AQbD-guided development of a robust and green SI-RP-HPLC method for impurity profiling of an efonidipine hydrochloride ethanolate and chlorthalidone combination, with LC-MS and NMR-based identification of degradation product in efonidipine.
Aug 2026· Journal of Chromatography A· Vol 1785, pp.
467321
· 0 citations· 27 references
Medicine
TL;DR
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.
Abstract
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.
An AQbD-based approach was employed to develop and validate a reliable, stability-indicating RP-HPLC method for quantifying sotagliflozin in API and tablet dosage forms. Sotagliflozin, a dual SGLT-1/2 inhibitor used in diabetes management and cardiovascular risk reduction, requires a sensitive and robust analytical method for quality control and stability assessment. Critical method parameters were screened using a Taguchi design and optimized through Box-Behnken Design (BBD). Chromatographic separation was achieved on an Agilent Zorbax SB-C18 column using an eco-friendly mobile phase comprising ethanol and 0.1% TFA (pH 2.25) in a 38:62% v/v ratio at a flow rate of 1 mL/min, yielding excellent system performance. The method was validated according to ICH guidelines, demonstrating good linearity (25-150 μg/mL), precision, accuracy, robustness, and sensitivity with low LOD and LOQ values. Forced degradation studies confirmed its stability-indicating capability, with notable degradation under acidic (11.03%), alkaline (14.13%), oxidative (12.39%), and thermal (14.49%) conditions, while minimal degradation occurred under hydrolytic and photolytic stress. LC-MS/MS enabled identification and structural characterization of four major degradation products. Greenness assessment using AGREE and MoGAPI tools yielded scores of 0.89 and 91, respectively, confirming the method's eco-friendly nature for routine pharmaceutical analysis.
Ankita Gupta, Akash Sharma, G. Devi et al.· Biomedical chromotography· 0 citations
Rigorous impurity monitoring of fixed-dose combination inhalation products containing fluticasone furoate, vilanterol, and glycopyrronium is essential for ensuring therapeutic efficacy and regulatory compliance. This study describes the development and validation of a stability-indicating reverse-phase liquid chromatographic (RP-HPLC) method designed for the simultaneous quantification of multiple degradation products. Separation was optimized using a BakerBond C18 stationary phase (5 µm, 250 × 4.6 mm) at 45 °C. The gradient elution utilized a potassium dihydrogen phosphate buffer (pH 2.2) and a binary organic phase of acetonitrile and methanol (75: 25, v/v) at a flow rate of 1.3 mL/min. To ensure detection sensitivity, UV wavelength switching was employed, monitoring at 210 nm for vilanterol and glycopyrronium derivatives and 248 nm for fluticasone-related species. Relative response factors (RRFs) were determined for thirteen characterized impurities, facilitating precise quantification in the absence of individual reference standards. Validation per ICH Q2(R2) guidelines confirmed the method’s selectivity, linearity (r2 > 0.998), and accuracy, with recoveries ranging from 80 to 120%. Precision studies yielded %RSD values between 6.02 and 13.76%, while stability testing confirmed sample integrity over 48 h. This robust analytical framework is highly suitable for routine quality control and impurity profiling of complex pulmonary fixed-dose combination formulations.
S. V. Kulkarni, Sharad P. Panchgalle, S. D. Deosarkar et al.· Discover Chemistry· 1 citation
Istradefylline, a selective adenosine A2A receptor antagonist used as an add-on therapy to levodopa therapy for Parkinson’s disease, requires reliable impurity profiling to ensure pharmaceutical quality and safety. In this study, a novel, stability-indicating, Quality by Design (QbD)-based and eco-friendly RP-HPLC method was developed and validated for the determination of Istradefylline and its related impurities in tablet dosage forms. Method robustness was systematically evaluated using a three-level, two-factor factorial design. Chromatographic separation was achieved on an XBridge C18 column (250 × 4.6 mm, 5 µm) using 0.01M ammonium formate buffer (pH 4.0) and acetonitrile at a flow rate of 0.8mL min⁻¹, with detection at 280 and 360nm. The method demonstrated excellent specificity, effectively separating Istradefylline from its impurities (IST-4, IST-5, IST-CIS, IST-10, and IST-40) without interference from excipients and degradation products. Linearity was established over the concentration range of 0.200–4.000µgmL⁻¹ (R² > 0.999), while accuracy, precision, and sensitivity met ICH acceptance criteria. Forced degradation studies confirmed the stability-indicating capability of the method. The method also complies with Green Analytical Chemistry principles through reduced solvent consumption and efficient chromatographic performance. Validation results demonstrated that the method is robust, reliable, and suitable for routine quality control, impurity profiling, and stability assessment of Istradefylline tablet formulations.
