In this study we systematically evaluated ethanol as a green mobile phase selectivity adjusting solvent in reversed-phase liquid chromatography using Abraham's solvation parameter model. Isocratic retention factors for varied compounds on a Zorbax SB-C8 column were compared across binary mixtures of water with acetonitrile, methanol, and ethanol. Methanol and ethanol have similar solvent strength, but different selectivity driven by differences predominantly in mobile phase cohesiveness and hydrogen-bond acidity. Additionally, ethanol-water systems successfully served as surrogate models for physiochemical property estimation. A 50 % (v/v) ethanol system is suitable for estimating infinite dilution activity coefficients, while extrapolated log kw values obtained for the composition range 20 - 60 % (v/v) ethanol provided a robust correlation model for octanol-water partition constants (r[2] = 0.909).

Acupoint sticking therapy (AST) is a traditional Chinese transdermal treatment widely used for chronic conditions such as asthma and dyspepsia. Despite its clinical efficacy, the material basis of AST, particularly the penetration dynamics of its active transdermal components, remains unclear, hindering further investigation into its therapeutic mechanisms. This study developed an integrated platform combining in vivo microdialysis with GC-Orbitrap-HRMS to characterize transdermal components and penetration profiles of AST, using the Jianpi Patch (JPT) as a model due to its proven efficacy in pediatric functional dyspepsia. Firstly, 204 volatile compounds in JPT were identified, including terpenoids, hydrocarbons, esters, ketones and alcohols. Furthermore, through real-time microdialysis at the Shenque (CV8) acupoint in rats, 66 compounds were identified as transdermal constituents by GC-Orbitrap-HRMS. Based on their transdermal profiles, these penetrating components were categorized into three distinct patterns: rapid penetration (peak within 1-2 h), delayed peak (gradual increase, maximum at 3-4 h), and sustained release (progressive rise, peak at 5-6 h). This work established a minimally invasive platform for screening the material basis of transdermal herbal formulations and provided new insights into the permeation behavior of AST.

In recent years, capillary gel electrophoresis (CGE) has become a significant analytical technique for separating and analyzing biomacromolecules such as proteins, peptides, and nucleic acids, due to its high efficiency, throughput, and resolution. The gel material, which serves as the core component of CGE, directly determines the separation performance through its molecular sieving properties. This review systematically examines the gelation mechanisms, separation principles, and key applications of various gel materials used in CGE for biomacromolecule analysis. It focuses on the specific characteristics and limitations of CGE when different gel materials are employed, outlines future development trends, and aims to provide a theoretical foundation for establishing standardized CGE methodologies suitable for analyzing biomacromolecules of diverse types and molecular weights.

The efficiency of solid-phase extraction (SPE) for determining low-concentration organophosphorus pesticides and atrazine in real samples depends on the adsorbent's ability to adsorb the analyte before entering the chromatography system. MOFs can be an excellent option as adsorbents for extracting analytes because of their large specific surface area and high adsorption capacity, particularly NH2-MIL-101(Cr). The aim of this study is to enhance extraction efficiency and sensitivity in a syringe-filter solid-phase extraction (SPE) method using a novel NH2-MIL-101(Cr) composite for the extraction of several organophosphorus pesticides (OPPs) and atrazine from environmental water samples. In this study, an NH2-MIL-101(Cr)/graphene nanocomposite was synthesized for the first time and packed into a syringe filter as an adsorbent, thereby increasing the method's sensitivity. Various characterisation techniques, including fourier-transform infrared spectroscopy (FTIR), Brunauer-Emmett-Teller (BET), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and scanning electron microscopy (SEM) were employed to verify the successful production of the nanocomposite. The variables influencing the extraction efficiency of NH2-MIL-101(Cr)/Graphene nano-powder-syringe filter-SPE/GC-MS, including sample volume, pH, elution solvent volume, and elution solvent type, were optimized. The findings indicated that the Cr-MOF/GNP nanocomposite exhibited enhanced adsorption efficacy and a reduced detection limit for all nine target pesticides compared with unmodified NH2-MIL-101 (Cr). The technique demonstrated a linear response range of 1.0 to 300.0 μg/L and achieved detection limits of 0.2 to 6.0 μg/L. The results demonstrated significant reproducibility, with outstanding recovery rates ranging from 83.0% to 103.0% across increasing concentrations from 15.0 to 60.0 μg/L and relative standard deviation values ranging from 3.2% to 6.3%.

