Gastrodia elata Blume is a vital herb requiring precise subtype discrimination for market authentication and traceability. However, the current analytical focus on small molecules limits the extraction of highly discriminative polysaccharide features, making chemometric authentication under class-imbalanced conditions highly challenging. To address these limitations, we established a robust multi-detector size-exclusion chromatography platform utilizing a 5 mM NH4Ac mobile phase to induce a mixed-mode separation, effectively resolving the cryptic structural heterogeneities and multidimensional properties of the bioactive polysaccharides. Furthermore, we systematically evaluated nine machine learning algorithms, comparing hierarchical versus direct architectures to mitigate class imbalance. For multimodal data fusion, we assessed chromatographic profile, physicochemical expert, and fused feature strategies, utilizing PCA, RFECV, and SFM for dimensionality reduction. Results demonstrated that the hierarchical architecture successfully resolved class imbalance. The KNN model utilizing PCA-reduced fused features achieved optimal performance (Accuracy: 0.8927, F1-Macro: 0.8515). More importantly, interpretable analysis using SHapley Additive exPlanations and PCA loadings revealed a robust chemical decision mechanism: the model prioritizes macroscopic molecular weight (PC1) to identify Gastrodia elata Bl. f. glauca S. Chow (WTM), while utilizing microscopic structural details (PC3/PC5) to distinguish Gastrodia elata Bl. f. elata (HTM) and their hybrids (WHTM). This interpretable chemometric strategy enables precise Gastrodia elata subtype authentication and offers a reliable reference for analyzing polysaccharide-rich herbs.

Bile acids (BAs) are recognized as established disease biomarkers, yet quantitative studies have largely focused on serum or plasma, with insufficient attention paid to intestinal contents that hold significant pathophysiological importance. Accurate quantification of intestinal BAs is key to elucidating disease mechanisms. However, this analysis remains highly challenging due to the complex matrix of intestinal contents and the presence of numerous bile acid isomers. This study focuses on developing and validating a rapid, accurate, and sensitive UPLC-MS/MS method for the simultaneous quantification of 42 BAs (covering 11 isomer groups) in intestinal contents, and its application to depression research. Samples were pretreated using lyophilization followed by composite solvent extraction. Chromatographic separation used a CORTECS[TM] Premier T3 column with gradient elution. Among the 11 isomer groups, baseline separation was achieved for most of them. The method validation results showed excellent linearity with r[2] ≥ 0.996; recoveries ranged 86.73% - 111.03%; stability tests under various conditions including room temperature storage for 24 h, 4 °C for 7 days, -80 °C for 90 days, and three freeze-thaw cycles yielded RSD ≤ 14.41%. Intra-day and inter-day precision RSD ≤ 16.10%. The lower limits of quantification ranged from 0.1 to 2.0 ng/mL. Applying to intestinal contents from a CUMS-induced depression SD rat model, the method revealed significant differences in 14 BAs compared with normal rats, among which Iso-HDCA was upregulated as much as 6-fold, suggesting that BAs detection should be given attention in exploring the mechanisms of depression.

Affinity purification is an essential technique for isolating highly purified proteins. However, the generation of affinity ligands requires significant time and financial investment. To address these limitations, we present a novel affinity chromatography method using in silico-designed cyclic peptides as ligands. Complement C1q (C1q), a plasma protein that plays a crucial role in the classical complement pathway, was specifically selected as the target protein. AlphaFold2 was used to design specific binding cyclic peptides based on biomolecular structure prediction. The pLDDT score for the designed complex was 85.28. Based on these designs, lariat-type cyclic peptides were synthesized. They were characterized by disulfide cyclization and biotinylation, and subsequently immobilized on streptavidin carriers. The binding kinetics between C1q and the synthetic peptides were measured via surface plasmon resonance (KD=7.79nM). Additionally, affinity column chromatography and other performance tests confirmed that the resulting column specifically captured C1q and allowed its elution from human plasma via a standard NaCl concentration gradient. Notably, the high selectivity was preserved even in the presence of plasma, thereby underscoring the practical robustness of the ligand. The purity of the purified C1q from plasma was 69.4%. By overcoming traditional constraints through (1) rapid and simple design, (2) high specificity, and (3) universal versatility without genetic modifications, this de novo design strategy represents a breakthrough in protein purification technologies.

