Drug Metabolism and Bioanalysis - Online First
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Pharmacokinetic Study: Liquid Chromatographic Assay of Apigenin and Analogues in Rat Plasma after Oral Administration
Available online: 01 September 2025More LessIntroductionVarious methods are available for estimating apigenin, but no methods have been applied to the pharmacokinetic study of its prepared analogues, such as polymorphs, co-crystals, and complexes. This study aims to develop a method for the pharmacokinetic assessment of apigenin and its analogues using HPLC-UV.
MethodsThe internal standardization approach was utilized to analyse apigenin and its derivatives using UV detection at 340 nm, with chromatographic separation achieved under isocratic conditions on a Phenomenex C18 column (250 mm × 4.6 mm id, 5 μm). The optimal mobile phase consisted of a mixture of 0.3% formic acid and methanol (30:70, v/v) at a 1.5 mL/min flow rate. The system demonstrated a significant and well-resolved peak for apigenin and the internal standard quercetin, with retention times of 4.2 and 2.9 minutes, respectively. The calibration curve’s linear regression analysis revealed a robust linear relationship over the concentration range of 2–10. 0 μg/mL; R2 was determined to be 0.9995.
ResultsThe findings indicated that the limit of quantification (LOQ) and limit of detection (LOD) were 0.0803μg and 0.0265μg, respectively. The pharmacokinetics in rats were evaluated using this approach. Cmax, the plasma concentration of apigenin and its analogs, was reached following an oral dose of 60 mg/kg/rat. The collected data were utilized to calculate all pharmacokinetic parameters.
DiscussionThe pharmacokinetic study revealed that apigenin analogues (A2–A7 and C1) significantly improved the oral bioavailability of apigenin (AP), as indicated by elevated Cmax and AUC0–24 values relative to AP alone. Specifically, analogues A6 and A7 reached peak plasma concentrations more swiftly, implying a quicker onset of action, whereas A3 exhibited a delayed Tmax, reflecting slower absorption.
ConclusionFollowing oral administration of a single dose of apigenin, this method was deemed suitable for quantifying the levels of apigenin and its derivatives in rats. This HPLC methodology could be an effective tool for determining apigenin analogues in plasma due to its excellent sensitivity, accuracy, linearity, and specificity.
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Assessment of Impacts of Clinically Effective Antihypertensive Herbal Medicines on the Activities of Major Drug Metabolizing Enzymes and Transport Proteins
Available online: 07 August 2025More LessIntroductionHypertension is one of the most common non-communicable diseases, and reports indicate that its prevalence is escalating globally due to several factors. Studies in different countries have shown an upsurge in the use of Herbal Medicinal Products (HMPs), including herbal antihypertensive medicines taken by people with hypertension, and 20-80% of hypertensive patients concurrently use these herbal medicines and conventional antihypertensive medicines or other drugs. The safety and efficacy of the concurrent use of herbal and orthodox medicines are of great concern because of the high possibility of herb-drug interactions.
MethodsAn extensive literature search was undertaken, and the information obtained was subjected to critical analysis. This review aimed to update the available data on HMPs with reproducible evidence-based antihypertensive efficacies. Additionally, the major phytochemical and bioactive constituents of these HMPs were identified along with a discussion of their potential to modulate activities of drug-metabolizing enzymes and drug transport systems, especially P-glycoprotein.
ResultsMore than 50 commonly used medicinal plants from different regions of the world have been documented for their anti-hypertensive activity. Most of these studies used animal models to authenticate the antihypertensive activities of the herbs. In contrast, a few studies on extracts of Hibiscus sabdariffa, Allium sativum, Apium graveolens, Nigella sativa, Linum usitatissimum, and Camellia sinensis involved clinical trials. Potentials for herb-drug interactions varied among the clinically effective HMPs due to the wide variability in their phytochemical constituents.
ConclusionSafety issues in using these HMPs were highlighted by the identification of beneficial or adverse, clinically significant herb-drug interactions.
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Study on the Forced Degradation Behaviour of Oteseconazole and Characterization of Its Degradants by LC-MS/MS
Authors: Meera Devi Mazhavancheril and K. G. BahetiAvailable online: 31 July 2025More LessIntroductionOteseconazole is a novel tetrazole antifungal agent used to treat Recurrent Vulvovaginal Candidiasis in women. Its mechanism of action is through the inhibition of cytochrome P450(CYP)51, thereby affecting the development and integrity of the fungal cell membrane. The main objective of the present work is to investigate the forced degradation behaviour of Oteseconazole, identify its degradants, and propose the fragmentation pathways of the degradants.