A novel amphiphilic sulfobetaine-type zwitterionic stationary phase, designated as Sil-C8-DSB, was fabricated via a facile thiol-ene click strategy by anchoring sulfobetaine-functionalized 1,4-diazabicyclo[2.2.2]octane ligands onto thiol-functionalized silica through hydrophobic octyl spacers. [1]H and [13]C NMR spectroscopy supported the proposed structures of the mono-quaternized intermediate and the C8-DSB ligand, while Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and elemental analysis supported the surface functionalization of Sil-C8-DSB. The retention behavior was investigated using polar, ionizable, and non-polar probe compounds. The effects of acetonitrile content, mobile-phase pH, salt concentration, and temperature indicated combined hydrophilic-interaction, ion-exchange, and reversed-phase retention contributions. Sil-C8-DSB resolved a four-component mixture containing neutral, basic, and acidic probes, whereas co-elution occurred on the commercial HILIC-Z and VP-ODS reference columns under the respective tested conditions. Application to a commercial Sprite sample yielded a sodium benzoate content of 140.2 mg L[-1], with recoveries of 97.05-99.14%. Same-column intra-day and inter-day retention-time RSDs were 0.21-0.52% and 0.05-0.76%, respectively, while a 30-day storage test showed no progressive loss of column efficiency. Following a 4 h stop-flow treatment in 50 mM aqueous ammonium acetate, retention times and theoretical plate numbers changed by <1%, with baseline separation maintained. These results support the mixed-mode selectivity, analytical applicability, and operational stability of Sil-C8-DSB under the evaluated conditions.

Characterizing adjuvanted vaccine formulations can be challenging due to sample polydispersity, shear sensitivity, and potential secondary interactions with column stationary phases. Asymmetric flow field-flow fractionation (AF4) coupled with multiple downstream detectors offers a low-shear, size-based separation alternative to size-exclusion chromatography (SEC) to aid in vaccine development. In the present study, an AF4 method was developed to separate a protein nanoparticle antigen and a commercial squalene-based nanoemulsion adjuvant (AddaS03™). Method optimization included cross flow gradient screening, membrane selection and conditioning, dn/dc determination for AddaS03, and recovery assessment. The antigen eluted as a monodisperse peak (Rh ∼15 nm across entire peak, Mw ∼1.8 MDa, Mw/Mn ∼1.00), while AddaS03™ was highly polydisperse (Rh ∼ 66 nm, Mw ∼738 MDa) with Rg/Rh consistent with spherical morphology (0.77-1.02). Recoveries were 71-74% for AddaS03™ and >90% for the antigen. An exploratory thermal stress study induced formation of low-molecular-weight species and aggregation of the antigen. Observed changes in fractograms for co-formulated samples were attributable to the formation of a smaller population of adjuvant-associated particles and a ∼20% decrease in apparent weight-average molar mass. The new subpopulation of adjuvant particles overlaps in size with the antigen, which limits the component-specific data interpretation in stressed co-formulated samples. The optimized AF4 method enables simultaneous separation and characterization of intact protein nanoparticle antigens and nanoemulsion adjuvants within the same formulation, supporting stability studies while offering a low-shear environment.

