J Chromatogr A [JOURNAL]
Sun
200 papers
Uncertainty-driven model-based search methods for method development in liquid chromatography.
Niezen LE, Cabooter D, Desmet G
J Chromatogr A · 2026 Aug · PMID 42127884 · Publisher ↗
Empirical retention modelling is a proven method development approach that uses a limited set of initial "scanning" experiments to build analyte retention models and predict optimal experimental conditions from a predict...
Empirical retention modelling is a proven method development approach that uses a limited set of initial "scanning" experiments to build analyte retention models and predict optimal experimental conditions from a predicted chromatographic response function (CRF) surface. Once initial models have been established, literature shows how smart algorithms can be used to further update the surface during their search for the optimal separation conditions. In this study, several search strategies were compared in silico using a set of 122 randomized mixtures. Each strategy was evaluated under varying degrees of peak detectability. Three main strategies were assessed: (i) a classic CRF-driven approach, wherein new search runs were conducted at the working point with the highest estimated CRF-value (ii) a new global uncertainty-driven strategy aimed at first improving all initial models simultaneously before attempting to maximize the objective, and (iii) a per-analyte variant of strategy ii). It was found that, as soon as peak detection was hindered (by peak overlap), the uncertainty-driven strategies outperformed the conventional CRF-driven searches. For example, when a resolution of R = 0.5 was needed to correctly identify and track the peaks, the uncertainty-driven searches only failed for 1 out of 122 samples (0.8 %), while the CRF-driven search failed in 14 % of the cases. When R = 1.0 was needed, failure rates increased to 4 % for the best uncertainty-driven approach, while the failure rate for the conventional CRF-driven approach grew to a very problematic 42 %, despite the retention time parameter estimation being conducted with the same model as the one used to generate the retention time data in the simulated chromatograms. To conclude, our results show that in the current setting uncertainty-driven strategies achieve much greater accuracy in identifying the desired separation than the CRF-driven approach. This improvement comes at the cost of a higher total analysis time, as a consequence of the richer retention time information gathered by the algorithm.
Synthesis of C18-modified radial mesoporous core-shell silica microspheres with tunable shell thickness for ultra-high performance liquid chromatography.
Huang R, Yang Y, Ming Y … +4 more , Quan K, Li Z, Qiu H, Chen J
J Chromatogr A · 2026 Aug · PMID 42127883 · Publisher ↗
With the advent of ultra-high performance liquid chromatography (UHPLC), core-shell silica microspheres (CSSMs) have emerged to balance system backpressure and column efficiency. As one of the methods for preparing CSSMs...
With the advent of ultra-high performance liquid chromatography (UHPLC), core-shell silica microspheres (CSSMs) have emerged to balance system backpressure and column efficiency. As one of the methods for preparing CSSMs, the biphasic synthesis method still faces challenges in precisely controlling the shell thickness. In this study, based on the classical biphasic method, we synthesized CSSMs with a radial mesoporous structure and tunable shell thickness through multi-parameter regulation. Tetrabutylammonium bromide (TBAB) was introduced for the first time as an electrolyte to enhance the ionic strength of the system, effectively promoting the directional deposition of silica oligomers on non-porous silica cores. Through the co-regulation of the amounts of TBAB and tetraethyl orthosilicate (TEOS), as well as the volume ratio of water to n-hexane, CSSMs with tunable shell thickness (59.54-251.93 nm), high specific surface area, and adjustable mesopore size were successfully synthesized. Furthermore, after modifying three CSSMs with different shell thicknesses (synthesized at varying water to n-hexane volume ratios) with octadecyltrichlorosilane (OTS), the resulting C18-modified stationary phases achieved baseline separation of 5 alkylbenzenes, 8 polycyclic aromatic hydrocarbons (PAHs), and 4 phthalate esters (PAEs) within 6 min. The stationary phase also exhibited excellent reproducibility and stability, further demonstrating the practical utility of CSSMs for UHPLC. This method provides a practical and reliable pathway for the rational design of ultra-high performance core-shell stationary phases.
Fabrication of trypsin-immobilized metal-organic framework-polymer monolithic spin column through a facile biomineralization strategy for efficient proteome digestion.
Zhang S, Xu K, Li B … +3 more , Li Z, Zhou W, Wang X
J Chromatogr A · 2026 Aug · PMID 42127882 · Publisher ↗
In this work, a novel trypsin-immobilized metal-organic framework (MOF)-polymer monolithic spin column was prepared by a facile biomineralization strategy, which intelligently utilizes the monolith to provide a porous su...
