Hydrogen sulfide (H₂S) is a gasotransmitter involved in the regulation of skin inflammation, making topical H₂S donors promising candidates for its management. Among these, GYY4137 has attracted considerable interest because of its slow H₂S-release profile. However, the lack of validated analytical methods for its direct quantification in biological matrices remains a significant limitation to study its delivery, pharmacological efficacy, and develop new treatment modalities. This study describes the development and validation of a high-performance liquid chromatography (HPLC) method for the quantification of GYY4137 in different skin layers. Porcine ear skin was used as a model for human tissue, and GYY4137 was extracted from the stratum corneum and viable epidermis/dermis using tape stripping, tissue homogenization, and sonication in water:methanol (1:1, v/v), providing consistent recovery (∼80%). Chromatographic separation was achieved using a C18 column, a mobile phase consisting of acetonitrile:phosphate buffer 10 mM (15:85, v/v) and detection at 240 nm. The method demonstrated adequate selectivity, linearity (0.5-10 μg/mL, R2 > 0.995), precision, and accuracy (<15%) across all matrices. The lower limit of quantification was 0.5 μg/mL. GYY4137 remained stable under sample preparation and storage at -20 °C, whereas partial degradation occurred at 4 °C (∼30% after one month). The method was successfully applied to in vitro skin penetration studies using Franz diffusion cells. GYY4137 delivery was time- and formulation-dependent, with propylene glycol and oleic acid enhancing skin penetration compared to aqueous solutions. Overall, the proposed HPLC method with UV detection provides a reliable and accessible analytical tool for routine quantification of GYY4137 and supports the development and optimization of H₂S-based topical therapies for inflammatory skin disorders.
It is crucial to determine equilibrium and kinetics of drug-receptor interactions for understanding thoroughly drug mechanism and drug efficacy. In this work, three antagonist drugs toward cysteinyl leukotriene receptor 1 (CysLTR1) were exemplified to measure the binding parameters by multiple affinity chromatographic approaches like frontal chromatography, injection amount-dependent method and nonlinear chromatography. The association equilibrium constants were measured as montelukast < zafirlukast < pranlukast and the dissociation rate constants were in the rank of montelukast > zafirlukast > pranlukast, which were correlated to the inhibition potencies of montelukast < zafirlukast < pranlukast to CysLTR1. Additionally, thermodynamic analysis in combination with molecular docking indicated that the three antagonists were spontaneously bound to CysLTR1 and their bindings were mainly driven by hydrogen bonds and van der Waals forces. Our work demonstrated that affinity chromatography can be utilized to evaluate the binding characteristics of ligand-receptor based on their retention behaviors, aiding the selection of the best ligands for further studies to become a drug molecule.
Meloxicam (MLX) and tenoxicam (TNX) remain analytically important across pharmaceutical, biological, veterinary, and environmental applications, yet their reliable determination is strongly influenced by matrix complexity and drug-specific physicochemical behavior. This review moves beyond conventional method-centered summaries by critically comparing MLX and TNX across matrices and linking their physicochemical, pharmacokinetic, metabolic, and stability characteristics to analytical performance. Sample-preparation strategies, chromatographic and mass-spectrometric platforms, spectroscopic techniques, and electrochemical methods are evaluated for selectivity, sensitivity, matrix tolerance, robustness, sustainability, and practical applicability. Particular emphasis is placed on unresolved limitations in parent drug–metabolite–degradation product discrimination, matrix-effect control, method transferability, and harmonized validation. Importantly, the review integrates emerging chemometric optimization, artificial intelligence, machine learning, automated workflows, and Green and White Analytical Chemistry as converging directions for next-generation analysis. Future progress should shift from isolated improvements in detection sensitivity toward matrix-adaptive, digitally assisted, sustainability-aware analytical systems that integrate automated sample preparation, intelligent data interpretation, robust validation, and lifecycle-based performance assessment. This integrated perspective identifies priorities for transforming the determination of MLX and TNX from fragmented, matrix-specific methodologies into more transferable, intelligent, and sustainable analytical frameworks.