Fluorinated liquid crystal monomers (FLCMs), widely used in electronic display technologies, have recently attracted increasing attention as emerging environmental contaminants, while their accurate determination in complex environmental matrices remains challenging. Herein, three crystalline imine-linked covalent organic frameworks (COFs) with systematically varied fluorination degrees (0F-COF, 1F-COF, and 3F-COF) were constructed and integrated as sorbents into an online fluorous solid-phase extraction coupled with high-performance liquid chromatography-ultraviolet detection (FSPE-HPLC-UV) platform. Structure-performance correlations revealed that 1F-COF achieved superior extraction performance by balancing pore structure with fluorine-regulated interfacial interactions, thereby enabling efficient enrichment of fluorinated analytes. Under optimized conditions, enrichment factors (EFs) of 135-272 were obtained, with detection limits of 0.15-0.33 ng mL[-1] and good linearity over 0.5-100 ng mL[-1]. The 1F-COF SPE cartridge retained stable performance over 60 extraction-elution cycles. The method was validated in water and soil samples with recoveries of 84.3-108.7%. This work establishes fluorine-regulated COFs as effective sorbents for online FLCM enrichment and provides a practical HPLC-UV strategy for environmental monitoring.

Affinity chromatography hyphenated to mass spectrometry (AC-MS) has emerged as a powerful technology for the simultaneous structural and functional characterization of therapeutic antibodies. However, the reliance on pH gradients in AC raises fundamental questions regarding the accurate ranking of proteoforms, in particular for target interactions requiring low pH ranges for elution. Here, we investigated the selectivity bias of light chain asparagine 30 deamidated proteoforms (LC-N30D) of trastuzumab in target AC-MS using the immobilized extra-cellular domain of human epidermal growth factor receptor 2 (HER2). By employing a middle-up strategy and cation exchange chromatography enrichment of Fab fragments, we successfully isolated and characterized enriched proteoforms. While HER2 AC-MS identified decreased binding of heavy chain aspartic acid 102 isomerization, the enriched LC-N30 deamidated (LC-N30D) Fab proteoform exhibited an apparent increased binding compared to Fab proteoforms with low modification content. Orthogonal surface plasmon resonance kinetics revealed a critical inversion in the binding behavior due to differences in mobile phase pH. While LC-N30D substantially reduces binding at physiological pH, the acidic separation conditions required for elution in AC, stabilized the HER2 interaction. Molecular dynamics simulations attributed the HER2 AC retention behavior to a protonation-dependent electrostatic stabilization of the LC-N30D-HER2 interface at low pH, which favored the LC-N30D Fab proteoform binding. These findings highlight a major limitation in pH-gradient AC, demonstrating that the elution mechanism may bias the translation to functional relevance. This study underscores the necessity of orthogonal validation when employing AC-MS for critical quality attribute assessment of biologics.

We developed and validated a method to quantify clofazimine, linezolid, levofloxacin, and moxifloxacin in breast milk. Inconsistencies in analyte behavior necessitated the development of separate sample preparation methodologies for the less polar analyte, clofazimine, and the more polar compounds linezolid, levofloxacin, and moxifloxacin. Clofazimine was extracted via online solid-phase extraction using a Synergi Hydro-RP column with a total run time of 7 min, while the polar compounds were extracted using a Restek Viva BiPh column for a total run time of 6.50 min. Both groups were separated by liquid chromatography on an Agilent Poroshell 120 EC-C18 column. Detection was performed using tandem mass spectrometry with electrospray ionization and multiple reaction monitoring. Calibration curves fitted weighted quadratic regression equations over the ranges of 0.0477-6.00 μg/mL for clofazimine, 0.191-24.0 μg/mL for linezolid, and 0.240-30.2 μg/mL for levofloxacin and moxifloxacin. The method demonstrated precision and inter/intra-day accuracies that readily met acceptance criteria according to the FDA and EMA guidelines. The average recoveries for clofazimine, linezolid, levofloxacin, and moxifloxacin were 68.0%, 94.1%, 104.5%, and 106.0%, respectively; mean precision ranged from 0.8% to 4.7%. The robust methodology, proven to be selective and reliable when tested in different lots of breast milk, was applied to the analysis of clinical samples collected from women on treatment for drug-resistant tuberculosis. Comparison of the observed drug breast milk concentrations with maternal plasma samples taken at the same timepoints provide much-needed data regarding the exposure of second-line TB drugs in pregnant and breastfeeding women and their infants.

