is relatively linear and the limits of detection and quantification for clobetasol propionate are very low at 0.93 µg/ml and 2.81 µg/ml respectively, it may be employed in a commercial setting. Retentions were verified and impurities were found for injecting the reference standard. Impurities A, B, J, L and M were found and their retention times were 7.91, 9.28, 18.75, 21.35 and 27.07 min. It was discovered that the approach was accurate and robust. [ABSTRACT FROM AUTHOR]

Ganirelix, a peptide‐based drug used to treat female infertility, has been in high market demand, which attracted generic formulation. A hitherto unknown impurity of ganirelix was observed in our formulation process, which reached ~0.3% in 6 months and led to a detailed investigation of its structure. In‐depth analysis of ESI‐MS/MS data of this impurity coupled with an artificial intelligence prediction tool led to a highly unusual putative structure, that is, N‐(2‐carboxyethyl)‐ganirelix (NCE‐GA), which was authenticated by chemical synthesis from ganirelix and NMR analysis and via corroborated HPLC and MS/MS data with the formulation‐derived impurity. [ABSTRACT FROM AUTHOR]

The present research aimed to comprehensively investigate the forced degradation of tepotinib through a combined in‐silico and experimental analysis. Tepotinib is a recently approved drug for metastatic non‐small cell lung cancer. A short and precise ultra‐high‐performance liquid chromatography (UHPLC) method utilizing a CSH C18 column (100 × 2.1 mm, 1.7 μm) was developed, using a mobile phase A as 0.1% formic acid and mobile phase B as HPLC‐grade acetonitrile. The flow rate was set at 0.2 mL/min, and detection was carried out at 260 nm. Forced degradation was carried out under acidic, basic, oxidative, and photolytic environments and also virtually with Zeneth software. Structural characterization of degradation products (DPs) was carried out using hyphenated techniques such as LC‐quadrupole time‐of‐flight‐tandem mass spectrometry in positive electrospray ionization mode. Further, toxicity was assessed with Derek and Sarah software. Analysis revealed the formation of five distinct forced DPs: DP‐1 (m/z 511), DP‐2 (m/z 527), DP‐3 (m/z 525), DP‐4 (m/z 512), and DP‐5 (m/z 509) under experimental conditions and eight in Zeneth prediction. Furthermore, nephrotoxicity alerts were observed for DP‐3 and DP‐5, whereas DP‐3 also showed mutagenicity alerts. This integrated blend of in‐silico and real experimental analyses was found to be effective in quality control for routing monitoring of degradation products of tepotinib. [ABSTRACT FROM AUTHOR]

Background: The goal of the proposed study was to develop and validate stability indicating mass compatible reverse phase UPLC method for impurity profiling of Darunavir Ethanolate and Ritonavir degradation impurities in fixed-dose drug combination products. Materials and Methods: The optimized chromatographic condition includes use of Zorbax Bonus C18 column (150x2.1 mm, 1.8 µm) with mobile phase A (Buffer (55): Methanol (45)) and mobile phase B (Acetonitrile: (30) and Methanol (70)), flow rate of 0.22 mL/min and detection at 240 nm with gradient program of 50 min. The method was validated as per ICH quality guideline Q2 (R1) including specificity by forced degradation study to confirm suitability for intended use. Results: The observed retention time for Darunavir was 11.4 min and for Ritonavir was 30.0 min. The Limit of Quantitation (LOQ) for all degradation impurities found is 0.05%, equivalent to the reporting threshold level. The method was found linear and accurate in the range of LOQ to 150% with an observed range of % accuracy of 90.1 to 106.3 for all known impurities. The method was found precise based on less than 10.0% RSD and robust for deliberate changes. Considering the use of a volatile mobile phase, the method can be applied for LC-MS-based analysis for mass identification. Conclusion: The developed UPLC method was applied for impurity profiling during the release and stability study of Darunavir Ethanolate and Ritonavir in fixed-dose drug combination products. [ABSTRACT FROM AUTHOR]