In this work, a novel trypsin-immobilized metal-organic framework (MOF)-polymer monolithic spin column was prepared by a facile biomineralization strategy, which intelligently utilizes the monolith to provide a porous support and the MOF to confer protective effect on trypsin. The proposed immobilized trypsin reactor showed higher enzyme-substrate affinity, better reusability, longer storage stability than the traditional covalently immobilized trypsin. Compared with free trypsin digestion, immobilization of trypsin by biomineralization exhibited enhanced pH and thermal stabilities with remarkably reduced digestion time, better economic performance due to reusability, without any loss of digestion activity. Finally, mouse liver proteomic analysis was carried out with emphasis on in-solution tryptic digestion and this immobilized trypsin spin column digestion, nearly twice the number of peptides and protein groups with varying molecular weights and isoelectric points were identified after spin column digestion within a much shorter hydrolysis time (from 14 h to just 1 h). Therefore, the biomineralized trypsin-MOF-monolithic spin column developed in our study appears to be an advanced tool in proteomic sample preparation, offering a combination of efficiency, speed, and robustness that is well-suited for both research and clinical areas.
An isotopologue-mixture-based internal standard strategy for LC-MS/MS quantification in plasma.
Guo P, Liu Y, Liu G … +2 more , Ma M, Chen B
J Chromatogr A · 2026 Aug · PMID 42127881 · Publisher ↗
Stable isotope-labeled internal standards (SIL-IS) are the gold standard for liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification in complex biological matrices. Their application can be limited by sy...
Stable isotope-labeled internal standards (SIL-IS) are the gold standard for liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification in complex biological matrices. Their application can be limited by synthetic complexity, high cost, and the lack of commercially available labeled standards for many bioactive analytes. Here, we establish and evaluate an internal-standard strategy based on hydrogen-deuterium exchange (HDX)-derived deuterated isotopologue mixtures. Using Pd/C, Pt/C, and AlCl catalytic systems, deuterated isotopologue mixtures (d-d) of eight structurally diverse bioactive compounds were prepared by HDX in deuterated methanol and characterized by H NMR and LC-MS. Isotopologues with a mass difference of Δm ≥ 3 were selected as internal-standard candidates to minimize overlap with analyte M + 1/M + 2 isotopic peaks. Isotopic-distribution screening and chromatographic evaluation showed that eligible d-d isotopologues co-eluted with their corresponding analytes (Δt < 0.01 min), with no appreciable chromatographic isotope effect (CIE). In plasma, LC-MS/MS validation showed linearity over 1-500 ng/mL (R ≥ 0.995), limits of detection of 0.003-0.080 ng/mL and limits of quantification of 0.01-0.25 ng/mL. Trueness ranged from 85% to 115%, while precision, expressed as relative standard deviation (RSD), was ≤8% intra-day and ≤12% inter-day. Ratio-based matrix effect (ME%) values ranged from 85% to 112%. Using clenbuterol hydrochloride as a model analyte, the isotopologue-mixture-derived internal standard showed calibration performance comparable to that of the commercial deuterated standard clenbuterol-d. Compared with quantification based on external calibration without internal-standard normalization, the proposed approach reduced quantitative deviation from 15 to 25% to <8%. Overall, the results support HDX-derived isotopologue mixtures as practical internal-standard sources and establish a selection-and-validation framework for LC-MS/MS quantification when commercial SIL-IS are unavailable.
Establishment of an integrated profiling-fingerprinting-chemometrics strategy for quality control and mechanistic investigation of Fuhuo Capsule.
Liang S, Yang J, Ge J … +2 more , Nie Y, Wang X
J Chromatogr A · 2026 Aug · PMID 42127880 · Publisher ↗
Fuhuo Capsule is widely prescribed for the treatment of osteonecrosis of the femoral head; however, its chemical composition and reliable quality markers have not been comprehensively elucidated. In this work, comprehens...
Fuhuo Capsule is widely prescribed for the treatment of osteonecrosis of the femoral head; however, its chemical composition and reliable quality markers have not been comprehensively elucidated. In this work, comprehensive chemical profiling of Fuhuo Capsule was conducted using ultra-high-performance liquid chromatography in combination with quadrupole Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-MS), enabling detailed identification of its principal constituents. On this basis, an integrated quality evaluation strategy incorporating high-performance liquid chromatography (HPLC) fingerprinting, chemometric analysis, and multi-component quantification was established. In total, 183 constituents were provisionally characterized based on the analytical data obtained, and 38 common chromatographic peaks were consistently detected across 11 batches. Ten characteristic constituents were further confirmed as representative quality markers. Multivariate statistical approaches such as hierarchical clustering (HCA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to analyze the acquired data, effectively discriminated batch-to-batch variability and collectively identified 11 differential chemical markers. An HPLC-based quantitation assay was set up afterwards for the simultaneous quantitation of ten selected marker components, which exhibited satisfactory linearity, precision, repeatability, and stability. Furthermore, network pharmacology analysis indicated that these key constituents exert their therapeutic effects by interacting with critical targets such as protein kinase B (AKT1), signal transducer and activator of transcription 3 (STAT3), and interleukin-1 beta (IL-1β), and by modulating pathways including the advanced glycation end product- receptor for advanced glycation end product (AGE-RAGE) and the mitogen-activated protein kinase (MAPK) signaling pathways. Overall, this integrated strategy provides a systematic framework linking chemical profiling, fingerprinting, chemometrics, and quantification with mechanistic insights through network pharmacology and molecular modeling, thereby enhancing both the quality control and mechanistic understanding of Fuhuo Capsule.
