Journal of Chromatography B最新文献_第10页

Preparation of sodium alginate-based temperature- and pH-responsive MOF/hydrogel microspheres and their adsorption and separation of proteins 基于海藻酸钠的温度和ph响应MOF/水凝胶微球的制备及其对蛋白质的吸附和分离。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-20 DOI: 10.1016/j.jchromb.2025.124831

Zhikun Li, Yuanyuan Li, Yulong Ma, Wenxin Ji, Yonggang Sun

The purification of biomacromolecules, particularly proteins, plays an increasingly vital role in the fields of medicine, biopharmaceuticals, and emerging technologies. Adsorbents with excellent adsorption capacity, superior cycling performance, and environmental compatibility are crucial for protein separation and adsorption. A temperature- and pH dual-stimulus responsive hydrogel material for the adsorption and separation of protein was successfully prepared by copolymerizing sodium alginate (SA) as the matrix with N-isopropylacrylamide (NIPAM), acrylic acid (AAC), and simultaneously introducing zirconium-based metal-organic frameworks (Zr-MOF). This hydrogel material exhibits excellent adsorption and controlled release properties for protein including bovine hemoglobin (BHB) and bovine serum albumin (BSA) with maximum adsorption capacities of 617.45 mg/g and 527.06 mg/g at 45 °C, respectively. Experimental results demonstrate that the adsorption and release behavior of the hydrogel toward proteins can be effectively regulated by adjusting environmental temperature or pH value in an aqueous medium without any acidic, alkaline, high-salt, or organic solvent. Additionally, the material exhibits good cyclic stability, maintaining more than 85 % of its initial protein adsorption capacity after five adsorption-desorption cycles. This study provides new insights into the development of smart responsive biomolecular adsorption materials and has significant application potential in the fields such as drug controlled release and biological separation.

生物大分子，特别是蛋白质的纯化，在医学、生物制药和新兴技术领域发挥着越来越重要的作用。吸附剂具有优异的吸附能力、优良的循环性能和环境相容性，是蛋白质分离和吸附的关键。以海藻酸钠（SA）为基体，与n -异丙基丙烯酰胺（NIPAM）、丙烯酸（AAC）共聚，同时引入锆基金属有机骨架（Zr-MOF），成功制备了一种温度和pH双刺激响应的蛋白质吸附和分离水凝胶材料。该水凝胶材料对牛血红蛋白（BHB）和牛血清白蛋白（BSA）具有良好的吸附和控释性能，在45℃下的最大吸附量分别为617.45 mg/g和527.06 mg/g。实验结果表明，在不含酸性、碱性、高盐或有机溶剂的水介质中，通过调节环境温度或pH值，可以有效调节水凝胶对蛋白质的吸附和释放行为。此外，该材料表现出良好的循环稳定性，在5次吸附-解吸循环后，其初始蛋白质吸附量保持在85%以上。该研究为智能响应型生物分子吸附材料的开发提供了新的思路，在药物控释和生物分离等领域具有重要的应用潜力。

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引用次数: 0

Development and evaluation of CD45-conjugated magnetic particles-based host cell depletion for enhanced metagenomic next-generation sequencing in bloodstream infection 基于cd45共轭磁颗粒的宿主细胞耗竭技术的开发和评估，用于增强血液感染的新一代宏基因组测序。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-15 DOI: 10.1016/j.jchromb.2025.124823

Weiwei Wu , Jingjing Song , Qiuyue Wu , Xin Wu , Ning Sun , Xinyi Xia

Metagenomic next-generation sequencing (mNGS) enables unbiased detection of human pathogens without prior assumptions. However, the direct detection of bloodstream infection pathogens is limited by host DNA interference, leading to the clinical adoption of microbial cell-free DNA mNGS (plasma cfDNA mNGS). This study developed a host cell depletion method based on immunomagnetic separation using CD45-conjugated magnetic particles (CD45-MPs, termed the IP method) to reduce host DNA interference and enhance mNGS performance for bloodstream pathogen detection. In simulated samples, known concentrations of pathogens (intact Escherichia coli and Candida albicans cells, fragmented Staphylococcus aureus genomic DNA) were spiked into whole blood samples, serially diluted 10-fold, and divided into whole blood, plasma, and IP-treated groups. These groups were analyzed using hematological analysis, microscopic smear, DNA concentration measurement, relative quantification of GAPDH using qPCR, and mNGS. Results showed that CD45-conjugated magnetic particles effectively removed host cells from whole blood (reducing cell count by 99.9%). The results of nucleic acid measurement and qPCR indicated that the supernatant from IP-treated samples contained significantly lower host DNA compared to whole blood and plasma groups. mNGS detection of simulated samples demonstrated that the IP method enabled detection of pathogens at concentrations as low as 100 CFU/mL for E. coli and S. aureus, and 50 CFU/mL for C. albicans. In clinical testing of 77 samples from patients with suspected bloodstream infections, mNGS combined with the IP method showed significantly higher positivity rates than plasma cfDNA mNGS. In conclusion, CD45-conjugated magnetic particles effectively deplete human cells and enhance the clinical performance of mNGS on the detection of bloodstream infections.

