Arenobufagin, a typical bufadienolide from toad venom, exhibits excellent anti-inflammatory activity; however, its inherent toxicity severely limits its further application. To improve its safety profile, this study employed glycosylation for the structural modification of arenobufagin. Given the complexity of the reaction matrix, counter-current chromatography (CCC) was selected for the efficient separation of the glycosylated products. First, response surface methodology was employed to optimize the glycosylation reaction conditions, which significantly enhanced the conversion rate. The glycosylation products were successfully separated using a solvent system composed of dichloromethane/methanol/water (8:4:8, v/v/v). From a 550 mg crude sample, 14.6 mg of arenobufagin-3-O-β-D-glucosyl (1→2)-β-D-glucoside (Arenobufagin-2G), 80.3 mg of arenobufagin-3-O-β-D-glucoside, and 17.5 mg of the unreacted arenobufagin substrate were obtained. Biological evaluation revealed that arenobufagin conjugated with two sugar moieties achieved a favorable balance between safety and anti-inflammatory efficacy. Mechanistically, Arenobufagin-2G suppressed inflammatory responses in Tohoku Hospital pediatrics-1-derived macrophages and attenuated lipopolysaccharide-induced nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 inflammasome activation. This study not only demonstrates that CCC is a powerful strategy for separating complex glycosylation products but also lays the foundation for the future research and development of safe arenobufagin-based therapeutics.

This study presents the applicability of a solid-phase microextraction (SPME) coating based on the CIM-80(Al) metal-organic framework (MOF), prepared by in situ solvothermal growth onto braided silver wires, for the untargeted analysis of passion fruit juices. After the characterization of the MOF-coated fiber by Fourier-transform infrared spectroscopy, scanning electron microscopy, and powder X-ray diffraction, it was applied in both headspace (HS)- and direct immersion (DI)- SPME in combination with gas chromatography-mass spectrometry. The studies were carried out in comparison with three commercial SPME fibers. Considering all the fibers studied, up to 52 and 26 compounds were tentatively identified via HS- and DI-SPME, respectively. Besides, all fibers presented adequate reproducibility and similar lifetime, even in the DI mode and despite the sample complexity. In HS-SPME, a total of 25 compounds were tentatively identified with the MOF-coated fiber. From these analytes, 10 compounds (∼42%) were exclusively detected with the MOF-coated fiber. In DI-SPME, 10 compounds were tentatively identified with the MOF-coated fiber, and four of these compounds (∼50%) were exclusively detected with this fiber. Overall, the results indicate that the MOF-coated fiber offers a superior selectivity for heavier, less volatile, and less polar analytes, therefore acting as a complementary tool for commercial coatings when intending metabolomic studies for food analysis. The dual approach with MOF-coated fiber and commercial SPME coatings provides more comprehensive information for food analysis.

As a kind of adsorption material with excellent properties, covalent organic frameworks (COFs) have a broad application prospect in the field of sample pretreatment. Herein, a magnetic COF was constructed by combining magnetic nanospheres with a stable azine-bonded COF via the monomer-mediated in-situ growth strategy for the enrichment and determination of sulfonamide antibiotics. The magnetic COF has good magnetic properties, an appropriate specific surface area, and high stability, and is an excellent adsorbent for magnetic solid-phase extraction. The adsorption mechanism study showed that the large conjugated system and the rich N element of the azine-bonded COF provided abundant π-π interactions and hydrogen-bonding sites, which were beneficial to its interaction with sulfonamide antibiotics. An analytical method for the detection of eight sulfonamides in fish and chicken was developed by combining magnetic extraction with capillary electrophoresis analysis. Under the optimal conditions, the proposed method showed good linearity between the peak areas and the concentrations of eight sulfonamides from 0.1 to 5.0 µg/mL, with correlation coefficients greater than 0.98 and limits of detection from 0.008 to 0.057 µg/mL. This work provides an alternative method to capture and detect sulfonamide antibiotics that is highly efficient, rapid, and sensitive.

