HSPiP, QbD, and Zeneth based green analysis and stability study of valsartan in the presence of mutagenic impurities: an in-silico study for predicting drug safety and quality control
Jul 2026· Journal of Analytical Science and Technology· Vol 17· 0 citations· 33 references
TL;DR
The newly developed HSPiP, QbD, and Zeneth enabled stability-indicating UFLC method was environmentally sustainable, sensitive, accurate, reproducible, simple, rapid, economic, and reliable to detect even the trace degradants of VAL.
Abstract
The stability of valsartan (Val) and its products (the presence of genotoxic impurities, such as N-nitrosodimethylamine as NDMA and N-nitrosodiethylamine as NDEA) is very crucial as it directly impacts safety and efficacy. Before, its product was recalled by various pharma industries due to the presence of the genotoxic impurities. It is always challenging to estimate the impurity profiling of the impurities during its shelf-life using a conventional methodology. The study addressed the HSPiP and QbD (quality by design)-driven the optimized mobile phase to improve system suitability, reliability, and sensitivity. The conventional analytical methods are less sensitive and selective in accelerated stress study for detecting the degradants and impurities. Therefore, the study addressed the combined effort of HSPiP, QbD, and in-silico tools for predicting the degradation profiling, optimizing the method, and assessing toxicity to circumvent the above limitations. Moreover, the AGREEprep and AGREE tools assessed the greenness of the method. HSPiP screened the right combination of solvents based on Hansen parameters. Zeneth evaluated the impact of the impurities on the stability. Furthermore, in-silico toxicity assessment predicted degradants to evaluate valsartan safety. The analytical method was optimized with high desirability (~ 0.98). For each stress condition, the degradants with their pathways, were predicted by employing the Zeneth software. Then, the stress study evaluated the precision of the degradants. The number of acidic, basic, oxidative, and photolysis degradants were found as 4, 3, 2, and 1, respectively, with the optimized method. The newly developed HSPiP, QbD, and Zeneth enabled stability-indicating UFLC method was environmentally sustainable, sensitive, accurate, reproducible, simple, rapid, economic, and reliable to detect even the trace degradants of VAL. x
The detection of nitrosamine-related impurities in pharmaceutical products has emerged as a critical issue for regulatory agencies owing to their potential mutagenic and carcinogenic risks. N-nitrosodibutylamine (NDBA), one of the identified nitrosamine contaminants, is regarded as likely to be carcinogenic to humans and may pose a cancer-related hazard, can be generated during drug synthesis, processing, or storage conditions. Therefore, the identification and quantification of such trace-level impurities are essential to ensure drug safety and compliance with regulatory requirements. The present study aimed to develop and validate a sensitive, selective, and reliable Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) method for the determination of NDBA impurity in Simvastatin and Ezetimibe tablet formulations. Separation was performed on a Zorbax SB-Phenyl column (150 × 4.6mm, 3.5µm) using a gradient system of 0.1% formic acid in water and 0.1% formic acid in methanol, with a flow rate of 0.5mL/min. Detection was achieved using a triple quadrupole mass spectrometer fitted with an APCI source operating in positive ion mode, employing multiple reaction monitoring (MRM). The selected MRM transition for quantification of NDBA was m/z 159 → 103. The proposed analytical method was validated in compliance with International Council for Harmonisation recommendations by evaluating parameters such as specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). The developed LC–MS/MS method was found to be sensitive, accurate, and suitable for routine quality control analysis and regulatory monitoring of NDBA impurity in Simvastatin and Ezetimibe tablet formulations.