新一代宏基因组测序（mNGS）能够在没有事先假设的情况下公正地检测人类病原体。然而，直接检测血液感染病原体受到宿主DNA干扰的限制，导致临床采用微生物无细胞DNA mNGS（血浆cfDNA mNGS）。本研究开发了一种基于免疫磁分离的宿主细胞耗尽方法，使用CD45-MPs （CD45-MPs，称为IP方法）来减少宿主DNA干扰，提高mNGS在血液病原体检测中的性能。在模拟样本中，将已知浓度的病原体（完整的大肠杆菌和白色念珠菌细胞，金黄色葡萄球菌基因组DNA片段）加入全血样本中，依次稀释10倍，并分为全血、血浆和ip处理组。通过血液学分析、显微镜涂片、DNA浓度测定、qPCR相对定量GAPDH和mNGS对各组进行分析。结果表明，cd45偶联磁颗粒能有效去除全血中的宿主细胞（使细胞计数减少99.9%）。核酸测定和qPCR结果表明，与全血和血浆组相比，ip处理样品的上清液中宿主DNA含量明显降低。模拟样品的mNGS检测表明，IP方法可以检测到大肠杆菌和金黄色葡萄球菌浓度低至100 CFU/mL，白色念珠菌浓度低至50 CFU/mL。在77例疑似血流感染患者的临床检测中，mNGS联合IP法的阳性率明显高于血浆cfDNA mNGS。综上所述，cd45偶联磁颗粒能有效消耗人体细胞，增强mNGS检测血流感染的临床性能。

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引用次数: 0

LC-MS-based metabolomics revealed promising role of leukotriene receptor antagonists against colorectal cancer 基于lc - ms的代谢组学揭示了白三烯受体拮抗剂对结直肠癌的良好作用。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-15 DOI: 10.1016/j.jchromb.2025.124824

Farah Hudaib , Sanaa Bardaweel , Wesam Darwish , Salah Abdelrazig , Lina A. Dahabiyeh

Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide. Current treatments are often limited by multidrug resistance and significant side effects, underscoring the urgent need for novel therapeutic strategies. Leukotriene receptor antagonists (LTRAs), including montelukast, zafirlukast, and pranlukast, have shown promising anticancer potential; however, their underlying molecular mechanisms remain insufficiently understood. In this study, the antiproliferative effects of montelukast, zafirlukast, and pranlukast on CRC cell lines (HCT-116 and Caco-2) were evaluated using cell viability and proliferation assays. Montelukast and zafirlukast were further examined using a liquid chromatography-mass spectrometry (LC-MS)-based metabolomics to assess any possible metabolic alterations in HCT-116 cells, along with flow cytometry to determine their effects on cell death. Montelukast and zafirlukast were found to significantly inhibit the cell proliferation of HCT-116 in a dose-dependent manner, with IC₅₀ values of 57.4 μM and 73.28 μM, respectively. Flow cytometry demonstrated that apoptosis was the predominant mode of cell death with both drugs. Neither montelukast nor zafirlukast showed any significant in vitro antioxidant effects. LC-MS Metabolomics revealed that montelukast and zafirlukast induced distinct metabolic changes with 47 and 34 significantly altered metabolites, respectively. Montelukast notably affected mannosamine, 5-oxo-L-proline, acetyl-phenylalanine, acetoin, and taurine, implicating pathways related to amino acid metabolism, redox homeostasis, and mitochondrial function. In contrast, zafirlukast impacted nucleotide-related metabolites, including inosine, adenine, cytidine, deoxyguanosine, and itaconate, highlighting nucleotide biosynthesis and immunometabolism disruptions. These findings demonstrate the antiproliferative potential of montelukast and zafirlukast in CRC and provide new insights into their distinct molecular mechanisms of action. This supports their potential repurposing as adjunctive therapies in CRC treatment.