Rapid enantioseparation and scalable purification processes of chiral drug molecules are crucial to drug development and quality control (QC) but still challenging. Supercritical fluid chromatography is one of the emerging approaches for quicker turnaround times and high throughput to solve these issues. In this study, we report an organized comparison of polar organic HPLC (PO-HPLC), conventional reversed-phase HPLC (RP-HPLC), and supercritical fluid chromatography (SFC) to perform the enantioseparation and gram-scale purification of antihistamine drugs Meclizine (I) and Buclizine (II) with a common polysaccharide-based chiral stationary phase (Chiralpak IJ column). Analytical performance was assessed by examining the retention behavior, resolution, peak symmetry, runtime, and solvent consumption. Effective baseline separations were obtained with polar-organic and reversed-phase HPLC, but longer times for analysis and more organic solvent usage were needed. Chiral SFC, on the other hand, provided quick analyses (5-6 min) with higher resolution (Rs > 2.2), improved peak shapes, and significantly lower organic solvent consumption. Preparative separations of all three techniques were demonstrated at the 1 g scale. Chiral SFC provided the highest overall productivity, with >99% enantiomeric excess and >90% recovery in less than 3 h, while greatly reducing post run work up. The analytical chiral SFC method validation was done according to ICH Q2 (R2) requirements, confirming its specificity, linearity, precision, accuracy, robustness, and solution stability. Taken together, the results illustrate chiral SFC as the most efficient and sustainable platform for analytical and preparative enantioseparation of meclizine and buclizine, with complementary roles for polar organic and reversed phase HPLC.

Given the complex composition of traditional Chinese medicine (TCM) formulas, quality evaluation cannot rely on a single component or a limited few. Therefore, a five-dimensional quality marker (Q-marker) identification system encompassing "effectiveness, specificity, transferability, measurability, and formula compatibility" was developed and subsequently applied to Fufang Huangqi Jianpi oral liquid (FFHQJPOL) as an example. Using UHPLC-Q-TOF/MS, 62 compounds were identified in the preparation. The measurability dimension was constructed based on 17 major constituents derived from 23 common peaks in the chromatographic fingerprints of 10 batches. Serum pharmacochemistry via UHPLC-Q-TOF/MS revealed 32 prototype components absorbed into the bloodstream, thereby establishing the transferability dimension. Network pharmacology approaches were applied to construct a "component-target-disease-pathway" network, defining the effectiveness dimension. A topological network of compounds and herbs was established to depict the distribution pattern of the candidate compounds across various herbs, thereby revealing specificity. The compound compatibility dimension was established based on the function and proportional weight of its active ingredients in the formula. Ultimately, through regression area (RA) and coefficient of variation (CV) calculation, 3-hydroxy-9,10-dimethoxypterocarpan, ononin, zizyphusine, calycosin-7-O-glucoside, and sinapine were initially identified as the key Q-markers. It is worth mentioning that astragaloside IV and astragaloside II were additionally incorporated into the Q-markers system based on UHPLC-Q-TOF/MS analysis and their known pharmacological activities, ultimately establishing seven key Q-markers for FFHQJPOL. This strategy provided a multi‑dimensional integrated paradigm for Q‑markers discovery in TCM compound preparations, facilitating the transition from single‑index to system‑integrated quality control.