Column length is traditionally considered a primary determinant of separation performance in liquid chromatography, with resolution expected to increase with increasing column length (efficiency). However, in gradient elution mode, this assumption neglects the potential intercorrelation between column length, gradient conditions, and selectivity. In this work, the role of column length in gradient separations is revisited. Model calculations were performed to analyze resolution as a function of column length and gradient time for systems exhibiting either "efficiency-limited" or "selectivity-limited" behavior. A selectivity sensitivity parameter (χ) was introduced to distinguish between these regimes. It is shown that in efficiency-limited systems, resolution increases monotonically with column length, consistent with classical chromatographic theory. In contrast, selectivity-limited systems exhibit a finite optimum column length due to gradient-induced compression of peak spacing, leading to conditions where shorter columns provide higher resolution than longer ones, at a specific gradient time. These findings demonstrate that column length optimization in gradient liquid chromatography requires simultaneous consideration of efficiency and selectivity. Although this study represents a proof-of-concept (based on model systems), it provides a foundation for the development of graphical tools and optimization strategies for practical method development.

Recent reports from families of individuals with Angelman syndrome (AS), a rare neurodevelopmental disorder characterised by severe neurological impairment and epilepsy, have described perceived neurological improvements following the topical application of a commercial turpentine oil (TO). These observations prompted the present analytical study, which aimed to characterise and quantify the terpenoid composition of 2 commercial TOs (Diamond and Creekwood), together with 6 additional commercial TOs, using gas chromatography-mass spectrometry (GC-MS). Analysis of 6 TOs reported to originate from Pinus pinaster revealed similar combined percentages of α- and β-pinene (≈75%), except for one oil that exhibited a more complex profile, a lower α-pinene content (≈30%), and the presence of eucalyptol, which is uncommon in TOs from this species. The Creekwood oil contained a higher proportion of α-pinene (≈76%) and a markedly lower β-pinene content (≈3%) than the Diamond oil (≈62% and ≈26%, respectively). Chiral GC-MS analysis further revealed contrasting enantiomeric distributions, with (-)-α-pinene predominating in the Diamond oil and (+)-α-pinene in the Creekwood oil, while (-)-β-pinene predominated in both samples. For quantitative analysis, the analytical method was successfully validated, demonstrating suitable detection (1.5-5.0 mg/L) and quantification (5.0-16.7 mg/L) limits, good linearity (r[2]>0.991), and acceptable precision and accuracy (<15%). These findings demonstrate substantial chemical variability among commercial TOs and provide a framework for future studies on the biological effects of pine resin terpenoids in neuronal models relevant to AS.

Vesicular stomatitis virus (VSV) is a promising platform for oncolytic virotherapy and vaccine development. However, its purification remains challenging due to the sensitivity of enveloped viruses to conventional downstream processing conditions. In this study, we investigated the transferability of our previously developed native elution immunoaffinity chromatography (IAC) concept to efficient VSV purification and systematically evaluated the key factors governing its performance while preserving virus integrity and infectivity. Polyclonal anti-VSV IgG antibodies of guinea pig and human origin were immobilized on hydrazide-activated monolithic columns and evaluated for virus capture and recovery. Although both columns exhibited high binding capacity, successful native elution was achieved only with human anti-VSV antibodies, which, in contrast to the guinea pig anti-VSV antibodies, exhibited substantially lower specific neutralization potency. Elution was performed under physiological conditions using high-molarity amino acid solutions. Among the tested eluents, arginine was found to be the most efficient, enabling high recovery of total and infectious virus particles. Process analysis revealed near-complete removal of host cell DNA following benzonase treatment, whereas a fraction of host cell proteins co-eluted with virus particles. Further experiments suggested that these proteins were primarily associated with particulate material rather than being present as soluble impurities. These results demonstrate that native elution IAC using arginine enables efficient purification of VSV under mild conditions, achieving high recovery of infectious virus and providing a strong foundation for developing scalable purification processes for enveloped viral therapeutics.