Pyrrolizidine alkaloids, including their free bases (PAs) and their N-oxides (PANOs), are plant-derived natural toxins, which often pose analytical challenges because they have many structurally similar isomers that are difficult to separate by conventional chromatography. To address this issue, we employed supercritical fluid chromatography-tandem mass spectrometry. A 3-hydroxyphenyl column with an ammonium formate-containing methanolic modifier gradient enabled simultaneous determination of the 40 PAs/PANOs, although some isomeric pairs were only partially resolved. We validated the developed method using nutmeg as a matrix. The calibration curves showed good linearity over the range of 1 to 100 μg/kg, with coefficients of determination of 0.997-1.000 for all PAs/PANOs. Recoveries were 84.9-101.3%, and intra- and inter-day precisions were 2.0-16.3% and 3.0-16.7%, respectively, at the spiking level of 1 μg/kg. These validation results indicate that the present method can quantify 40 PAs/PANOs in herbs and spices. We also determined PAs/PANOs in commercially available herbs and spices (oregano, savory, thyme, marjoram, cumin, dill seed, and sage) to confirm the applicability of the developed method. At least four PAs/PANOs were quantified in all samples other than dill seed. Relatively high levels of PAs/PANOs (>1,000 μg/kg) were noted in oregano and cumin. These results suggest our developed analytical method is a reliable and useful means for determining PAs/PANOs in herbs and spices.
Chiral high-performance liquid chromatography (HPLC) remains one of the most important analytical techniques for investigating the stereochemical behavior of bioactive compounds. Although many commercial chiral stationary phases are available and new selector chemistries are continually introduced, successful enantioseparation alone does not ensure analytical applicability. In pharmaceutical and biomedical practice, chromatographic methods must perform reliably with complex sample matrices, low analyte concentrations, stringent validation requirements, and increasingly frequent coupling with mass spectrometric (MS) detection. Although achieving adequate enantioseparation remains a major challenge for many structurally complex analytes, increasing attention is being devoted to developing robust, validated, and application-oriented analytical methods suitable for pharmaceutical and biomedical analysis. Instead of presenting another catalog of chiral stationary phases or reported enantioseparations, this review examines chiral HPLC from a method development perspective. It discusses how chromatographic selectivity, sample preparation, matrix effects, detection strategy, elution order assignment, stereochemical stability, and validation requirements interact throughout the analytical workflow. Special emphasis is given to the transition from initial column screening to validated methods suitable for pharmaceutical quality control, stereoselective pharmacokinetic studies, drug-protein binding investigations, forensic toxicology, and chiral metabolomics. By integrating selector chemistry with practical analytical requirements, the review provides a framework for application-driven method development and highlights current trends shaping the future of chiral HPLC in biomedical and pharmaceutical sciences.
Diabetes mellitus is rapidly increasing in India, where coexistence of hemoglobinopathies such as HbE and β-thalassemia complicates accurate HbA1c estimation due to potential analytical interferences, particularly in cation-exchange high-performance liquid chromatography (CX-HPLC)-based assays. Capillary electrophoresis (CE) has been proposed as an alternative method with improved variant resolution in such populations. In this study, we evaluated the analytical comparability of CX-HPLC and CE for HbA1c measurement in hemoglobinopathy cases from Eastern India. A retrospective analysis was conducted on 3500 patient samples screened for hemoglobinopathies at Nirnayan Health Care Private Limited, Kolkata (January 2025). Hemoglobin variant analysis was performed using the Bio-Rad Variant II system, while HbA1c estimation was carried out using CX-HPLC (Bio-Rad Variant II Turbo; HbA1c mode ∼ 1.5 min) and CE (Sebia Minicap Flex Piercing; HbA1c mode ∼ 2.5 min). Statistical analyses, including Pearson correlation, Bland-Altman analysis and box-plot visualization, were performed using GraphPad Prism 9. Of the total samples, 327 showed hemoglobinopathies, predominantly HbE trait (n = 157) and β-thalassemia carriers (n = 129), along with other variants. HbA1c values measured by CX-HPLC and CE exhibited excellent correlation (r = 0.9799, p < 0.0001) with minimal bias and clinically acceptable limits of agreement. Subgroup analyses showed strong agreement in HbE carriers (r = 0.9745) and β-thalassemia carriers (r = 0.9086) with comparable glycaemic classification across non-diabetic, pre-diabetic, and diabetic ranges, although minor classification shifts were observed near diagnostic thresholds in a small subset of cases. HbA1c measurement was unreliable in homozygous hemoglobinopathies due to the absence or marked reduction of HbA, irrespective of the analytical method. These findings demonstrate that modern CX-HPLC and CE provide analytically comparable HbA1c results even in the presence of common hemoglobin variants. Both methods may be used interchangeably for HbA1c testing in variant-rich Indian populations.
