Journal of Chromatography A最新文献

Monoclonal antibodies (mAbs) are prone to post-translational modifications during manufacturing and storage, which can affect product quality. Modifications within the complementarity-determining regions (CDRs) impairing antigen interaction are considered as critical quality attributes. A modular workflow was developed using analytical antigen affinity chromatography to enable functional separation of mAb variants based on their antigen affinity. The workflow was optimized using trastuzumab and its antigen, human epidermal growth factor receptor-2 (Her2). A linear pH gradient (pH 7.4 - 2.5) was employed to separate binding-deficient variants, which were fractionated and characterized using surface plasmon resonance (SPR), size exclusion chromatography (SEC), ion exchange chromatography (IEC), reducing capillary gel electrophoresis (CGE), and peptide mapping. Critical modifications, such as HC-D102 isomerization and LCN30 succinimide (Asu) formation, were identified as contributors to binding loss, with HC-D102 isomerization showing the most significant impact. Binding was reduced by up to 6 % in deficient fractions. The workflow was applied to two additional mAbs, demonstrating its adaptability. For mAb1 variants, binding activity decreased 10 % with HCCDR isomerization and Asu formation being critical, while for mAb2, distinct variants with binding activity ranging from 2 % to 66 % were resolved. In mAb2, Asu formation in HCCDR and LCCDR was identified as the primary modification contributing to binding loss and was found to be irreversible under physiological-like conditions (37 °C, pH 7.4). This approach enables the targeted identification of critical modifications, supports early risk mitigation as well as control strategies during development, and ensures comprehensive characterization of mAb variants.

A MXene-based molecularly imprinted polymer (MXMIP) composite was developed for the enrichment and detection of puerarin and its metabolites in complex biological samples. As an ideal substrate material, MXene can make the recognition sites within the MIP matrix more easily exposed compared to conventional MIP. Through the operation of the "dual screening" mechanism, MXMIP combines interaction enhancement with molecular recognition, significantly enhancing the adsorption capacity and efficiency of this material. The successful synthesis of the composite was confirmed by comprehensive characterization of structure and properties. MXMIP achieved a maximum adsorption capacity of 3.70 mg∙g^-1, higher than conventional MIP (2.24 mg∙g^-1) and MXene alone (1.74 mg∙g^-1). The adsorption capacity of MXMIP was 1.65 times that of conventional MIP and 2.12 times that of MXene. In selective adsorption experiments, MXMIP shows excellent specificity toward puerarin, with selectivity factors ranging from 1.33 to 10.4 relative to structural analogs, and achieved an imprinting factor of 6.46. The hydrogen bonding interactions between MXMIP and puerarin were confirmed by ultraviolet spectroscopy, Fourier transform infrared spectroscopy, and density functional theory simulations. MXMIP was successfully applied to enrich and detect puerarin and its metabolites in biological samples. LC-MS/MS analysis showed that the mass charge ratios of MXMIP adsorbed samples were 415.09, 447.13, 253.05 and 255.23, respectively, corresponding to puerarin and its metabolites (dihydroxypuerarin, daidzein and dihydrodaidzein). This study provides a new approach for synthesizing highly selective molecularly imprinted polymers that effectively address the problem of binding site interception.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Its occurrence and development are closely related to abnormal lipid metabolism, especially the metabolic changes of sphingolipids. Stable isotopes are considered a powerful tool for studying complex lipid metabolism networks. However, due to the structural complexity of lipids, achieving comprehensive and targeted detection remains challenging. This study innovatively combined stable isotope labeling technology with "modeling-prediction" strategy to achieve high targeted coverage detection and accurate quantification of isotope peaks. Moreover, a combined "static-dynamic" analysis approach was employed in this study to investigate and compare the impacts of glucose and palmitic acid on sphingolipid level in HepG2 cells and LO2 cells. A total of 220 sphingolipids were detected, among which 95 showed significant differences in response to glucose, 86 to palmitic acid, and 85 to the combination of both. Notably, 35 sphingolipids were identified as common differentially expressed sphingolipids across all conditions. Stable isotope labeling revealed that sphingolipids synthesized with glucose as the substrate had a broader isotopic distribution and higher 13C labeling than those with palmitic acid, suggesting that glucose contributes more broadly to sphingolipid-associated carbon pools through multiple metabolic routes than exogenous palmitate under our conditions. By integrating static and dynamic analyses, sphingolipid alteration profiles in hepatocellular carcinoma were further characterized, enabling the prediction of trends in sphingolipid metabolite synthesis and consumption. In summary, this study is expected to provide new insights into the study of complex sphingolipid metabolic networks.