A sensitive three-step online enrichment strategy including large-volume sample stacking, dynamic pH junction, and sweeping was successfully developed and validated for the simultaneous online enrichment and separation of phthalides and phenolic acids in Chuanxiong Rhizoma (CX) by coupling reversed migration cyclodextrin-modified micellar electrokinetic chromatography (RM-CD-MEKC) with a diode array detector. The samples were prepared in a low-conductivity, high-pH matrix consisting of 3 mM borax buffer at pH 7.5. The background electrolyte was a high-conductivity, low-pH buffer containing 10 mg·mL[-1] sulfobutylether-β-cyclodextrin, 80 mM sodium dodecyl sulfate, and 25 mM sodium dihydrogen phosphate in 20% methanol (pH 3.5). Under the optimized conditions, four major active components (senkyunolide A, senkyunolide I, ferulic acid, and chlorogenic acid) of Ligusticum chuanxiong Hort. were efficiently separated within 17 min, achieving good resolution and 10- to 44-fold enrichment. The limits of detection ranged from 0.09 to 0.25 µg·mL[-1] while the limits of quantification ranged from 0.31 to 2.24 µg·mL[-1]. The relative standard deviations for intra-day and inter-day precision were all below 3.45%, demonstrating good precision and method stability. Overall, the proposed RM-CD-MEKC combined with a three-step enrichment technique provides a simple, rapid, and environmentally friendly enrichment approach for the simultaneous determination of phthalides and phenolic acids in CX samples.

Radial flow stream (RFS) splitting has been reported to reduce operating pressure and apparent theoretical plate height (increasing column efficiency) while increasing detector response relative to conventional high-performance liquid chromatography column operation. However, the mechanisms responsible for these effects have remained unclear. Here, pressure measurements, computational fluid dynamics, and reduced-order outlet models were used to examine how radial outlet partitioning in the new end fitting alters column behavior. Most of the measured pressure reduction arose from post-column effects, although a smaller in-column contribution remained. Simulations showed that the radial outlet disrupted the conventional outlet-flow funnel and preferentially captured wall-adjacent flow. When the outlet models were applied to a published model of radial column heterogeneity, apparent column plate height decreased by approximately 19% and peak height increased by approximately 18%, consistent with typical experimental results. Experimental sensitivity improvements also persisted under detector conditions that were independent of flow rate. These results indicate that the reported benefits under RFS splitting mode arise from a combination of post-column pressure effects and selective sampling of the radial solute distribution.

Wild-simulated ginseng (linxiashen, LXS) powder, farm-grown ginseng (yuanshen, YS) powder, and farm-grown ginseng decoction pieces (YSDP), as well as the Baoyuan decoction prepared from them (BYD-LXS, BYD-YS, and BYD-YSDP) by boiling, exhibit certain metabolic differences in their chemical composition. To elucidate the holistic molecular profile and differences, a reversed-phase UHPLC-Q-TOF-MS[E] metabolomics strategy with an ACQUITY HSS T3 stationary phase was applied to characterize and discover differential chemical markers. In total, 293, 663, 311, and 645 compounds (including 22, 13, 24, and 28 rare ginsenosides) were annotated in LXS/YS, BYD-LXS/BYD-YS, YS/YSDP, and BYD-YS/BYD-YSDP respectively. Based on PCA and OPLS-DA, 87, 222, 95, and 168 chemical markers (including 8, 7, 13, and 3 rare ginsenosides) were annotated in LXS/YS, BYD-LXS/BYD-YS, YS/YSDP, and BYD-YS/BYD-YSDP decoctions, respectively. Secondary metabolites such as triterpene saponins were found to be present at higher levels in LXS decoction and BYD-LXS decoction. The amount of components eluted from YSDP decoction was significantly higher than that from the decoction of YS powder. In addition, Support Vector Machine and Back Propagation Neural Network models with feature selection nested within cross-validation were constructed to classify LXS/YS, BYD-LXS/BYD-YS, YS/YSDP, and BYD-YS/BYD-YSDP decoctions, achieving 100%, 100%, 100%, and 94.44% classification accuracies, respectively. This study provides a basis for the quality assessment of ginseng and Baoyuan decoction, as well as for the development and utilization of rare ginsenosides.

Stemona alkaloids possess multiple stereogenic centers, giving rise to structurally diverse isomeric forms that remain challenging to differentiate by conventional mass spectrometric approaches. In this study, six tuberostemonine isomers were annotated and discriminated using LC-MS/MS in combination with molecular networking-guided spectral similarity analysis. Based on their MS/MS spectral features, these isomers were classified into two distinct groups. Gaussian fitting further revealed characteristic differences in fragmentation energy profiles and relative fragment-ion intensities, enabling refined differentiation of closely related isomeric alkaloids. The relative abundances of these isomers were subsequently compared across commercial "Bai-Bu" herbal materials. These results provide experimental evidence and an MS-based analytical strategy for the identification, discrimination, and quality evaluation of isomeric Stemona alkaloids.