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.
Demonstration of biological drug product quality prior to first-in-human dosing is a regulatory requirement to ensure patient safety and product performance. Protein aggregates, including dimers and higher-order species, are classified as product-related impurities and must be adequately controlled. However, conventional Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC) may not provide selective aggregate quantitation in samples prepared in diluent containing human serum albumin (HSA). To overcome this limitation, we adapted and optimized a mobile affinity selection chromatography (MASC) method that incorporates a fluorescent selector in the mobile phase with specific affinity for the Fc region of antibody molecules. Following SE-HPLC separation, fluorescence detection enables elective monitoring of Fc-containing species. This strategy effectively eliminates interference from HSA, which lacks an Fc region and therefore is not fluorescently labeled. The method demonstrated good accuracy and linearity for quantification of aggregates in complex HSA-containing matrices. It is suitable for routine application in pharmacy manual qualification and in-use stability studies, providing a robust and selective solution for aggregate analysis.
Atirmociclib is a selective cyclin-dependent kinase 4 (CDK4) inhibitor currently under clinical investigation for the treatment of advanced malignancies, particularly hormone receptor positive (HR+) or human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Here, we report the first validated liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical method for the quantification of atirmociclib in mouse plasma, validated in compliance with international council for harmonisation-M10 (ICH-M10) guidelines. Sample preparation involved a rapid and straightforward protein precipitation approach using acetonitrile, yielding consistent recovery (>80%) across low to high concentration levels. Chromatographic separation was achieved on a Kinetex C18 column (50 mm length × 2.1 mm internal diameter, 5 μm particle size) using gradient elution with 5 mM ammonium acetate in water (mobile phase A) and 0.1% v/v formic acid in acetonitrile (mobile phase B). The analyte (atirmociclib) and internal standard (warfarin) were detected using Q1/Q3 (m/z) mass transitions 464.2/303.9 and 309.0/163.0 respectively. The method demonstrated excellent selectivity, accuracy, precision, and linearity over a concentration range of 1-1000 ng/mL. Additionally, reliable quantification was maintained for samples exceeding the upper limit of quantification following up to 10-fold dilution. All validation parameters, including stability in biological matrices and solution, met the predefined acceptance criteria. This sensitive, and reproducible method was successfully applied to support preclinical pharmacokinetic study of atirmociclib in mouse, providing a valuable analytical tool.
Traditional Chinese Medicine (TCM) exerts its therapeutic effects through multiple bioactive constituents acting on multiple targets, while the analysis of trace-level compounds in complex herbal matrices remains challenging for quality control. Conventional extraction procedures may require substantial amounts of volatile organic solvents and prolonged treatment, motivating the development of more efficient and miniaturized sample-preparation strategies. Ionic liquids (ILs), characterized by negligible vapor pressure and tunable physicochemical properties, have therefore attracted considerable interest as designer extraction media. This review critically summarizes recent advances in ionic-liquid-based extraction and microextraction for the analysis of bioactive constituents in TCM. Phase-based liquid-phase microextraction formats, including ionic liquid-based dispersive liquid-liquid microextraction (IL-DLLME), homogeneous liquid-liquid microextraction (IL-HLLME), aqueous biphasic systems (IL-ABS), single-drop microextraction (IL-SDME), and supported or membrane-based LPME, are distinguished from energy-assisted solid-liquid extraction strategies such as ionic liquid-based ultrasound-assisted extraction (IL-UAE) and microwave-assisted extraction (IL-MAE). Ionic-liquid-modified solid-phase and magnetic solid-phase extraction, as well as hybrid and multiphase configurations, are also discussed. Representative TCM applications are organized according to extraction methodology rather than analyte class, with emphasis on the relationships among IL structure, extraction mechanism, analyte properties, selectivity, and multi-component coverage. The coupling of IL-based extraction with chromatographic, mass-spectrometric, spectroscopic, and capillary-electrophoretic techniques is further examined. Current challenges, including viscosity, potential toxicity, IL recovery, instrumental compatibility, reproducibility, and scale-up, are critically evaluated together with future opportunities involving task-specific ILs, magnetic and polymeric IL materials, and data-driven solvent design.