The article by Bennani et al. raises important considerations regarding the assessment of uracil (U) and dihydrouracil (UH2) stability for DPD phenotyping. While the analytical method is robust, concerns arise about the experimental design used for stability evaluation. Specifically, the use of frozen plasma spiked with analytes does not adequately reflect the behavior of endogenous compounds in fresh biological samples. This may lead to an underestimation of pre-analytical variability. In contrast, studies performed on freshly collected plasma have demonstrated significant increases in uracil concentrations at room temperature, highlighting the influence of ongoing biological and enzymatic processes. These discrepancies have direct implications for clinical interpretation and have led to updated guidelines emphasizing strict pre analytical conditions. Therefore, the current approach to stability assessment may not fully capture real-world conditions encountered in routine practice. A detailed discussion of these methodological differences and their impact on DPD phenotyping is provided in the letter.

Minicircle DNA (mcDNA) is a non-viral vector known for its superior therapeutic potential and safety, making it a promising biopharmaceutical platform for gene therapy, DNA vaccination, and molecular biology applications. However, its structural similarity to parental plasmid (PP), the presence of isoforms (e.g., open circular mcDNA and supercoiled mcDNA), and host-derived impurities pose significant challenges for accurate detection and quantification, which are key steps for effective quality control. This work reports, for the first time, the development and validation of an anion-exchange chromatographic method for quantifying p53-mcDNA using a customized TSKgel® DNA-NPR column. Chromatographic parameters, including pH, elution gradient, gradient time, and flow rate, were optimized. Real samples from complex Escherichia coli lysates containing p53-mcDNA, p53-PP, miniplasmid (mP), and RNA were used, demonstrating applicability for process monitoring. The method demonstrated consistent linearity (R[2] = 0.9994) over the 10-100 µg/mL range for total DNA, with accuracy meeting FDA guidelines. The method was validated in terms of selectivity, linearity, accuracy, precision, and sensitivity over the concentration range of 10-100 µg/mL, with the lower limit of quantification (LLOQ) and detection (LOD) established at 10 µg/mL and 1 µg/mL, respectively. Intra- and inter-day precision studies confirmed reproducibility, with coefficients of variation below 6%. Overall, the chromatographic method here developed and proposed effectively separates p53-mcDNA from its parental plasmid and may support process monitoring during upstream and downstream operations.

Clobazam (CLB) and its active metabolite N-desmethylclobazam (N-CLB) exhibit substantial interindividual variability in paediatric patients with refractory epilepsy, highlighting the value of therapeutic drug monitoring (TDM). This study aimed to develop and validate a rapid and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of CLB and N-CLB and to investigate clinical factors associated with their concentrations in plasma. The method required 50 μL of plasma and achieved chromatographic separation within 3 min. The linear ranges were 25-800 ng/mL for CLB and 237.5-7600 ng/mL for N-CLB, and the method was validated according to the core elements of the ICH M10 guidelines. The validated method was applied to 61 paediatric patients with refractory epilepsy, whose concomitant antiseizure medications and CYP3A4 and CYP2C19 genotypes were evaluated. Multivariate analysis revealed that the daily CLB dose was positively associated with CLB concentration (β = 5.75; 95% CI: 3.01-8.49), whereas the use of VPA was associated with lower CLB concentration (β = -88.37; 95% CI: -134.43 to -42.31) and the use of STP was associated with higher CLB concentrations (β = 206.29; 95% CI: 137.11-275.47). For N-CLB, poor CYP2C19 metabolism (β = 4354.34, 95% CI: 3259.61-5449.07) and STP use (β = 3005.10, 95% CI: 1740.35-4269.84) were associated with higher concentrations. The method is rapid, requires a small plasma volume, and is suitable for routine simultaneous TDM of CLB and N-CLB. Integrating TDM with CYP2C19 genotyping may support individualized CLB therapy and safety monitoring in paediatric patients with refractory epilepsy.