Quantification of residual protein from biocatalysts employed in the synthesis of pharmaceutical drug substances using reversed-phase HPLC.
Swoyer MR, Flynn KM, Brown TA … +7 more , Clayman PD, Gill EL, Roberson AD, Shick JM, Hartnett EA, Grinias JP, Godinho JM
J Chromatogr A · 2026 Aug · PMID 42114345 · Publisher ↗
Biocatalysis, the use of enzymes to catalyze chemical transformations, is becoming an increasingly important focus in the manufacture of pharmaceutical drug substances. The use of biocatalysts lends marked improvements i...
Biocatalysis, the use of enzymes to catalyze chemical transformations, is becoming an increasingly important focus in the manufacture of pharmaceutical drug substances. The use of biocatalysts lends marked improvements in process efficiency and sustainability, aligning with industry-wide initiatives for improving greenness. Despite this advantage, biocatalysts introduce additional challenges that must be addressed to ensure patient safety and product quality. Notably, control of biocatalyst residuals must be demonstrated through process optimization and nuanced analytical techniques. Analytical techniques have long been established for the quantification of protein, however in this context many fall short in terms of accuracy, user-friendliness, reproducibility, and susceptibility to matrix interferences. To address these shortcomings, an analytical approach using RPLC with UV absorbance detection to quantify biocatalyst residuals in the presence of drug substances and/or synthetic intermediates is presented. Preliminary data for the broad application to small molecule and oligonucleotide drug substance modalities were demonstrated. Further, the approach was validated as a method to quantify residual protein in a late-stage small molecule intermediate synthesized using a biocatalyst. The approach provides a user friendly, robust and broadly applicable solution that overcomes critical analytical limitations, thereby supporting the continued expansion of biocatalytic routes in pharmaceutical development and manufacturing.
Porous organic framework materials for the separation of natural products: A comparative study of separation mechanisms in metal-organic frameworks and covalent organic frameworks.
Zhu T, Li X, Liu B … +3 more , Zheng W, Chen N, Zhang L
J Chromatogr A · 2026 Aug · PMID 42114344 · Publisher ↗
The efficient isolation and purification of natural products are critical for fundamental research, drug discovery, and quality control. However, the complexity of sample matrices and the structural diversity of target a...
The efficient isolation and purification of natural products are critical for fundamental research, drug discovery, and quality control. However, the complexity of sample matrices and the structural diversity of target analytes present significant challenges to conventional separation media. Traditional sorbents, including silica-based and polymeric phases, exhibit limitations regarding pore-structure tunability, surface chemistry customization, and stability, which can restrict selectivity and resolution. Conversely, Metal-Organic Frameworks (MOFs) and Covalent Organic Frameworks (COFs) possess ordered pore topologies and tailorable surface functionalities, providing a robust platform for elucidating structure-retention relationships. This review systematically compares the progress of these two materials in natural product separation from a separation science perspective. At the preparation level, solvothermal methods and post-modification strategies play a key role in regulating pore regularity and the surface microenvironment. At the mechanistic level, MOFs rely on metal-site-driven coordination and Lewis acid-base interactions, supplemented by physical adsorption, making them suitable for broad-spectrum enrichment; COFs, on the other hand, rely on a synergistic network of multiple weak interactions within their all-organic frameworks. By introducing chemical recognition sites such as boronic acid affinity, they achieve highly selective separation of molecules with specific functional groups. Analysis using flavonoids as a typical case study indicates that MOFs excel at high-capacity capture driven by metal coordination, while COFs demonstrate superior precision in recognition conferred by their covalent frameworks; the mechanisms of the two are complementary. This paper discusses key development directions, including stability, green large-scale synthesis, and rational design, providing a systematic theoretical framework for the development of next-generation separation materials.
Natural deep eutectic solvents for stilbenoid extraction from vine by-products: Solubilization and extraction efficiency study.
Serafin W, Corral MM, Valls-Fonayet J … +4 more , Gaubert A, Richard T, Cluzet S, Gaudin K
J Chromatogr A · 2026 Aug · PMID 42114274 · Publisher ↗
Natural deep eutectic solvents (NADES) have emerged as sustainable alternatives to conventional solvents for polyphenol extraction; however, their rational selection remains a major challenge due to the vast number of po...