Molecularly imprinted polymers with affinity toward gentamicin were synthesised according to the principles of green chemistry, in an aqueous solution at ambient temperature. Polyacrylic acid, a polymer that changes chain conformation depending on the pH, was incorporated into the polymer network. This approach allowed the development of a material regeneration method using changes in the alkalinity of the solution. The conditions of static sorption (1.45 mg/g and 0.69 mg/g for MIP and NIP, respectively, pH 5.5) and desorption (1.04 mg/g and 0.64 mg/g for MIP and NIP, respectively, pH 9.5, 4 °C) were selected. Next, the obtained polymers were placed in solid-phase extraction columns and their performance was optimised. Bed conditioning (1 × 24 h, pH 9.5, 4 °C; 2x continuous mode pH 9.5, 4 °C; 1 × 15 min pH 5.5, 22 °C) sorption (1-5 h) and desorption (4 × 30 min) were established. In the presence of amoxicillin and norfloxacin, selectivity in tap water was determined (selectivity factor in the range of 3.03-3.07, respectively).

Fluorotelomer alcohols (FTOHs) are volatile per- and polyfluoroalkyl substances (PFAS) used in consumer products that contribute to human exposure and environmental contamination. Traditionally, FTOHs are measured in consumer products by solid-liquid extraction coupled with gas chromatography-mass spectrometry (GC-MS). Herein, we optimized a direct thermal desorption (DTD)-GC-MS method to screen for five n:2 FTOHs (n = 4, 6, 8, 10, 12) in 13 food contact materials purchased between 2020 and 2026. Method optimization established a 20 min desorption at 200 °C, achieving an average recovery of 114% and detection limits ranging from 20-53 pg for FTOHs. The method was further extended to include secondary FTOHs, fluorotelomer iodides, fluorotelomer acrylates, fluorotelomer olefins (FTOs), and fluorotelomer methacrylates. The DTD of three popcorn bags and an 'ecofriendly' paper plate at 200 °C generated 6:2 FTOH and 6:2 FTO at concentrations above the quantifiable range due to the thermal degradation of a 6:2 fluorotelomer side-chain fluoropolymer (SCFP). Even after multiple rounds of desorption, 6:2 FTOH and 6:2 FTO were continuously released, albeit at decreasing concentrations, and served as an indicator for the presence of SCFPs. Simulated heating experiments at a realistic temperature of 115 °C showed that a popcorn bag released 150 ± 46 ng/g of 6:2 FTOH within 2 min, highlighting the potential for human exposure to volatile PFAS via inhalation or ingestion through migration into food. Overall, DTD-GC-MS provides a rapid, solvent-free approach for screening volatile PFAS and identifying SCFPs in food contact materials.

The retention behavior of oligonucleotides (ONs) in anion-exchange chromatography (AEX) is often described using simplified assumptions that lack systematic experimental validation. In this work, the isocratic retention factors of 19 ONs ranging from 10- to 120-mer were measured over a broad range of sodium chloride concentrations on the 5.0μm Protein-Pak[TM] Hi Res Q Column (4.6 × 100 mm), from slight exclusion to strong retention (-0.1<k<30). A purely empirical but robust five-parameter model was shown to accurately capture the complete dependence of the retention factor on salt concentration across all ON sizes and salt ranges. The validity of this model was confirmed through excellent agreement between simulated and experimental gradient chromatograms, demonstrating that both retention times and peak shapes of complex ON mixtures can be reliably predicted. These results provide a robust and experimentally grounded framework for modeling ON retention in AEX without relying on simplified stoichiometric assumptions. The data further show that the commonly invoked "on-off" retention mechanism (defined as when the retention factor is decreasing stepwise from infinity to zero at a specific salt concentration) does not apply to ONs up to at least 120-mer, as retention varies smoothly with salt concentration even for large oligomers. Retention extrapolation for larger ONs even suggests that such behavior may only emerge beyond approximately 650-mer. Because ON retention is strongly correlated with size, peak elution is advantageously ordered rather than random, enabling efficient use of peak capacity. However, non-uniform peak spacing, nucleotide sequence, and size-dependent broadening limit full baseline resolution locally for all ONs. For instance, the numerical gradient simulations enable the users to immediately identify optimal combinations of column length (65 cm), gradient time (2 h), and salt concentration range (551 mM to 754 mM NaCl) to maximize peak capacity (∼ 260) and resolve all 111 ONs in a 10- to 120-mer mixture. This work delivers a strong foundation for the development of improved AEX columns (in terms of length and particle size) and advanced gradient methods, including S-shaped salt profiles, to achieve the highest resolution separation of highly complex ON mixtures up to 120-mer.