MethodsGood chromatographic separation of Oteseconazole was achieved using an HPLC System equipped with a PDA detector, and an X-Bridge Phenyl column (150 x 4.6mm, 5µm) with a mobile phase comprising acetonitrile and trifluoroacetic acid buffer (50:50) run in isocratic mode at 268 nm. For mass analysis, the HPLC system was connected to a SCIEX QTRAP 5500 mass spectrometer, operated in positive ion electrospray ionization interface mode.
ResultsAn accurate, specific, and time-efficient stability-indicating RP-HPLC method was developed for the estimation of Oteseconazole and its degradation products. The developed method was validated with Linearity in a range of 1.25µg/ mL – 7.5 µg/mL, accuracy (%RSD 0.06), system precision (%RSD 0.256), method precision (% RSD 0.49), LOD(0.3µg/mL), and LOQ(1µg/mL). Upon forced degradation studies according to ICH guidelines, Oteseconazole was found to be stable in photolytic, hydrolytic, and thermal conditions but degraded readily in acidic, alkaline, peroxide, and reduction environments.
DiscussionThe developed method was robust and can be used in routine analysis to quantify Oteseconazole. The degradation behaviour of Oteseconazole was studied by performing forced degradation studies according to the ICH guidelines, and it was found to be stable in photolytic, hydrolytic, and thermal conditions, but degraded more readily in acidic, alkaline, peroxide, and reductive environments. The degradation products were characterized by LC-MS/MS, and their fragmentation pathways were proposed.
ConclusionOteseconazole remained stable in photolytic, hydrolytic, and thermal conditions, but showed significant degradation in acidic, alkaline, peroxide, and reductive environments. The degradation products were characterized by LC-MS/MS and characterized as 5-(4-chlorophenyl)-2-(2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)propyl)pyridine (acid impurity, DP1), sodium 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)propyl)pyridin-3-yl)phenolate (alkali impurity, DP2), 2-(2-(2,4-difluorophenyl)-1,1-difluroropropyl)-5-(4-hydroperoxyphenyl)pyridine (peroxide impurity, DP3) and 4-(6-2(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-tetrazol-1-yl)propyl)pyridin-3-yl)phenyl hydrogen sulfate (reduction impurity, DP4), and their fragmentation pathways were proposed. The developed method was accurate, specific, less time-consuming, and could be used in routine analysis for the quantification of Oteseconazole.
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Green Ecofriendly RP-HPLC Method for Estimating Bifonazole in Transethosomes: Application of Box Behnken Design
Authors: Umeshkumar Patel, Vinayak Mastiholimath and Pallavi ChiprikarAvailable online: 31 July 2025More LessIntroductionThe antifungal drug bifonazole, a substituted imidazole, exhibits broad-spectrum in vitro action against Gram-positive bacteria, yeasts, moulds, dermatophytes, and dimorphic fungi.
MethodsThe analysis was carried out using a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm particle size) maintained at a constant temperature of 25°C. Methanol and 0.1% trifluoroacetic acid were utilized as the mobile phase. Detection was performed at 256 nm with a UV detector, and quantification was achieved using the standard method. The method validation was carried out for various parameters as per ICH guidelines. The developed eco-friendly HPLC method was employed to assess the vesicle size and entrapment efficiency of the transethosomes. The greenness assessment tool was applied to assess the environmental sustainability of the proposed HPLC method.
ResultsThe retention time was under 5 minutes, confirming the method's appropriateness for routine analysis. Linearity was observed in the concentration range of 5 to 25µg/ml, LOD and LOD were 2µg/ml and 8µg/ml respectively. Intraday and interday precision were within ICH limits (<2). The method was successfully used to analyse vesicle size (178nm) and entrapment efficiency (77.6±0.72%.) of the nanoformulation. The greenness assessment tool yielded a value of 0.71.
DiscussionThe developed method is both analytically sound and eco-friendly, making a significant improvement in the estimation of bifonazole. The developed method's LOD and LOQ values were sufficiently low to facilitate the detection and measurement of bifonazole in pharmaceutical formulations frequently utilized in research applications, thereby supporting its potential utility in analytical and research settings.
ConclusionOverall, this green, eco-friendly HPLC method is well-suited for routine analysis of bifonazole, demonstrating excellent cost-effectiveness, sensitivity, precision, and robustness.
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