Natural deep eutectic solvents (NADES) have emerged as sustainable alternatives to conventional solvents for polyphenol extraction; however, their rational selection remains a major challenge due to the vast number of possible component combinations and the lack of rapid screening approaches. This study aimed to develop a rapid and cost-effective ultraviolet-visible (UV-Vis)-based screening method to assess stilbenoid solubility in NADES without requiring pure standards, and to use this approach to guide the selection and optimization of a NADES-assisted extraction process. Twenty NADES were screened using a fast UV-Vis method applied to a stilbenoid-rich plant extract. The results showed moderate correlations with high-performance liquid chromatography with diode-array detection analysis (r = 0.49-0.61), enabling efficient preselection of 12 promising solvents. Among them, betaine/levulinic acid (1:2 molar ratio) exhibited the highest extraction efficiency. Process optimization using a Box-Behnken design identified the solid-to-liquid ratio as the most influential parameter, with optimal conditions corresponding to 20 min extraction time and 39% water content under ultrasound-assisted extraction. The optimized method was applied to Cabernet Sauvignon cane powders of different harvest years and particle sizes, yielding up to 2.6 g kg⁻¹ dry weight of (E)-resveratrol and 2.5 g kg⁻¹ of (E)-ε-viniferin in the most recent samples. Comparable extraction performance was obtained relative to previously reported NADES and a conventional ethanol/water system. Overall, this study demonstrates that solubility-based UV-Vis screening provides a rapid, scalable, and standard-free strategy for NADES selection, offering a practical alternative to conventional trial-and-error approaches and supporting the development of efficient and sustainable extraction processes.
Systematic regulation of pore microenvironments: Thermodynamics and mass transfer kinetics of functionalized UiO-66 chromatographic stationary phases.
Wu H, Jia J, Zhao C … +3 more , Luo Y, Pan Y, Luo J
J Chromatogr A · 2026 Aug · PMID 42114273 · Publisher ↗
To address the challenges posed by the ill-defined regulatory mechanisms of pore microenvironments in metal-organic framework (MOF) chromatographic stationary phases, this study employs continuous flow synthesis to fabri...
To address the challenges posed by the ill-defined regulatory mechanisms of pore microenvironments in metal-organic framework (MOF) chromatographic stationary phases, this study employs continuous flow synthesis to fabricate a series of functionalized UiO-66 wall-coated open-tubular column with precisely controlled thicknesses. This approach aims to decouple physical morphological interference and systematically elucidate intrinsic separation principles. In the thermodynamic dimension, the retention of aromatic compounds is synergistically governed by framework shape selectivity and π-π interactions. Conversely, for permanent gases where size-sieving is restricted, thiol functionalization achieves successful baseline separation through physical confinement and the induction of specific dipole-quadrupole interactions. Grand Canonical Monte Carlo (GCMC) simulations quantitatively confirm a significant elevation in the isosteric heat of adsorption at the molecular scale, which corroborates the macroscopic chromatographic performance. Long-term stability tests (RSD < 0.28 %) further validate the physicochemical robustness endowed by the synergy between Zr-O coordination bonds and covalent modification groups. This work provides a definitive methodological basis for the theoretical customization and engineering-scale fabrication of high-performance MOF stationary phases.
Update of descriptors for organosilicon compounds compatible with the 2025-WSU compound descriptor database for use with the solvation parameter model.
Poole CF
J Chromatogr A · 2026 Aug · PMID 42107307 · Publisher ↗
Descriptors compatible with the calibration models for the WSU-2025 compound descriptor database are assigned for 24 organosilanes of general structure RSi, 15 linear organosiloxanes of general structure RSiO(RSiO)SiR wi...
Descriptors compatible with the calibration models for the WSU-2025 compound descriptor database are assigned for 24 organosilanes of general structure RSi, 15 linear organosiloxanes of general structure RSiO(RSiO)SiR with m = 0-4, 11 cyclic organosiloxanes of general structure (RSiO) with m = 3-6, hexamethyldisilazane, hexamethylcyclotriazane, hexamethyldisilthiane, and heptamethylcyclotetrasiloxanol. The R group for organosiloxanes includes hydrogen, chlorine, methyl, vinyl, ethyl, octyl, and trimethylsiloxy and, in addition, for organosilanes acetate, methoxy, ethoxy, phenyl, nonafluorohexyl, 3-aminopropyl, 3-isocyanatopropyl, 3-glycidoxypropyl, and 3-methacryloxypropyl. Descriptors were assigned using the Solver method from experimental measurements of gas chromatographic retention factors at several temperatures on up to 12 varied stationary phases and liquid-liquid partition constants in a minimum of three totally organic biphasic systems (n-hexane-acetonitrile, n-heptane-N,N-dimethylformamide, and n-heptane-2,2,2-trifluoroethanol). Organosilicon compounds are characterized by weak dispersive interactions compared with organic compounds of a similar size, weak dipole-type interactions unless substituents contain embedded polar functional groups, an absence of hydrogen-bond acidity, except for organosilanes with -NH-, -NH or -OH functional groups (Si-H bond is not hydrogen-bond acidic), and generally relatively large but variable hydrogen-bond basicity moderated for Si-O compounds by the type of substituents co-bonded to the silicon atom. The solvation parameter model provides varied results for the estimation of the octanol-water, octanol-air, and water-air partition constants for six volatile organosiloxanes, likely resulting from the uncertainty in their experimental values since the partition constants fall outside the range for convenient laboratory measurements.