Propranolol hydrochloride (PPH) is a medication of beta‐adrenergic receptors. It is used to treat pediatric hemangiomas that are growing and need systemic therapy. A reversed‐phase high‐performance liquid chromatography (HPLC) was made to separate and estimate the amounts of ten known organic impurities of propranolol in bulk drugs, tablets, and capsule dosage forms. The chromatographic separation was achieved on a C18 column (100 × 4.6 mm, 2.7 μm) using a binary gradient mixture of pH 2.3 phosphate buffer and organic modifiers of acetonitrile, methanol, and isopropyl alcohol as the mobile phase. The stability‐indicating character of the proposed method was proven using stress testing study. The test method was validated for specificity, limit of detection, limit of quantitation, linearity, precision, accuracy, and robustness. For the propranolol and its ten organic impurities, the limit of quantitation, linearity, and recoveries were found in a range of 0.0123–0.4266 μg/mL (R2 > 0.9982) and 89.2–98.9%, respectively. The proposed liquid chromatography method is found to be highly suitable for impurity profiling of propranolol in bulk drugs and pharmaceutical formulations (tablets and capsules). [ABSTRACT FROM AUTHOR]

Omeprazole (OME) is a proton pump inhibitor used to treat gastroesophageal reflux disease associated conditions. The current study presents an Analytical Quality by Design-based approach for the development of a CE method for OME impurity profiling. The scouting experiments suggested the selection of solvent modified Micellar ElectroKinetic Chromatography operative mode using a pseudostationary phase composed of sodium dodecyl sulfate (SDS) micelles and n -butanol as organic modifier in borate buffer. A symmetric three-level screening matrix 37//16 was used to evaluate the effect of Critical Method Parameters, including Background Electrolyte composition and instrumental settings, on Critical Method Attributes (critical resolution values, OME peak width and analysis time). The analytical procedure was optimized using Response Surface Methodology through a Central Composite Orthogonal Design. Risk of failure maps made it possible to define the Method Operable Design Region, within which the following optimized conditions were selected: 72 mM borate buffer pH 10.0, 96 mM SDS, 1.45 %v/v n -butanol, capillary temperature 21 °C, applied voltage 25 kV. The method was validated according to ICH guidelines and robustness was evaluated using a Plackett-Burman design. The developed procedure enables the simultaneous determination of OME and seven related impurities, and has been successfully applied to the analysis of pharmaceutical formulations. [Display omitted] • A CE method for the analysis of Omeprazole and its impurities was set up. • Analytical Quality by Design principles were employed to develop the procedure. • The knowledge space was explored through a 37//16 symmetric screening matrix. • In Response Surface Methodology a Central Composite Orthogonal Design was used. • Probability maps allowed the Method Operable Design Region to be defined. [ABSTRACT FROM AUTHOR]