An anionic covalent organic framework (COF) was constructed using 2,5-diaminobenzenesulfonic acid as an ionic building unit. The incorporation of sulfonate functionalities endows the framework with permanent anionic sites, thereby enabling selective ion-exchange interactions with cationic species. The adsorption performance toward berberine hydrochloride (BBH) was systematically investigated by optimizing pH, ionic strength, adsorbent dosage, extraction time, and elution conditions. Rapid adsorption equilibrium was achieved within 10 min, and the kinetic data were well described by a pseudo-second-order model, indicating a chemisorption-dominated process. Under optimal conditions (pH 11, 303 K), the maximum adsorption capacity reached 1082.4 mg g[-1]. The material maintained high adsorption efficiency in the presence of competing cations, demonstrating pronounced selectivity arising from electrostatic interactions. Moreover, it retained over 90% of its initial adsorption capacity after five adsorption-desorption cycles, confirming its structural stability and recyclability. These results highlight the effectiveness of introducing ionic functionalities into COF architectures to achieve high-capacity and selective adsorption of alkaloid compounds, providing a viable platform for the separation and analysis of BBH in complex herbal matrices. The design and synthesis of novel COF porous adsorption materials will play a crucial role in the separation and analysis of natural products.

Countercurrent chromatography (CCC) is a liquid-liquid separation method that is predominantly used for the preparative isolation of natural products. Key indicators for the development of CCC methods are the partition coefficient (K value) between the two liquid phases and the properties of the chosen biphasic solvent system. Based on this information, it is possible to predict elution volumes and peak widths with the probabilistic model for immiscible separations and extractions (ProMISE). However, a common challenge in reliably predicting CCC elution profiles is a persistent loss of stationary phase (i.e., bleeding), which leads to a decrease in the percentile share of stationary phase (Sf value) in the column as the separation progresses. In this study, two carotenoids were isolated and used as test compounds to investigate the effect of bleeding with the n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v) solvent system. After showing that the stationary phase bleeding rate was linear (and not exponential), experiments were carried out both in head-to-tail and in tail-to-head mode. Next, a simple calibration method was developed to predict elution volumes and peak widths with the ProMISE2 software in CCC separations with linear bleeding. Finally, based on the collected data, a method was developed to accurately predict the elution volume and peak width during a (long) CCC run, particularly for analytes with comparatively high elution volumes.

The robustness of a test for characterizing hydrophilic interaction liquid chromatography (HILIC) columns against variations in aqueous mobile-phase composition was evaluated using mobile phases of acetonitrile/aqueous mixture (9:1, v/v) in the pH range of 3.6-6.8 of the aqueous part. Ten columns (commercially available and home-made columns) were examined under five mobile phases differing in anion species (formate or acetate), NH4 [+] concentration and salt concentration. The specific hydrophilicity of the columns was assessed via the retention factor of uridine (k(U)), and the relative retention characteristics (i.e., retention selectivity: α) were evaluated using structure-related probe pairs. The k(U)-values exhibited low variability under low-salt conditions (20 mM in the aqueous part; CVs = 0.01-0.08), but higher variability under high-salt conditions (100 mM in the aqueous part; CVs = 0.03-0.18). Under comparable pH conditions (ammonium formate at pH 4.5 vs. ammonium acetate at pH 4.7), k(U)-values were consistently higher in the former, indicating that differences in NH4 [+] concentration affect hydration at the stationary-phase interface and thereby influence retention. This suggests that k(U), commonly used as an indicator of hydrophilic retention, can be affected significantly by partition interactions that are controlled by ionic concentration rather than the pH value of the mobile phase. In contrast, the characteristic α-values primarily associated with solute retention via partitioning exhibited high robustness (CVs ≤ 0.03). Notably, α(k(U)/k(2'-deoxyuridine)) values correlated with the estimated phase ratio, suggesting that it reflects the effective volume of the hydrated layer on the surface of the packing materials. Furthermore, the relationships between α(k(1-β-D-arabinofuranosyl uracil)/k(U)) and α(k(vidarabine)/k(adenosine)) indicated that nucleobase‑dependent interactions contribute significantly to stationary‑phase retention. Overall, these results demonstrate that each k(U) and α, particularly those associated with partition-dominated retention, exhibit generally high robustness within this test framework and provide a basis for more robust and transferable HILIC column characterization.