Despite the growing therapeutic importance of peptides such as glucagon-like peptide-1 (GLP-1) analogues, their analytical characterization has received comparatively limited attention. Reversed-phase liquid chromatography (RPLC) remains the reference technique for peptide purification and analysis; however, the pronounced structural and physicochemical diversity of therapeutic peptides, spanning linear, macrocyclic, and disulfide-rich scaffolds, underscores the need for complementary separation strategies. This study aimed to expand the analytical toolbox for therapeutic peptide characterization by evaluating ion-exchange chromatography (IEX). Four IEX columns were assessed: two strong cation exchangers (ProPac 3R SCX and BioPro IEX SF Accura) and two strong anion exchangers (ProPac 3R SAX and BioPro IEX QF Accura), all tested in 2.1 × 50 mm formats to reduce sample consumption and analysis time. Initial evaluations using standard protein IEX methods revealed they were unsuitable for the target therapeutic peptides. To optimize the separation, salt gradients at pH 2.0 (cation exchange chromatography) and pH 10.0 (anion exchange chromatography) were evaluated alongside alternative pH-gradients, specifically a classic phosphate-citrate mixture and a polyamine-based tri-buffer mixture (pH 2.5-9.0). Among the approaches tested, salt-gradient elution consistently provided superior performance. The prevalence of secondary hydrophobic interactions significantly impacted chromatographic performance, necessitating the addition of at least 30% acetonitrile (ACN) to mitigate peak broadening and improve peptide recovery. To evaluate stability-indicating capability, the developed IEX methods were benchmarked against an in-house reference RPLC purity method through forced degradation studies. Finally, we explored the use of IEX retention behavior to estimate peptide pI values, illustrating both the potential and the current limitations of ion-exchange chromatography for the characterization of therapeutic peptides.

Surface thermodynamics of [CMIM][MeSO4] (n = 2, 4, 6, 8) ionic liquids were investigated by inverse gas chromatography at infinite dilution over 313.15-353.15 K. Three calculation approaches were compared for the dispersive surface energy (γd[s]): the conventional Schultz method, the Dorris-Gray method, and the Schultz method with Hamieh's temperature-dependent molecular area correction. Lewis acidity-basicity constants (KA, KB) were derived by the Brookman-Sawyer, Flour-Papirer, Donnet-Park, and Schultz approaches within the Gutmann framework, and interpreted at the molecular level by ESP analysis based on the Murray-Politzer framework. The three γd[s] methods exhibited distinctly different temperature dependences: the Dorris-Gray method gave the smallest temperature coefficient, the conventional Schultz method was intermediate, and the Hamieh-corrected Schultz method showed the largest. The relative magnitudes among the three methods varied with temperature, with the Schultz and Hamieh values converging at elevated temperatures. The four Lewis acid-base approaches gave consistent rankings of apparent surface basicity in the order Brookman-Sawyer > Schultz > Flour-Papirer > Donnet-Park, with all four ionic liquids exhibiting amphoteric Lewis behavior dominated by basicity, decreasing in the order EMIM > BMIM > HMIM > OMIM with increasing alkyl chain length. The complementary ESP analysis provided convergent evidence for the [Formula: see text] anion as the dominant Lewis basic surface feature. Adsorption free energies ranged from approximately 22 to 43 kJ/mol, consistent with physisorption with partial specific interaction contributions.

The structure and functional properties of new large-pore, cellulose-based particles for biomolecule chromatography are evaluated. The particles are approximately 90 µm in diameter and contain interconnected pores with an average diameter of 3.6 µm and a macro-porosity of 0.76. Smaller pores, approximately 4 nm in diameter, are also present within the cellulose backbone. Because of the large macro-pore size and porosity, the particles have a high internal hydraulic permeability (0.08 μm[2]), similar to the permeability of typical monoliths. Thus, when these particles are packed in a column, a significant fraction of the mobile phase (about 0.018) flows within the particles themselves, resulting in a large convective enhancement of intraparticle transport rates. Experimental measurements with different proteins show flow rate-independent HETP for pulse injections of IgG, thyroglobulin, and IgM under non-binding conditions, residence time-independent dynamic binding capacity for frontal loading of IgG and IgM in the range 1-0.25 min, and constant peak width for gradient elution of IgG at residence times between 2 and 0.25 min. The results are consistent with a perfusion chromatography model, which accurately predicts frontal loading and gradient elution results using parameters obtained from the non-binding HETP data. The model can be used to assess performance when using these materials for different biomolecules and bioparticles. Compared to existing resins, dynamic binding capacities of the new resin are smaller for smaller proteins and at long residence times, but comparable to those obtained with monoliths for larger proteins and short residence times.