This article described the development and validation of a method for spiramycin related substances based on hybrid particle column. The chromatographic conditions were as follows: water - 0.2 mol/L dipotassium hydrogen phosphate (the pH value adjusted to 9.5 using a 1 mol/L KOH solution) - acetonitrile - methanol (10: 60: 28.5: 1.5, v/v/v/v) as mobile phase A, water - 0.2 mol/L dipotassium hydrogen phosphate (pH 9.5) - acetonitrile - methanol (10: 30: 57: 3, v/v/v/v) as mobile phase B and gradient elution was performed. Compared with previous analytical methods, this method has strong specificity, excellent sensitivity and stability, which could be used for the daily testing of related substances of spiramycin. Furthermore, impurities above 0.1 % were characterized using two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (2D LC-QTOF-MS/MS) and there were 6 impurities reported for the first time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Technical advances in the field of quality analysis allow an increasingly deeper look into the impurity profile of drugs. The ability to detect unexpected impurities in addition to known impurities ensures the supply of high-quality drugs and can prevent recalls due to the detection of harmful unexpected impurities, as has happened recently with the N -nitrosamine and azido impurities in losartan (LOS) drug products. In the present study, the LC-MS/HRMS approach described by Backer et al. was applied to an even more complex system, being the investigation of 35 LOS drug products and combination preparations purchased in 2018 and 2022 in German pharmacies. The film-coated tablets were analysed by means of four LC-MS/HRMS method variants. For the separation a Zorbax RR StableBond C18 column (3.0 ×100 mm, particle size of 3.5 µm, pore size of 80 Å), a gradient elution and for mass spectrometric detection a qTOF mass spectrometer with electrospray ionization in positive and negative mode was used. An information-dependent acquisition method was applied for the acquisition of high-resolution mass spectrometry data. The combination of an untargeted and a targeted screening approach revealed the finding of eight impurities in total. Beside the five LOS related compounds, LOS impurity F, J, K, L, M, and related compound D from amlodipine besilate, LOS azide and an unknown derivative thereof were detected. Identification and structure elucidation, respectively, were successfully performed using in silico fragmentation. Differences in the impurity profiles of drug products from 2018 and 2022 could be observed. This study shows that broad screening approaches like this are applicable to the analysis of drug products and can be an important enhancement of the quality assurance of medicinal products. [Display omitted] • Analysis of losartan finished dosage forms by means of HRMS. • Structural elucidation by means of MS and MS/MS data. • Verification of proposed structural formulae by in silico fragmentation. • Comparison of the impurity profiles of losartan tablets purchased in 2018 and 2022. • Development of a procedure which provides a broad look into the impurity profile of drugs. [ABSTRACT FROM AUTHOR]

[Display omitted] • An Advanced, environmentally friendly stability indicating analytical method for Pretomanid analysis integrating Quality by Design (QbD) and Green Chemistry Principles. • The regulatory focus on quality in drug development emphasised for the drug stability in different circumstances and PMD found highly sensitive towards hydrolysis, oxidation, and photolytic conditions. • The eco-friendly method developed for PMD degradation products in stress studies unveils vital insights into its stability profile, contributing to the development of robust pharmaceutical formulations. • The developed method's eco-friendliness is confirmed through complex GAPI and AGREE assessments, aligning with the increasing focus on green chemistry and highlighting its broader impact on environmentally conscious drug development. Regulatory agencies prioritize the safety and effectiveness of pharmaceuticals. The focus here is to highlight the crucial need for control and oversight of Pretomanid (PMD), a synthetic nitroimidazooxazine utilized in treating multidrug-resistant tuberculosis. This study aims to develop a simple, sensitive, LCMS compatible, and environmentally friendly HPLC Stability Indicating Analytical Method following ICH Q1A (R2) and Q3 guidelines. The approach involves the application of Quality by Design (QbD) principles and adherence to green chemistry practices. A comprehensive risk assessment was conducted using cause-and-effect diagram and risk assessment tool. The QbD approach includes screening and optimization phases. Plackett-Burman design was performed for screening selected 11 primary parameters. Central composite design was used for optimisation of selected critical variables based on critical method parameters (CMPs) and Critical method attributes (CMAs). The method was optimised using Contour plot, 3D plot, ANOVA, Desirability value and Design space. Superior component separation was achieved through an isocratic elution using Kromasil C18 (250 × 4.6 mm; 5 μm) column, mobile phase composition of (50:50; v/v) ACN: 10 mM Ammonium Acetate pH 4.5 adjusted with Glacial Acetic acid and a flow rate of 0.9 mL/min having runtime of 30 mins. The stress studies revealed that the drug is highly sensitive to hydrolysis, oxidation, and photolytic conditions. Experimentation results demonstrated that acetonitrile served as the most suitable diluent. Additionally, the correlation coefficient (r2) exceeded 0.996, while the RSD values (n = 6) varied from 0.76 % to 1.93 % across the LOQ–150 % range. Specificity studies revealed no interference between peaks from impurities and known active analytes. Through stress studies, major degradation products were identified. The method's eco-friendliness was assessed using ComplexGAPI and AGREE, confirming its environmentally friendly nature. [ABSTRACT FROM AUTHOR]