Deschloroketamine (DXE) is a dissociative new psychoactive substance whose enantiomers and metabolites may exhibit distinct pharmacological and toxicological profiles. Reliable analytical methods enabling the chiral determination of DXE and its metabolites in biological matrices are thus essential. In this study, we evaluated supercritical fluid chromatography coupled to tandem mass spectrometry (SFC‑MS/MS) and capillary zone electrophoresis with ultraviolet detection (CZE‑UV) for the enantioselective analysis of DXE and its major metabolites in rat brain tissue. SFC using polysaccharide‑based chiral stationary phases provided baseline separation of the hydroxylated and demethylated metabolites, but severe peak distortion of DXE enantiomers prevented their quantification. Therefore, quantitative analysis was performed by CZE employing carboxymethyl‑β‑cyclodextrin as a chiral selector and a high‑sensitivity Z‑shaped detection cell. Analysis of brain tissue samples obtained following high-dose administration revealed near equimolar concentrations of the DXE enantiomers and a higher concentration of (R)-norDXE relative to (S)-norDXE. In this particular case, the combined use of SFC‑MS/MS and CZE‑UV provided a practical workflow for the chiral profiling of DXE and its metabolites in complex biological matrices.

Cationic cellulose paper is a promising adsorbent for acidic compounds in water. However, cellulose modified with glycidyl trimethyl ammonium chloride (EPTAC) often exhibits compromised structural integrity and readily disperses in water. In this study, a Gemini quaternary ammonium salt (Gemini-QAS) was synthesized via nucleophilic substitution of N,N,N',N'-tetramethyl-1,6-hexanediamine with epichlorohydrin and subsequently used for the etherification of cellulose paper. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and zeta potential analyses confirmed the successful incorporation of the Gemini-QAS onto the cellulose substrate. Tensile testing demonstrated that the Gemini-modified cellulose paper (GCP) exhibited enhanced mechanical strength in both dry (25.16 MPa) and wet (1.54 KN/m) states, and the wet tensile strength ratio was 0.29, whereas EPTAC-modified cellulose paper showed decreased wet tensile strength (0.51 KN/m) and ratio (0.16). Batch experiments demonstrated that the optimal adsorption and desorption conditions for GCP on 12 weakly acidic non-steroidal anti-inflammatory drugs (NSAIDs) were pH 7.0, an adsorption time of 30 min, and an eluent of 2% formic acid-methanol. Then, these 12 NSAIDs were extracted by GCP from real water samples and quantified by high-performance liquid chromatography-tandem mass spectrometry. The analytical method achieved limits of detection ranging from 0.02 to 0.64 µg/L and limits of quantification between 0.07 and 2.11 µg/L. Precision, expressed as intra-day relative standard deviation (RSD), remained below 10%, and inter-day RSD below 12%. The relative recoveries ranged from 71% to 102% at spiking levels of 10 and 50 µg/L, while recoveries at 1 µg/L ranged from 65.1% to 89.7% due to more pronounced matrix effects. This work successfully resolves the poor aqueous stability and fiber dispersion issues of EPTAC-modified cellulose, providing a green, low-cost, and robust paper-based sorptive phase for trace NSAID monitoring in environmental water, which aligns with the principles of sustainable analytical chemistry.