Extracellular vesicles (EVs) are lipid-bound nanoparticles secreted by cells that participate in intercellular communication during physiological and pathological processes. Among EV subpopulations, exosomes (30-150 nm) are widely studied for liquid biopsy diagnostics and therapeutic delivery applications. However, existing EV isolation methods often suffer from low throughput, long processing times, and poor scalability. Hydrophobic interaction chromatography (HIC) using polyester (PET) capillary-channeled polymer (C-CP) fiber stationary phases has previously been demonstrated as a rapid (<15 min), low-cost (∼$5 per column) EV purification platform in a microbore column format. This study extends the previously reported microbore-scale platform to an analytical-scale column configuration (2.1 mm i.d. × 250 mm) evaluated across multiple fiber packing densities, which serves as an intermediate scale-up step toward preparative-scale operation. Using urine-derived EVs, dynamic binding capacities approaching ∼1012 particles per column and a 2.3-fold increase in EV capture relative to the microbore format were achieved. EV yields exceeded 65% with process throughputs of up to ∼1011 EVs min-1 alongside ∼96% reduction in co-isolated protein content. Column reproducibility (%RSD = 3.1%) and stable multi-cycle (n = 10) performance further supported platform robustness. The results demonstrate that scaling up the C-CP fiber column format while maintaining efficient transport behavior supports high-yield, high-purity EV isolation at increased throughput, advancing this platform toward practical preparative-scale purification workflows.
Semaglutide is a potent long-acting glucagon-like peptide-1 receptor agonist with outstanding therapeutic efficacy for type 2 diabetes and obesity. Because of its widespread clinical use, there is an increasing demand for analytical techniques to identify semaglutide in pharmaceutical formulations and biological matrices. The current study provides an overview of analytical methodologies for determining semaglutide across various disciplines. The material may be analysed using the following techniques: chromatography, spectroscopy, electrophoresis, and mass spectrometry. Some of these approaches include reversed-phase high-performance liquid chromatography, liquid chromatography/tandem mass spectrometry, ultraviolet-visible spectrophotometry, Fourier-transform infrared spectroscopy, Raman spectroscopy and others. The reported methodological characteristics, including validation parameters are discussed. In addition, a comparison and discussion of analytical performance, approaches, advantages, and disadvantages are presented. Chromatographic techniques are presented as the most effective, sensitive, and reliable analytical methods for semaglutide determination, but alternative approaches are also described. The current study may provide a comprehensive and informative guide for further investigations into semaglutide analysis and related research.
In this paper, the effect of hypoxia on the content of active ceramides in tissues was investigated for the first time, and the pharmacokinetic changes of ceramide C24:1 with hypoxia-protective effects were compared under both normoxic and hypoxic conditions. A UPLC-MS/MS method was developed to determine five ceramides in mouse tissues and plasma. The performance criteria for sensitivity, linearity, matrix effect, recovery, stability, precision, and accuracy were evaluated and found to be within the FDA-recommended guidelines. This method was successfully employed to quantify both endogenous and exogenous ceramides in tissues and plasma. The results revealed tissue-specific changes in endogenous ceramide levels under hypoxia and showed that hypoxia increased the systemic exposure and prolonged the retention of exogenous C24:1 while reducing its clearance. These findings support further investigation of ceramides as potential biomarkers or therapeutic targets for hypoxia-related diseases and provide important in vivo pharmacokinetic data for the development of novel neuroprotective drugs.