A comprehensive assessment of the total flavonoid content and antioxidant activities of 13 medicinal materials was conducted utilizing high-performance liquid chromatography with ultraviolet-visible detector (HPLC-UVD) and spectral-effect relationship analysis. The contents of seven flavonoids were quantified, and chemometric methods including hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied to differentiate the samples. One-way analysis of variance (ANOVA) identified specific herbs for which both flavonoid profiles and antioxidant capacity varied significantly with geographical origin, underscoring the impact of provenance on bioactive quality. The results revealed significant variations in flavonoid composition, with isoquercitrin, quercetin, kaempferol, isorhamnetin, and hyperoside being identified as the key contributors to quality. A structure-activity relationship analysis was employed to elucidate how specific molecular features, such as hydroxylation and glycosylation patterns, influenced antioxidant efficacy. In vitro antioxidant assays (2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging) ranked Houttuyniae Herba as the most potent, followed by Rubi Fructus and Albiziae Flos. Grey relational analysis (GRA) identified isoquercitrin, astragalin, and hyperoside as the primary flavonoids correlated with antioxidant capacity. This work provides a scientific basis for quality assessment and bioactive component screening in multi-source medicinal materials.

The monitoring of aflatoxin B₁ (AFB₁) precursors was a significant step to prevent the production of AFB₁. In this research, a novel magnetic fluorinated covalent organic framework (F-COF@Fe₃O₄) was prepared for the extraction of AFB₁ and its two precursors of sterigmatocystin (ST) and averantin (AVN). Multiple interactions (i.e., F-O, F-F, etc.) between F-COF and analytes were confirmed via the theoretical calculation and the instrument characterization, which brought desirable extraction capacity of F-COF@Fe₃O₄ towards three targets. Thus, a purification and preconcentration method was established for the determination of AFB₁, ST and AVN with the linearity of 0.02-100 μg L^-1 and LODs of 0.012-0.017 μg L^-1. Through the application in real samples, results presented that extraction recoveries were 70.03-101.20 %, and matrix effects were 5.29-19.37 %. The applicability of this method opened a new avenue to early warn the production of AFB₁ in agricultural products.

Accurate monitoring of vehicular exhaust gases is fundamental for emission reduction and the advancement of cleaner technologies. However, conventional methods for analyzing these gases have limitations related to sensitivity, interferences, and, in some cases, the need for derivatization. In this study, the aim is to develop a chromatographic method based on gas chromatography with barrier discharge ionization for the rapid, sensitive, and simultaneous determination of H₂, CO, CH₄, and CO₂. An exhaust sample from a motorcycle was collected in a 1 L Tedlar bag, aliquots were diluted with atmospheric air using an adjustable volume graduated flask, and 0.1 mL was analyzed by GC-BID. The method demonstrated excellent selectivity, precision (1.27 to 6.22%), and accuracy (90.81 to 102.5%). Application to exhaust gas samples revealed that CO₂ (106,388 ppm_v) is predominant in the studied sample, and CO concentrations (54,189 ppm_v) were about 11 times above the regulatory limit, consistent with the older vehicle model analyzed. This method represents an efficient and sustainable alternative for emission analysis and environmental monitoring.