V. Bypaneni, Padmakar Gandla, Murugesan Palanivelu et al.· Saudi Journal of Medical and...· 0 citations
Diabetes mellitus is a long-term metabolic disorder that often requires combination therapy to achieve effective blood glucose control. In the present study, a simple, economical, eco-friendly, and stability-indicating RP-HPLC method was developed and validated for the simultaneous estimation of Lobeglitazone Sulphate and Glimepiride in combined tablet dosage forms. The chromatographic separation was carried out using an Inertsil C18 column with a mobile phase consisting of methanol and phosphate buffer (pH 4.5) in a 65:35 (v/v) ratio at a flow rate of 0.8 mL/min. Detection was performed at 230 nm, and the drugs were eluted at retention times of approximately 2.04 minutes and 7.45 minutes, respectively. The method was validated according to ICH Q2(R2) guidelines and showed excellent linearity, accuracy, precision, specificity, robustness, and sensitivity, with correlation coefficients of 0.999 and %RSD values below 2%. Forced degradation studies under different stress conditions confirmed the stability-indicating capability of the method. In addition, the method followed green analytical chemistry principles and achieved an AGREE score of 0.78, indicating good environmental sustainability. Therefore, the developed RP-HPLC method is reliable and suitable for routine quality control and stability analysis of these antidiabetic drugs in pharmaceutical formulations.
A quality by design (QbD) approach can potentially lead to a more robust/rugged method development due to
emphasis on the risk assessment and management. Aim of present investigation was to develop a new simple,
rapid and sensitive RP-HPLC method by employing Box- Behnken design (BBD) approach for determination of
telmisartan and azelnidipine in prepared tablets. Box-Behnken design (BBD) was employed to optimize the
chromatographic conditions for HPLC method development, taking mobile phase composition (% methanol),
flow rate and λmax as independent variables and retention time and tailing factor as the measured responses.
The mobile phase composition was methanol: water (80:20 v/v) on a Cosmosil C18 (250mm x 4.6 ID, Particle
size: 5 µm) column. In isocratic mode, it had a flow rate 0.9 mL/min and eluted analyte was detected at 256 nm.
Analysis of variance (ANOVA) confirmed that the three factors were significant. Validation parameters followed
the International Council for Harmonization (ICH) guidelines for the new HPLC method. Forced degradation
(acid, base, oxidation, thermal and photolytic) studies were also performed according to ICH guidelines. The
developed method was validated as per recommended ICH guidelines which revealed the high degree of linear,
precise, accurate, sensitive and robust method over the existing RP-HPLC method. The developed BBD-based
method helped in generating a design space and operating space with knowledge of all method performance
characteristics, and RP-HPLC method takes less time and can be used in routine for quality control of bulk and
marketed formulation of TELM and AZEL.
Kolte Manisha, S. Wadher, Priyanka Wankhede· International Journal of Dru...· 0 citations
The US FDA-approved oral finasteride and topical minoxidil for alopecia have adverse effects and fewer therapeutic outcomes, highlighting the need for alternative topical delivery approaches. This study aimed to develop and validate a robust and sustainable reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of cyclosporin A (CsA) and luteolin (Lu) in combined drug solutions and two compositionally distinct nanoformulations, namely nanosized emulsions and nanocapsules. The novelty of the study lies in integrating AQbD-assisted simultaneous CsA and Lu quantification with its application to two compositionally distinct nanoformulations, and a comprehensive assessment of analytical greenness and blueness. The AQbD approach was employed to systematically optimise chromatographic conditions. The optimised method employed a Phenomenex Luna C18 column (250 × 4 mm), acetonitrile-0.1% formic acid in water (pH 2.9), 206 nm wavelength, a flow rate of 1.2 ml/min, and a column temperature of 59 °C. CsA and Lu exhibited retention times of 9.14 and 6.04 min, respectively. The method demonstrated excellent linearity (R2 = 0.9994 and 0.9996 for CsA and Lu, respectively), precision (%RSD <2%), and robustness, and was validated in accordance with ICH Q2(R2). Drug content and entrapment efficiency ranged from 69.80 to 86.17% and 57.66 to72.38%, respectively. Regardless of the two compositionally distinct nanoformulations, the Lu release was higher than the CsA release (57.42 ± 5.98% vs 12.24 ± 1.40%). Analytical sustainability was supported by an Eco-Scale score of 84, AGREE score of 0.63, GAPI assessment, and BAGI score of 75.
Ankita Das, A. Sree, A. Chabru et al.· Journal of chromatography. B...· 0 citations
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