• Effects of ion-pairing agent and counter ion on peak capacity were investigated. • The system configuration was fine-tuned to enhance resolving power. • ACN in IP-RPLC provides better chromatographic resolution than MeOH. • Column comparison was conducted at the kinetic performance limit (1000 bar). • Sub-minute separation and high-resolution analysis with coupled columns are demonstrated. A generic performance comparison strategy has been developed to evaluate the impact of mobile-phase additives (ion-pairing agent / counter ion systems), distinct stationary phases on resulting resolving power, and MS detectability of oligonucleotides and their critical impurities in gradient IP-RPLC. Stationary-phase considerations included particle type (core-shell vs. fully porous particles), particle diameter, and pore size. Separations were carried out at 60°C to optimize mass transfer (C-term). The incorporation of an active column preheater mitigated thermal mismatches, leading to narrower peaks and overcoming peak splitting. Acetonitrile as organic modifier outweighed methanol in terms of peak-capacity generation and yielded a 30% lower back pressure. Performance screening experiments were conducted varying ion-pairing agents and counter ions, while adjusting gradient span achieved an equivalent effective retention window. Hexafluoromethylisopropanol yielded superior chromatographic resolution, whereas hexafluoroisopropanol yielded significantly higher MS detection sensitivity. The 1.7 µm core-shell particle columns with 100 Å pores provided maximum resolving power for small (15–35 mers) oligonucleotides. Sub-min analysis for 15–35 polyT ladders was achieved operating a 50 mm long column at the kinetic performance limits. High-resolution separations between a 21-mer modified RNA sequence oligonucleotides and its related (shortmer and phosphodiester) impurities and complementary strand were obtained using a coupled column set-up with a total length of 450 mm. [ABSTRACT FROM AUTHOR]

[Display omitted] • A triple-action cramp relief over-the-counter pharmaceutical formulation of paracetamol, caffeine and pyrilamine maleate. • Novel eco-friendly HPTLC-densitometry and HPLC-UV methods for mixture analysis and purity assessment. • Successful applicability in assaying and quality control of the market tablets. • Dissolution profiling of Midol® Complete caplets as an in-vivo efficiency indicator. • Affirming methods sustainability via implementing various greenness and whiteness assessment tools. Over the years, several single-pill combinations have been produced and utilized as an alternative option for treatment of the signs and symptoms of Primary dysmenorrhea. The presence of multiple drugs together poses a challenge for analysts regarding analysis, making it a significant hurdle to overcome. A combination of paracetamol (PAR), caffeine (CAF), and pyrilamine maleate (PYR) is commonly recommended as an over-the-counter triple-action treatment to alleviate mild to moderate premenstrual syndrome symptoms. This study presented two liquid chromatographic methods that simultaneously determine PAR, CAF, and PYR in Midol® Complete caplets along with three officially recognized impurities, namely, p-aminophenol, theophylline, and p-anisaldehyde. The initial approach employed high-performance thin-layer chromatography alongside a densitometric scanning technique. The experimental setup utilized silica gel HPTLC plates as the stationary phase, and ethyl acetate: aqueous ammonium hydroxide (10.0:0.1, v/v) mixture as the developing system. The densitometric scanning was conducted at a wavelength of 210.0 nm. The second approach, developing high-performance liquid chromatographic method coupled with ultraviolet detection. Inertsil ODS-3 C 18 column (250 × 4.6 mm, 5 μm) was used to separate the six compounds effectively. For the experimental procedure, an isocratic elution method was chosen with a mobile phase consisting of a mixture of 0.02 M potassium dihydrogen phosphate buffer acidified with o -phosphoric acid (pH 3.0), and methanol (80:20, v/v) at a flow rate of 1.0 mL/min. UV detection was carried out at a wavelength of 210.0 nm. The two proposed methods have been validated in accordance with ICH recommendations. In addition, the suggested HPLC-UV technique was used to track the release profile of the cited drugs during the in-vitro study. The developed methods were assessed and compared for their environmental impact using various greenness evaluation tools. Additionally, the recently introduced RGB12 model was used to study the whiteness features of the suggested methods. The proposed methods yielded desirable outcomes and are sustainable, simple, affordable, and low-cost, which highly recommends their use in quality control labs. [ABSTRACT FROM AUTHOR]