Sustainable depletion of high-abundance serum proteins using aqueous biphasic systems based on glycine-betaine analogue ionic liquids for the detection of masked biomarkers.
Pereira MM, Pedro SN, E Silva FA … +3 more , Mohamadou A, Coutinho JAP, Freire MG
J Chromatogr A · 2026 Aug · PMID 42107306 · Publisher ↗
The detection of protein biomarkers in human serum using more sustainable analytical strategies remains constrained by the predominance of high-abundance proteins, particularly human serum albumin (HSA) and immunoglobuli...
The detection of protein biomarkers in human serum using more sustainable analytical strategies remains constrained by the predominance of high-abundance proteins, particularly human serum albumin (HSA) and immunoglobulin G (IgG), which limits access to low-abundance targets. To address these limitations, aqueous biphasic systems (ABS) comprising glycine-betaine analogue ionic liquids (AGB-ILs) are introduced as a green sample preparation strategy for the selective depletion of high-abundance serum proteins. After synthesizing and characterizing AGB-ILs, including their thermal properties and ecotoxicity, these were combined with a citrate buffer and screened for ABS formation. HSA and IgG were efficiently depleted in a single step via interfacial precipitation, allowing the detection of previously suppressed analytes such as transferrin using size-exclusion high-performance liquid chromatography with UV detection (SE-HPLC-UV). This demonstrates that effective sample pretreatment can enable the use of simpler analytical techniques compared to immunoassay- and mass spectrometry-based approaches. The sustainability profile of the proposed approach was evaluated using the AGREE (Analytical GREEnness), AGREEprep (Analytical GREEnness for sample PREParation), and BAGI (BioAnalytical Greenness Index) metrics, confirming low toxicity, reduced reagent consumption, and potential for improved workflow integration. Ultimately, this work extends the applicability of IL-based ABS for protein depletion, establishing bio-inspired ILs as promising tools for green bioanalytical workflows.
Development of multiple heartcutting two-dimensional liquid chromatography with ion-pairing reversed-phase and hydrophilic interaction separations for analysis of impurities in therapeutic oligonucleotides.
Meston D, Sorensen M, Maloney T … +1 more , Stoll D
J Chromatogr A · 2026 Aug · PMID 42105443 · Publisher ↗
As the clinical applications of RNA-based medicines continue to grow, innovative analytical approaches are essential to assess quality attributes of these complex therapeutic molecules. Synthetic oligonucleotides (ONs) s...
As the clinical applications of RNA-based medicines continue to grow, innovative analytical approaches are essential to assess quality attributes of these complex therapeutic molecules. Synthetic oligonucleotides (ONs) such as small interfering RNA and antisense oligonucleotides are an expanding sub-class of genetic medicines that typically consist of single- or double-stranded RNA or DNA molecules ranging from 20 to 30 nucleotides in length. Characterization of synthetic ONs presents considerable analytical challenges due to their relatively large molecular size, polyanionic nature, and the potential for numerous synthetic modifications and impurities. It is becoming increasingly clear that conventional chromatographic methodologies are not always sufficient to fully resolve the full impurity profile present in these samples. 2D-LC is a potentially effective solution, enabling rapid resolution of species that co-elute from the first dimension using a complementary second dimension separation. In this work we explore the potential utility of pairing HILIC separation in the second dimension as a complement to a first dimension ion-pairing reversed-phase separation. Using a 23-mer surrogate ON and several closely related structures representing potential impurities, we find that the selectivities of the IPRP and HILIC separations are highly complementary, and thus suitable for pairing in a 2D separation format. Moreover, the mass loadability of the two separations are similar under the conditions studied, which gives the user flexibility when choosing which separation to use in the first dimension. We then demonstrate that an iterative retention modeling approach can be used to systematically discover shallow second dimension gradients needed to resolve species that co-elute from the first dimension column. In an unexpected twist, we found that the interface conditions must be optimized when using Active Solvent Modulation, because the weakest diluent (i.e., high ACN for a HILIC separation) does not always yield the best second dimension separation. Finally, we developed IPRP-HILIC methods for two different ON sequences that demonstrate improved resolution of previously co-eluting impurities and sufficient sensitivity for detection at 0.1 % relative to the full length product.
Optimisation and application of the metabolite extraction method of vitreous humour samples.