Agarose microspheres form a dense gel network through strong intermolecular hydrogen bonding, which limits the accessibility of internal hydroxyl groups. This compact network can hinder the access of epichlorohydrin to internal hydroxyl groups and reduce activation efficiency, thereby constraining ligand loading and protein separation performance. To overcome this limitation, a sequential anhydride esterification-deesterification strategy was developed to construct agarose microspheres with an expanded network structure, high epoxy-group density and high-capacity cation-exchange functionality. Acetic anhydride (AA), succinic anhydride (SA), and octenyl succinic anhydride (OSA) were used to temporarily esterify agarose and regulate its network structure. Among the three anhydrides, OSA produced the most pronounced network expansion, despite its relatively low degree of substitution, which was attributed to the stronger structural perturbation associated with its bulky amphiphilic octenyl-succinyl substituent. Subsequent alkaline treatment removed the temporary ester groups and restored the previously esterified hydroxyl groups, while BDDE-mediated covalent crosslinking helped retain the expanded network structure. Under the optimized epoxidation conditions, OSAAM exhibited an epoxy-group density of 67.7 μmol/g, 49.8% higher than that of native AM treated under the same optimized conditions (45.2 μmol/g). After sulfonate functionalization, the OSAAM-SP medium exhibited an ion-exchange capacity (IEC) of 233.7 μmol/g, a static adsorption capacity of 377.57 mg/g (wet resin) in the absence of salt, a dynamic binding capacity of 206.7 mg/mL, and a recovery of 98.21%. The resulting medium was successfully applied to the purification of lysozyme from quail egg white. These results demonstrate that sequential regulation of the polysaccharide network and restoration of temporarily esterified sites can enhance subsequent functionalization and chromatographic performance.

In this study, a tetraaldehyde ligand was prepared via the Duff formylation of 4,4'-methylenediphenol using trifluoroacetic acid and hexamethylenetetramine, which was further condensed with (1R, 2R)-1,2-cyclohexanediamine to produce a [2 + 4]-type chiral porous organic cage (POC). The obtained POC was covalently bonded on thiol-modified silica via thiol-ene click reaction to fabricate a new chiral stationary phase (CSP). This new CSP was tested for chiral separation in normal-phase (NP) and reversed-phase (RP) modes. 15 racemic samples were enantioseparated in NP mode, and 16 in RP mode. Importantly, this new CSP packed column exhibits favorable enantioseparation complementarity with our previously reported two POC columns ([3 + 6]-type NC1-R and [4 + 6]-type CC19-R columns). It can effectively resolve several racemates that were unresolved or poorly separated on our earlier reported two POC columns, significantly broadening the scope of separable racemates and improving the versatility of POC-based columns. Under NP-HPLC mode, 9 and 8 of the 15 tested racemates achieved no enantiomeric separation on the NC1-R column and CC19-R column, respectively. Under RP-HPLC mode, 11 and 12 of the 16 tested racemates achieved no enantiomeric separation on the NC1-R column and CC19-R column, respectively. The combined use of these chiral POC columns can separate more chiral compounds than any single column used alone. Furthermore, the newly fabricated POC column is also able to resolve a number of racemates that cannot be fully resolved or are unresolvable on two typical commercial chiral HPLC columns, Chiralcel OD-H [CSP: cellulose tris(3,5-dimethylphenylcarbamate)] and Chiralpak AD-H [CSP: amylose tris(3,5-dimethylphenylcarbamate)] columns. In addition, the influences of column temperature, injection mass, and mobile phase composition on enantioseparation were systematically explored. After hundreds injection runs, the relative standard deviations (RSDs) (n = 5) of retention time and resolution values (Rs) were below 0.83% and below 1.39%, respectively. In addition, the RSDs (n = 3) of retention times and Rs values for column-to-column were below 7.06% and 8.33% respectively. These results demonstrate the good repeatability and stability of the prepared column. The present work confirms that the [2 + 4]-type chiral POC holds great potential as a CSP for HPLC, which not only greatly expands the scope and applicability of POC-based CSPs for chiral separation, but also advances the development of POCs as a new category of HPLC CSPs.