The first application of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection method for high performance liquid chromatography (HPLC) in the ultraviolet (UV) spectral range of 250-400 nm is reported. A custom-designed HPLC-BBCEAS system, incorporating a laser-driven light source (LDLS) and high reflectivity cavity mirrors (R ≥ 0.99), was employed for the separation and quantification of the polycyclic aromatic hydrocarbons (PAHs) anthracene and pyrene at near-UV wavelengths (300-400 nm). Minimum detectable absorption coefficients, αmin(t) of 9.8 × 10[-6] cm[-1] Hz[-]½ and 9.6 × 10[-6] cm[-1] Hz[-]½ were achieved for anthracene and pyrene at 338 nm and 333 nm, respectively, with 6.8 × 10[-6] cm[-1] Hz[-]½ observed for anthracene at 356 nm. These values correspond to 25-fold and 18-fold improvements in sensitivity compared to a conventional Perkin-Elmer HPLC-UV system. In the deep-UV region (250-300 nm), the separation and detection of vitamin D2 and D3 were demonstrated using both the LDLS and a 280 nm LED with R ∼ 0.96 cavity mirrors. LDLS-based measurements yielded αmin(t) values of 5.1 × 10[-4] cm[-1] Hz[-]½ and 4.0 × 10[-4] cm[-1] Hz[-]½ for D2 and D3, respectively, while the LED measurements exhibited enhanced sensitivity with values of 7.7 × 10[-5] cm[-1] Hz[-]½ and 5.8 × 10[-5] cm[-1] Hz[-]½. These findings demonstrate that HPLC-BBCEAS enables broadband, multiplexed, and highly sensitive detection of UV-absorbing analytes and extends the practical detection range of HPLC into the deep-UV region.

Anti-doping and forensic science detection rely fundamentally on target-based methodologies, especially for metabolite screening and identification. Traditional manual screening is constrained by low efficiency and knowledge bias. Conversely, although current metabolomics and machine-learning approaches efficiently calculate lots of potential biomarkers using statistical methods, these findings necessitate further rigorous screening to isolate true biomarkers that satisfy the requirements of both anti-doping and forensic science. To bridge the gap between machine learning and manual screening, we developed an innovative reverse-metabolomics strategy: utilizing LCHRMS for Phase II conjugate automated prediction as well as GCHRMS for targeted Phase I tentative validation. In the study of 6β‑chloro-testosterone (6β-Cl-T) metabolism, while LCHRMS (via Compound Discoverer software) initially predicted 244 putative phase II metabolites, the integration of GCHRMS, following the release of Phase I metabolites and subsequent derivatization, acted as a high-precision filter to further elucidate these candidates. 74 of the 244 candidates were putatively validated as candidate biomarkers. Eliminating high-throughput false positives, this workflow boosts data confidence in anti-doping and clinical metabolomics.

In this study, an ionic liquid-functionalized covalent organic framework (IL@COF) composite was synthesized by immobilizing different ILs onto a COF support, which possesses a hierarchical porous structure with a high specific surface area (1067.8 m[2]/g). The IL@COF was subsequently fabricated into 13 mm discs using ethyl cellulose as an excipient. After systematic characterizations, they were applied for the simultaneous adsorption of iconic Tanshinone IIA and Salvianolic acid B in Salvia miltiorrhiza from Danshen oral liquid, promoting its component analysis, quality control, and basic research on the chemistry-bioactivity relationship of such a compound preparation. The adsorption process followed the pseudo-second-order kinetic model and Langmuir isotherm model, indicating chemisorption-dominated monolayer adsorption on energetically homogeneous active sites. Moreover, the adsorption of Tanshinone IIA was exothermic and enthalpy-driven, while the adsorption of Salvianolic acid B was driven by both enthalpy and entropy, with a significant desolvation effect. Surface electrostatic potential (ESP) calculations and independent gradient model based on Hirshfeld partition (IGMH) weak interaction analysis revealed that hydrogen bonding, π-π stacking, and van der Waals forces are the primary non-covalent interactions governing the recognition and binding of the target compounds to the IL. In addition, the developed discs and separation method exhibited satisfied recyclability and friendly greenness, respectively.