Pietrowska K, Gosk W, Godzien J … +5 more , Grochowski ET, Konopinska J, Kretowski A, Dmuchowska DA, Ciborowski M
J Chromatogr A · 2026 Aug · PMID 42105442 · Publisher ↗
Vitreous humour (VH) is a unique, avascular ocular structure whose biochemical composition may reflect retinal pathophysiology. Although VH has been studied using metabolomics, it remains less explored compared to other...
Vitreous humour (VH) is a unique, avascular ocular structure whose biochemical composition may reflect retinal pathophysiology. Although VH has been studied using metabolomics, it remains less explored compared to other biological matrices, and existing sample preparation protocols show considerable variation. In fact, most available reports are application-oriented studies that describe the sample preparation conditions used, without providing a rationale for why these particular parameters were chosen, consideration of alternative options, or offering a critical evaluation or systematic comparison of methodologies. Therefore, in this study, we systematically compared and optimised procedures for VH preparation prior to untargeted LC-MS analysis. Key steps, homogenisation, extraction, and preconcentration, were evaluated based on feature coverage, signal intensity, reproducibility, and practical aspects such as throughput and cost. The optimal protocol involved sample disruption using liquid nitrogen combined with ultrasound, protein precipitation and metabolite extraction with acetone, followed by evaporation and resuspension in methanol/water (2:1). This protocol was subsequently applied to VH samples from patients with epiretinal membrane (ERM, n = 14) and macular hole (MH, n = 13), conditions with different clinical manifestation but often combined into a single control group in ophthalmic studies. Univariate and multivariate analyses revealed no statistically robust metabolic differences between ERM and MH samples in this cohort. Simulation-based power analysis indicated that, under the present sample size and multiple-testing correction, the study was primarily powered to detect only large effects; therefore, subtle differences cannot be excluded. These findings support the use of ERM and MH as a pragmatic combined comparator group in LC-MS-based vitreous metabolomics, while warranting validation in larger cohorts. The optimised preparation method provides a practical framework for future metabolomic investigations of VH in ophthalmic research.
Tailored interfacial modulation for constructing high-affinity imprinted polymers for protein isolation in complex biological matrices.
Xu X, Ge Y, Wang L … +5 more , Wang C, Xie Y, Yang L, Shi Y, Liu J
J Chromatogr A · 2026 Aug · PMID 42102637 · Publisher ↗
The precise recognition of high-value proteins from complex biological matrices remains a critical challenge in separation science. In this work, a tailored interfacial modulation strategy was developed to construct high...
The precise recognition of high-value proteins from complex biological matrices remains a critical challenge in separation science. In this work, a tailored interfacial modulation strategy was developed to construct high-performance lysozyme-imprinted polymers. The combination of interfacial microenvironment regulation and a dual-monomer approach enabled the formation of stable and specific recognition sites. The obtained polymers exhibited a competitive adsorption capacity (395.13 mg/g) and a high imprinting factor (8.78), with equilibrium reached within 30 min. They showed good selectivity toward lysozyme over competing proteins and stable performance over repeated adsorption-desorption cycles. The practical applicability was further confirmed by the one-step isolation of lysozyme from a real biological matrix. Overall, this work provides a stepwise strategy to modulate the interfacial microenvironment for efficient protein separation.
Development of an ion-pairing reagent-assisted LC-MS/MS method employing fragmentation with stepped collision energy for monophosphoryl lipid A containing 3-deoxy-D-manno-octulosonic acid and its derivatives.
Hong J, Yoon J, Jin H … +3 more , Oh H, Chung HS, Kim KH
J Chromatogr A · 2026 Aug · PMID 42102636 · Publisher ↗
As interest in lipopolysaccharide (LPS)-based vaccine adjuvants continues to grow, clarification of the 3-deoxy-D-manno-octulosonic acid (Kdo)-lipid A structure, a key component of LPS that maintains outer membrane integ...
As interest in lipopolysaccharide (LPS)-based vaccine adjuvants continues to grow, clarification of the 3-deoxy-D-manno-octulosonic acid (Kdo)-lipid A structure, a key component of LPS that maintains outer membrane integrity and ensures bacterial viability, is essential. Separately, LPS functions as an immune stimulant by activating Toll-like receptor 4 and caspase-4 in human cells. While Kdo-lipid A acts as an endotoxin, its derivative, Kdo-monophosphoryl lipid A (Kdo-MPLA), shows reduced toxicity and has been explored as a therapeutic agent, including a vaccine adjuvant. Structural variations in their acyl chains and phosphate groups determine both biological activity and safety, emphasizing the need for accurate molecular characterization. However, its inherent heterogeneity and amphiphilic structure-arising from phosphate groups, lipid chains, and the Kdo sugar units-causes significant analytical challenges in reverse-phase liquid chromatography. Herein, we developed a liquid chromatography mass spectrometry method using triethylamine as an ion-pairing reagent. The complex Kdo-MPLA mixtures, extracted from Escherichia coli, were successfully separated and detected. Furthermore, the use of stepped collision energy enabled the acquisition of mass spectra with wider fragment coverage, providing detailed structural information such as Kdo sugar units as well as the specific locations and lengths of O-acyl chains. By incorporating an ion-pairing reagent, we successfully established a reliable LC-MS/MS method for Kdo-lipid A species with great selectivity and sensitivity. This approach provides detailed structural information as an identification tool for engineered Kdo-lipid A species, with potential to elucidate biosynthetic variations and establish a foundation for engineering lipid A analogs as next-generation LPS-based vaccine adjuvants.