Kalpesh Upadhyay, J. Mehetre, Tushar Mehta et al.· Asian Journal of Pharmaceuti...· 0 citations
The literature does not have any sustainable high-performance thin-layer chromatographic (HPTLC) methods for concurrently identifying tenofovir (TEN) and emtricitabine (ECT). Therefore, the proposed study develops and verifies a sustainable reverse-phase HPTLC methodology for determining TEN and ECT concurrently in their fixed-dose combination (FDC) products. The wavelength at which TEN and ECT were simultaneously identified was 255 nm. A 70:30 v/v binary mixture of ethanol and water served as the green development system. The method was validated for accuracy, precision, robustness, sensitivity, and specificity according to ICH guidelines. The validated method was applied for the simultaneous determination of TEN and ECT in commercial FDC tablets. The method's greenness, blueness, and whiteness profiles were evaluated using eight different tools: "the analytical eco-scale (AES), chloroform toxicity (ChlorTox), analytical GREEnness (AGREE), modified green analytical procedure index (MoGAPI), complex MoGAPI, blue applicability grade index (BAGI), carbon footprint reduction index (CaFRI), and click analytical chemistry index (CACI)". For both medications, the devised technique was linear in the range of 25-1000 ng/band. In addition, the created approach was proven to be accurate (% recoveries = 99.12-100.70 for TEN and 100.57-101.83 for ECT), precise (% RSD = 0.85-0.99 for TEN and 0.88-0.97 for ECT), sensitive (LOD = 8.50 ng/band for TEN and 8.39 ng/band for ECT, LOQ = 25.51 ng/band for TEN and 25.19 ng/band for ECT), and robust (% RSD = 0.82-0.86 for TEN and 0.94-0.99 for ECT). Using the current method, the amount of TEN in commercial FDC tablet brands A and B was 98.58 ± 1.21% and 101.13 ± 1.30%, respectively. The amount of ECT in FDC brands A and B was 99.82 ± 1.29 and 100.64 ± 1.37%, respectively. The findings of all the greenness, blueness, and whitening tools, such as AES (93), ChlorTox (0.72 g), AGREE (0.77), MoGAPI (85), complex MoGAPI (90), BAGI (85), CaFRI (89), and CACI (87), demonstrated that the current method had the notably sustainable profiles. The greenness parameters of present method were better than reported HPTLC approaches. The findings of the study suggested that the recommended method may be applied to precisely evaluate TEN and ECT in commercial formulations.
P. Alam, Faiyaz Shakeel, Mohammed H. Alqarni et al.· BMC Chemistry· 0 citations
Introduction / Objective: The selectivity, potency, and stability of antiviral compounds, including Ritonavir (RTV),
require high-precision analytical testing and impurity profiling for confirmation. As a widely used pharmacokinetic
enhancer in COVID-19 therapy, the regulation and clinic validation of impurities and their toxicity are of the utmost
importance. The main goal of the present work was to develop and confirm a simple, accurate, and stability, indicating
HPLC method for determining the Ritonavir content and its related impurity (Impurity, A Free Base) includes theoretical
and experimental toxicity assessments. Methods: Chromatographic separation was performed on a Phenomenex C18
column (250 4.6 mm, 5 m). The mobile phase was 20:80 of water and acetonitrile, flow rate 1.0 mL/min. The method
validation tested different things using ICH Q2(R1) rules, Accuracy, Precision, Specificity, and Linearity. The toxicity
assessment for Impurity A was done by the insilico prediction tools (PROTOX 3.0) and checked with an in vitro study of
toxicity in PBMC cells. Results: The method demonstrated (R = 0.9999) in a range of 1.64 to 29.94 g/mL, with recovery
rates from 92% to 99% and a relative standard deviation below 2%. Ritonavir and its impurity- A with distinct retention
times is 4.3 min and 2.3 min respectively. In silico studies stated that the drug has a medium acute oral toxicity as low
with a LD50 values of more than 2000 mg/kg with mild hepatotoxic and neurotoxic while MTT results showed an IC50
value >500 µg/mL, suggesting low cytotoxicity. Discussion: The validated HPLC method is a reliable, accurate, and
economical way for the routine analysis of Ritonavir and its contaminants. The use of computational and in vitro toxicity
assessments used to identify impurity hazards and comply with quality control standards of antiviral drugs. Conclusion:
A consistent, precise, and cost-effective HPLC method was successfully developed and validated for Ritonavir and its
related impurity. The integration of in silico and in vitro toxicity studies strengthens impurity risk assessment and supports
the safe and effective quality control of antiviral pharmaceutical products.