In this work, a novel sulfonic acid-functionalized microporous organic network-coated SiO₂ composite (SiO₂@MON-SO₃H-2) was synthesized for efficient solid-phase extraction (SPE) of quaternary ammonium alkaloids (QAAs) from complex traditional Chinese medicine (TCM) samples. The material exhibits a high specific surface area (381.4 m² g^-1), superhydrophilicity, and excellent stability. These properties, combined with synergistic interactions such as electrostatic attraction, hydrophobic effects, and π-π stacking, enabled the adsorbent to demonstrate outstanding extraction performance toward four typical QAAs. The developed method owns wide linear ranges (0.88-1000 μg L^-1) with good linearity (R² > 0.999), high sensitivity (limits of detection: 0.26-1.02 μg L^-1), satisfactory precision (intra- and inter-day RSDs < 6.3%), high enhancement factors (> 91), and insignificant matrix effects (86.6-115.3%). The material maintained excellent reusability, with recoveries remaining above 89% after 50 extraction-desorption cycles. When applied to spiked herbal matrices, the method provided recoveries of 92.7-112.0%. SiO₂@MON-SO₃H-2 also gave higher recovery than commercial C18, activated carbon, macroporous adsorption resin AB-8, and the cation-exchange resin Amberlite 732. The present work demonstrates the prospect of SiO₂@MON-SO₃H-2 for trace QAAs extraction from complex samples and provides a reliable approach for TCM analysis and quality control.

Lipids produced by different organisms are preserved as fossils in sediments and soils. These so-called lipid biomarkers and related organic proxies represent valuable tools to reconstruct past changes in ecosystems. For example, C₃₇ alkenones produced by microalgae (Isochrysidales) represent biomarkers specific for these organisms. Furthermore, an index based on C₃₇ alkenones can be used as proxy to estimate past changes in surface water temperature. However, before analysis by gas or liquid chromatography and mass spectrometry, biomarkers extracted from the sediment are treated by solid phase extraction (SPE), a process which is time-consuming and requires the use of organic solvents which can be harmful to health and environment. Here, we compared a routinely applied manual SPE (mSPE) analytical method with an automated SPE (aSPE) method using a PrepLinc platform automated sample preparation system (J2 Scientific). The analysis of marine sediments from the North Sea and the Southeast Pacific conducted with both mSPE and aSPE resulted in mainly statistically similar results, although care has to be taken when compound co-elution occurs. aSPE resulted in a higher recovery rate, but consumed more solvents, mainly for rinsing. The use of the PrepLinc platform for automated SPE, which can process up to 27 samples in a single run, allows saving not only time, but also fume hood space as the PrepLinc platform has a completely closed circuit. The PrepLinc platform represents, thus, a valuable instrument to perform automated SPE for the analysis of environmental lipid biomarkers with a high repeatability.

Developing sustainable and efficient separation technologies is crucial for achieving a green transformation in the bioprocessing industry. This study proposes an innovative chromatographic material that utilizes abundant pine wood as raw material. It employs a dextran grafting strategy to functionalize the substrate, resulting in the preparation of high-capacity anion-exchange monolithic columns. This method serves as a robust sustainability strategy, converting low-value biomass into high-value separation media. The optimized monolithic material exhibits a static adsorption capacity of 122.1 mg g^-1 for bovine serum albumin and a dynamic capacity of over 97 mg g^-1. Leveraging the natural convective channels within wood, the material achieves rapid binding kinetics and exhibits excellent column permeability of 1.12 × 10^-10 m². Its exceptional reusability (over 100 cycles) and long-term stability further demonstrate its practicality and economic viability. We validated its practical application value by successfully purifying target proteins from complex mixtures. This work integrates fundamental material innovation with scalable bioprocessing technology to provide a sustainable, high-performance, and cost-effective platform for purifying proteins and other high-value bioproducts.