Analysis of weight-loss compounds in health supplements by ultrasonic extraction, dispersive sorbent cleanup, and liquid chromatography tandem mass spectrometry quantification.
Nguyen KH, Hoang AQ, Vu TT … +9 more , Vu AP, Le VN, Nguyen QH, Cao CK, Dinh VC, Nguyen TO, Nguyen QH, Nguyen TAH, Le THH
J Chromatogr A · 2026 Aug · PMID 42102635 · Publisher ↗
Current society is facing the global surge in obesity rates, making people more aware of the importance of body weight control and increasing the consumption of weight-loss supplements due to their over-the-counter statu...
Current society is facing the global surge in obesity rates, making people more aware of the importance of body weight control and increasing the consumption of weight-loss supplements due to their over-the-counter status. However, certain producers have intentionally adulterated supplements with prescription weight-loss drugs without label declaration, necessitating suitable analytical methods for routine analysis of illegal weight-loss compounds in health supplements. In this study, a rapid, effective, and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 11 wt-loss compounds in three supplement forms (i.e., hard capsule, soft capsule, and tea powder). A suitable analytical procedure was selected via the optimization of LC-MS/MS parameters and sample preparation conditions. The samples were ultrasonicated with methanol, and the extracts were cleaned up using dispersive solid-phase extraction with C18 and carbon sorbent. The method was validated for specificity, linearity, method detection and quantification limits, precision, and accuracy, producing good satisfaction. The method was applied to analyze 82 health supplement samples collected from the Vietnamese market, in which seven samples were found to be adulterated with at least one of the target compounds. Sibutramine, phenolphthalein, and benzylsibutramine were detected in 7/82, 5/82, and 2/82 samples with concentration ranges of 0.44-16.4, 0.54-2.14, and 0.14-1.43 mg/g, respectively. These results indicated the method applicability to the quality control of health supplements in developed and emerging countries.
Simultaneous determination of fumonisins and hydrolyzed fumonisins in corn products and animal feed by automated dual-magnetic solid phase extraction.
Suo D, Feng Y, An Z … +3 more , Xiao Z, Wang R, Fan X
J Chromatogr A · 2026 Aug · PMID 42102634 · Publisher ↗
Fumonisins(FBs) and their hydrolyzed derivatives (HFBs), which are water-soluble mycotoxins, are commonly found in corn products and animal feed, yet data on their co-occurrence remain limited. In this study, a simple an...
Fumonisins(FBs) and their hydrolyzed derivatives (HFBs), which are water-soluble mycotoxins, are commonly found in corn products and animal feed, yet data on their co-occurrence remain limited. In this study, a simple and sensitive method based on dual-magnetic solid phase extraction with two types of magnetic materials was developed for the simultaneous determination of FB, FB, HFB, and HFB in corn products and animal feed. The method achieved limit of quantification of 1-2 μg/kg for FBs and HFBs in different samples, with recoveries ranging from 85.3% to 105% and relative standard deviations below 15%. This approach was applied to analyze 145 samples of corn products and feed to assess the co-concurrence of both FBs and HFBs, thereby providing valuable data for risk assessment.
Green ultrasound-assisted extraction of flavonoids from Scutellariae Radix using deep eutectic solvents: A hybrid BBD-ANN-GA optimization strategy and mechanistic insights.
Hu Q, Qi R, Feng X … +6 more , Jin D, Sun L, Zeng Z, Liang F, Xiong Z, Liu Z
J Chromatogr A · 2026 Jul · PMID 42102581 · Publisher ↗
Deep eutectic solvents (DESs) have emerged as promising green alternatives to conventional organic solvents for the sustainable extraction of bioactive compounds from medicinal plants. In this study, a DES-based extracti...
Deep eutectic solvents (DESs) have emerged as promising green alternatives to conventional organic solvents for the sustainable extraction of bioactive compounds from medicinal plants. In this study, a DES-based extraction system enhanced by ultrasound was employed to extract flavonoids from Scutellariae Radix (SR). A hybrid optimization strategy integrating Box-Behnken design (BBD), artificial neural network (ANN) modeling, and genetic algorithm (GA) was utilized to maximize the extraction efficiency. Under the optimized conditions, the predicted yield of flavonoids reached 194.58 mg/g, which was in close agreement with the experimental value (194.03 mg/g). The purified DES extracts exhibited strong antioxidant activity. After five consecutive extraction cycles, the DES maintained its structure stability and retained a high extraction yield with only a 11.39% decrease. The characterization of the solid extraction residue indicated the microstructure of SR was disrupted by the DES, facilitating the release of flavonoids. In addition, mechanistic studies based on quantum chemical calculations revealed strong hydrogen bonding and orbital interactions between the DES and flavonoids as key contributors to the extraction process. These results demonstrate the potential of DESs as tunable and reusable media for the efficient extraction of natural products, supported by both experimental data and molecular-level insights.