Asphodelus aestivus L. is a traditionally used medicinal plant with significant therapeutic and nutritional value. This study investigated the phytochemical composition, antioxidant activity, phenolic and flavonoid contents, cytotoxic effects, and nutritional profile of different organs of A. aestivus L. extracted with aqueous, methanol, and 80% ethanol. Major bioactive compounds were further evaluated using DFT analysis, molecular docking, and network pharmacology approaches, including GO-BP and KEGG pathway enrichment analyses. LC-ESI-MS/MS analysis identified chlorogenic acid, p-coumaric acid, vanillin, trans-ferulic acid, and gentisic acid as the major phenolic compounds, with chlorogenic acid being the predominant constituent across most plant organs and extracts. Leaf extracts, particularly the methanol extract, exhibited the strongest antioxidant activity in both DPPH and ABTS assays, whereas stem and flower extracts showed the weakest activities. Among all plant organs, leaf extracts showed the greatest accumulation of phenolics and flavonoids. ICP-MS analysis demonstrated a mineral-rich profile in the leaves, dominated by K, Ca, Na and Mg. Among the extracts, the aqueous leaf extract showed the highest cytotoxicity against A549 cells while exhibiting low toxicity toward healthy L929 cells. DFT analysis revealed comparable electronic properties among the major compounds, with sinapic acid exhibiting the strongest electron-donating ability. The docking analysis identified chrysin and chlorogenic acid as the compounds with the most favorable predicted interactions toward AChE and BChE, respectively; GO-BP and KEGG analyses revealed enrichment of inflammation, apoptosis, and cancer-related pathways targeted by the identified phenolic compounds. Overall, the integrated in vitro and in silico findings demonstrate that A. aestivus L. represents a valuable natural resource with significant potential for the development of functional foods, nutraceuticals, and novel therapeutic agents.
This study investigated the optimization, development, and validation of an accurate, precise, and sensitive reversed-phase high-performance liquid chromatographic technique with variable-wavelength detection (RP-HPLC-VWD) method for the rapid determination of four active ingredients (APIs) of hydroquinone (HYQ), methylparaben (MP), fluocinolone acetonide (FLN), and propylparaben (PP) in pharmaceutical dosage forms. The optimized chromatographic conditions consisted of a mobile phase of a mixture of acetonitrile and a sodium heptane-1-sulfonate buffer (pH 3.5) with a BDS C18 column (5 μm, 250 mm × 4.6 mm) and wavelength detection at 254 nm. The method revealed excellent linearity (R2 > 0.999 for all analytes), accuracy (recovery ranging from 99.0 to 101.5% across three concentration levels), high precision, with a repeatability RSD% of <0.3% for all APIs, and robustness/ruggedness confirmed within the ICH-recommended limit of <2.0% (RSD% range from 0.218% to 1.367%). To enhance process efficiency, Lean Six Sigma methodology was implemented, incorporating the process capability index (Cpk), which yielded values greater than 1.0, indicating a capable and stable process. The RP-HPLC-VWD method for HYQ/MP/FLN/PP achieves an excellent balance between superior analytical performance and practicality, while also revealing a good environmentally sound approach. The accompanying tools complement each other perfectly, with high performance of RAPI (95.0%)/ BAGI (77.5%) highlighting the strengths and acceptable satisfaction level of EPPI (EI = 76.7% and PPI = 55.0%) and Multi-color Assessment platform (MA = 61.1%) and Red-Green-Blue (72.5%) as a whiteness evaluation tool.
A sensitive and robust micro-solid phase extraction LC-tandem mass spectrometry (μSPE-LC-MS/MS) method was established for quantifying 23 per- and polyfluoroalkyl substances (PFAS) in mouse feces and liver. Method development identified interferences due to the complex fecal matrix, especially at unit-mass resolution detection, where co-eluting endogenous compounds caused transition-specific interferences. Conventional C18 stationary phases failed to adequately resolve these matrix components, particularly for perfluoroalkyl sulfonic acids (PFSAs), resulting in compromised selectivity. To reduce these matrix effects, alternative stationary phases, including Phenyl-Hexyl (Phe) and Pentafluorophenyl (PFP) columns, were systematically evaluated. The PFP column, operated with acetonitrile in the mobile phase, demonstrated superior chromatographic selectivity and effectively eliminated co-eluting interferences. Notably, transition-specific interferences previously observed for PFOS on C18 columns were resolved, allowing accurate quantification using both m/z 499 → 80 and 499 → 99 transitions. Using the improved selectivity of the PFP column, automated μSPE cleanup combined with isotope-labelled internal standards achieved high extraction efficiencies (96-104%), ensuring reliable accuracy and precision (recoveries 96-99%, RSD <5%) for most analytes despite matrix complexity. These findings demonstrate the importance of chromatographic selectivity, rigorous cleanup, and isotopic correction for quantifying PFAS in highly complex biological matrices using unit-resolution mass spectrometry. The developed workflow provides a reliable analytical platform for toxicokinetic studies and may be applicable to other challenging biological matrices.