Aclidinium bromide (ACL) is a long-acting muscarinic receptor antagonist used for the long-term treatment of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate the degradation of aclidinium bromide under stress and stability testing conditions, for which we developed and validated the first stability-indicating, specific, precise, accurate, and robust assay and related substances HPLC methods. Nine of the compounds used as reference standards were synthesized and fully characterized by 1H and 13C NMR, MS, and FTIR techniques. Two of these molecules, namely ACL-dimer and ACL-desphenyl, are novel compounds and reported herein for the first time. Hydrolysis of aclidinium resulted in major degradation via the formation of ACL-desDTG and DTGA metabolites. ACL-desphenyl and phenol were observed only under oxidative conditions at very low levels (< 0.10%), while ACL-hydroxy, known as a metabolite of aclidinium and confirmed by LC/QDa and LC/Q-TOF m/z data, formed under oxidative stress-testing conditions, UV light, and daylight. The identification of two impurities formed only when aclidinium bromide was treated with hydrogen peroxide was done by LC/QDa and LC/Q-TOF studies and the novel structures were proposed as ACL-bromophenoxy and ACL-bromothiophenyl formed via bromination of the phenyl and thiophene ring on the aclidinium, respectively. The stress and photostability testing studies showed that the aclidinium bromide drug substance is not sensitive to elevated temperature (105 °C, 10 days), slightly sensitive to daylight and UV-radiation, and it showed significant degradation under all hydrolysis and oxidizing conditions. The related substances HPLC method reported herein is also capable to monitor the potential genotoxic impurity 3-bromopropoxybenzene (3-BPB), a class 2 impurity according to ICH M7, and ensures that the impurity remains below the threshold of toxicological concern (TTC) limit, making it safe for patients. [Display omitted] • Stability-indicating related substances & assay HPLC methods for aclidinium bromide. • Evaluation, synthesis, and characterization of nine compounds by NMR, MS, and FTIR. • Degradation of aclidinium bromide studied under stress test conditions. • Identification of degradation impurities by LC-MS & discussion of formation pathways. • Four new compounds described for the first time in the literature. [ABSTRACT FROM AUTHOR]

• 2D-LC separation of oligonucleotides using IPRP separations in both dimensions. • Elution conditions are systematically discovered through iterative retention modeling. • 2D-LC separations provide both the selectivity and sensitivity needed for impurity profiling. • Application to therapeutic siRNA single strands from solid phase oligonucleotide synthesis. Oligonucleotides constitute an emerging and highly complex bioanalytical challenge and it is becoming increasingly clear that 1D methodologies are unable to fully resolve all possible impurities present in these samples. 2D-LC therefore constitutes a perfect solution wherein critical pairs can be sampled from a steep gradient 1D and separated in a shallower 2D gradient. Herein, we provide a facile 2D-LC method development approach to quickly generate high selectivity gradients utilizing ion pairing reverse phase (IPRP-IPRP). In particular we demonstrate how to iteratively generate a 12 % gradient from two training runs and then to utilize that data to predict retentions of analytes with a 2 % gradient with retention prediction errors as low as 3 and 11 %, respectively. This iterative method development workflow was applied to impurity profiling down to 1:1000 for the full-length product and phosphorothioate modified impurities. Additionally, we demonstrated the elucidation of critical pairs in complex crude pharmaceutical oligonucleotide samples by applying tailored high selectivity gradients in the second dimension. It was found that the iterative retention modeling approach allows fast and facile 2D-LC method development for complex oligonucleotide separations. [ABSTRACT FROM AUTHOR]