In-depth profiling of essential oils from Portuguese forest trees using GC-based methods: biogenic volatile characterization and thermal degradation relevant to fire risk.
Gonçalves OC, Martins C, Rocha SM … +3 more , Mestre AS, Neng NR, Nogueira JMF
J Chromatogr A · 2026 Jul · PMID 42102580 · Publisher ↗
This study reports the characterization of biogenic volatile organic compounds (BVOCs) present in the essential oils (EOs) extracted from major tree species in Portugal: Eucalyptus globulus Labill., Pinus pinaster Aiton,...
This study reports the characterization of biogenic volatile organic compounds (BVOCs) present in the essential oils (EOs) extracted from major tree species in Portugal: Eucalyptus globulus Labill., Pinus pinaster Aiton, Quercus robur L., and Quercus suber L. EOs were obtained by hydrodistillation of dried leaves and analysed using gas chromatography-quadrupole mass spectrometry (GC-qMS) and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-ToFMS). EO yields ranged from 0.38 % to 2.37 % (w/w, dry weight basis). GC-qMS analysis identified 34 compounds in E. globulus, 36 in P. pinaster, 34 in Q. robur, and 19 in Q. suber. The major constituents were 1,8-cineole (60.1 %) in E. globulus, abietadiene (21.6 %) in P. pinaster, kaur-16-ene (17.7 %) in Q. robur, and eugenol (5.4 %) in Q. suber. GC × GC-ToFMS showed a substantially higher analytical capacity, allowing the identification of 98 compounds in E. globulus, 57 in P. pinaster, 105 in Q. robur, and 35 in Q. suber, including 25, 13, 77, and 24 compounds reported for the first time, respectively. The dominant BVOC classes were oxygenated monoterpenoids (66.9 %) in E. globulus, hydrocarbon diterpenoids (29.8 %) in P. pinaster, hydrocarbon diterpenoids (18.0 %) in Q. robur, and miscellaneous compounds (26.4 %) in Q. suber. Beyond their industrial and therapeutic relevance, these terpenoids may act as potential fuels during forest fires. Thermal carbonization experiments (100-600 °C) further identified key BVOC classes and burning compounds, highlighting their possible contribution to flammability under extreme atmospheric conditions.
Elucidating the underlying mechanism of the total breakthrough phenomenon in reversed-phase liquid chromatography.
Miertz M, Dewil R, Cabooter D
J Chromatogr A · 2026 Jul · PMID 42096851 · Publisher ↗
The increasing adoption of two-dimensional liquid chromatography (2D-LC) for the separation of complex samples has driven the development of various modulation strategies to overcome solvent-strength mismatch when combin...
The increasing adoption of two-dimensional liquid chromatography (2D-LC) for the separation of complex samples has driven the development of various modulation strategies to overcome solvent-strength mismatch when combining orthogonal separation mechanisms, allowing to fully exploit the separation potential of 2D-LC. Conventional approaches rely on dilution or reduction of fraction volumes prior to their transfer to the second dimension column. Such approaches often require additional hardware, leading to complicated optimization procedures and potential losses in sensitivity or separation power. Recently, total breakthrough (TB) has been proposed as an alternative strategy to deal with solvent-strength mismatch in 2D-LC. TB occurs when a large volume of solvent with a high-elution strength is injected in a (second-dimension) column, resulting in the appearance of a non-retained breakthrough peak and a retained, symmetrical analyte peak. It has been theorized that TB results from interactions between analytes and unbonded silanols on the stationary phase, but no mechanistic study of this phenomenon has been undertaken so far. In this work, the influence and role of residual silanols on the occurrence of the TB phenomenon was evaluated for reversed phase liquid chromatography (RPLC) columns with different types of endcapping. Furthermore, the impact of different silanol populations was studied by varying the pH of the employed mobile phase via suitable additives. It was demonstrated that a small and comparably acidic population of residual silanols is key in the emergence of the TB phenomenon, while complete protonation of these silanols or the presence of (less acidic) silanols inhibited the emergence of TB. TB could be induced on different stationary phase chemistries, suggesting that the phenomenon is mostly governed by the type of endcapping and the applied mobile phase conditions. Understanding the underlying mechanism allows identifying optimal conditions for the emergence of this phenomenon and will enable a more effective implementation as modulation strategy in 2D-LC in the future.
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