Balaji Ramakrishnan, J. S, Esakkimuthukumar Mariappan et al.· International Journal of Dru...· 0 citations
For many years, it is known as Nitrosamines are responsible for carcinogenicity in human. These nitroso-impurities were found detected in plastics, food items, cigarette, insect killer, water and alcoholic beverages. In 2018 EMA and USFDA claimed the presence of N-nitrosodimethylamine in valsartan used for the treatment of hypertension. Consequently, to assess the adverse effects of nitrosamine generation during storage, in the course of production, or through contaminated supply chains, regulators have issued many guidelines. Hence, there is a crucial need for accurate evaluation technique to measure nitroso-impurities in pharmaceutical products. According to the Carcinogenic Potency Categorization Approach, the allowable intake is 400 ng/day, which translates to an allowable limit of 16 ppm for a 25 mg daily dose. A study was then performed at 1.5 ppm relative to sample concentration, which is less than 10% of this limit. The main aim of this development to optimize and prove suitability of a new liquid chromatography-tandem mass spectrometry method for quantitatively analyzing 3-amino-N-nitrosopiperidine (NTPA) in Alogliptin Benzoate (AGP), achieving detectable response of 0.03 ppm and quantifiable response value of 0.1 ppm relative to sample concentration. Current method was found to be linear with the coefficient of regression of 0.9979. Also, spiking study was performed by the calculating percentage spiked NTPA in the drug, which ranged from 96.67% to 100.14%. The results confirmed that the methodology was reliable, precise, robust and reproducible to quantify NTPA at 1.5 ppm in samples of 2.75 mg/mL concentration.
Mithun M Gharat, Pallavi T. Roy, Amit N Gosar et al.· Biomedical chromotography· 0 citations
Efonidipine Hydrochloride Ethanolate (EHE) and Chlorthalidone (CTD) are co-formulated for the management of hypertension owing to their complementary pharmacological actions. Ensuring accurate impurity profiling in such combination formulations is vital for maintaining drug stability, safety and regulatory compliance. However, conventional analytical techniques often face challenges related to sustainability and robustness. Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) remains a key analytical approach for impurity determination, yet the integration of Quality by Design (QbD) principles into its development is still evolving. The present study focuses on developing a novel, sustainable RP-HPLC method for impurity profiling of EHE and CTD tablets using a QbD approach and green chemistry. Chromatographic separation was achieved on a Zorbax SB C8 column (150 mm × 4.6 mm, 5 µm). Critical method parameters such as mobile phase composition and pH were systematically optimized through QbD to enhance method robustness. A photolytic degradation product of EHE was identified as Efonidipine Related Compound A, and a plausible photolytic degradation pathway was proposed. A photolytic degradation product was identified in the LC-MS and its structure was confirmed through NMR spectroscopy. The developed green method exhibited excellent resolution, precision and reproducibility across multiple batches, allowing for effective separation and quantification of impurities. This green QbD-optimized, environmentally sustainable RP-HPLC method provides a reliable and scalable solution for impurity profiling in complex pharmaceutical formulations, aligning with modern quality control and regulatory expectations.
Pranav Bang, V. Choudhari, V. Gharge· Journal of Chromatography A· 0 citations
Background: The rise of pathogenic fungi capable of infecting people is a major public health problem. Flucytosine is an effective antifungal agent, but its clinical use is constrained by rapid clearance and dose-related adverse effects. Nanoemulsion-based systems offer a promising strategy for drug delivery and therapeutic performance. The objective of this work was to develop and optimize a flucytosine-loaded nanoemulsion using a Quality by Design (QbD) approach based on the Box-Behnken design, and to evaluate its physicochemical characteristics. Methodology: A Box-Behnken design (BBD) was used to optimize formulation factors, including lipid concentration, Smix (surfactant-to-cosurfactant) ratio, and homogenization time, using high-speed homogenization. The impacts on particle size, polydispersity index, and entrapment efficiency (EE) were investigated. UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) were used to assess the compatibility and stability of the improved formulation (F11) with the chosen excipients. Results and Discussion: The optimized formulation (F11) had a particle size of 318.18 nm and a PDI of 0.136, a zeta potential of -22.2 mV, and high entrapment efficiency (96.36%), indicating good stability and uniformity. Preformulation and compatibility studies confirmed the absence of drug-excipient interactions. Statistical analysis demonstrated that formulation variables significantly influenced critical quality attributes. Conclusion: The study demonstrates that QbD-driven optimization can successfully develop a stable and efficient flucytosine nanoemulsion with enhanced drug-loading capacity and potential to improve antifungal therapy. However, further studies, including in vitro drug release, in vivo evaluation, long-term stability, and clinical validation, are required to confirm its performance in drug delivery applications.
Rajat Srivastava, A. Rawat· Journal of Applied Pharmaceu...· 0 citations