结直肠癌（CRC）仍然是全球癌症相关死亡的主要原因之一。目前的治疗往往受到多药耐药和显著副作用的限制，强调迫切需要新的治疗策略。白三烯受体拮抗剂（LTRAs），包括孟鲁司特、扎非鲁司特和普鲁司特，已经显示出有希望的抗癌潜力；然而，其潜在的分子机制仍未得到充分的了解。在这项研究中，孟鲁司特、扎非鲁司特和普鲁司特对CRC细胞系（HCT-116和Caco-2）的抗增殖作用通过细胞活力和增殖试验进行了评估。使用基于液相色谱-质谱（LC-MS）的代谢组学进一步检测孟鲁司特和扎非鲁司特，以评估HCT-116细胞中任何可能的代谢改变，并使用流式细胞术确定它们对细胞死亡的影响。发现孟鲁司特和扎非鲁司特以剂量依赖的方式显着抑制HCT-116的细胞增殖，IC₅0值分别为57.4 μM和73.28 μM。流式细胞术显示凋亡是两种药物的主要细胞死亡方式。孟鲁司特和扎非鲁司特均未显示出明显的体外抗氧化作用。LC-MS代谢组学显示孟鲁司特和扎非鲁司特诱导了明显的代谢变化，分别有47种和34种代谢物显著改变。孟鲁司特显著影响甘露胺、5-氧- l-脯氨酸、乙酰-苯丙氨酸、乙酰蛋白和牛磺酸，涉及氨基酸代谢、氧化还原稳态和线粒体功能相关的途径。相反，zafirlukast影响核苷酸相关代谢物，包括肌苷、腺嘌呤、胞苷、脱氧鸟苷和衣康酸，突出核苷酸生物合成和免疫代谢中断。这些发现证明了孟鲁司特和扎非鲁司特在结直肠癌中的抗增殖潜力，并为其独特的分子作用机制提供了新的见解。这支持了它们作为CRC治疗辅助疗法的潜力。

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引用次数: 0

Facile construction of hydrophilic core-shell structured magnetic covalent organic framework for efficient magnetic solid-phase extraction of five glucocorticoids from milk sample 亲水性核壳结构磁性共价有机骨架的快速构建，用于高效磁固相萃取牛奶样品中的五种糖皮质激素。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-12 DOI: 10.1016/j.jchromb.2025.124822

Zhi-Peng Li , Chen-Ri Su , Long-Xin Yang , Han-Yue Li , Han Zhang , Ji-Fei Yang , Xian-Hua Wang , Yun-Chao Liu , Rui-Xue Ran

Glucocorticoids play a key role in a variety of physiological processes, but their extensive use in the environment has brought potential health hazards. Herein, it is of great necessity to develop a rapid and efficient method for the detection of glucocorticoids. In this work, a hydrophilic core-shell structured magnetic covalent organic framework (HMCOF) was fabricated via a post-modification strategy for the efficient magnetic solid-phase extraction (MSPE) of five glucocorticoids from tap water and milk samples. The HMCOF featured a Fe₃O₄@SiO₂ magnetic core encapsulated by a porous COF shell modified with polyethylene glycol, endowing it with a hydrophilic outer layer, porous structure and sufficient paramagnetism. The adsorption studies showed that HMCOF exhibited high adsorption capacities (50.77–80.25 mg g⁻¹) for glucocorticoids. Notably, HMCOF retained 80 % of its adsorption capacity after 5 cycles, confirming its reusability. Under the optimal conditions of MSPE, the HMCOF-based MSPE-UPLC method was developed to test five glucocorticoids, which demonstrated good linearity (5–150 ng mL⁻¹, R² ≥ 0.9991), low detection limits (0.2–1.4 ng mL⁻¹) and satisfactory spiked recovery rates (89.5–114.2 %) with intra-day variability below 4.3 % and inter-day precision within 6.3 %. The method underscores the potential of HMCOF serving as an adsorbent for MSPE, providing a promising approach for the analysis of glucocorticoids within food products.

糖皮质激素在多种生理过程中发挥着关键作用，但其在环境中的广泛使用带来了潜在的健康危害。因此，开发一种快速有效的糖皮质激素检测方法是十分必要的。在这项工作中，通过后修饰策略制备了亲水性核壳结构磁性共价有机骨架（HMCOF），用于从自来水和牛奶样品中高效地提取五种糖皮质激素。HMCOF的特点是Fe₃O₄@SiO₂磁芯被聚乙二醇修饰的多孔COF壳包裹，使其具有亲水性外层、多孔结构和足够的顺磁性。吸附研究表明，HMCOF对糖皮质激素具有较高的吸附量（50.77 ~ 80.25 mg g-1）。值得注意的是，HMCOF在5次循环后仍保持了80%的吸附容量，证实了其可重复使用。在最佳MSPE条件下，建立了基于hmcof的MSPE- uplc检测5种糖皮质激素的方法，线性良好（5 ~ 150 ng mL-1, R2≥0.9991），检出限低（0.2 ~ 1.4 ng mL-1），加标回收率（89.5 ~ 114.2%），日内变异性小于4.3%，日内精密度在6.3%以内。该方法强调了HMCOF作为MSPE吸附剂的潜力，为食品中糖皮质激素的分析提供了一种有前途的方法。

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引用次数: 0

Hypoglycemic potential of American ginseng saponins: inhibition of α-amylase and improvement of insulin resistance 西洋参皂苷的降糖潜能：抑制α-淀粉酶和改善胰岛素抵抗。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-10 DOI: 10.1016/j.jchromb.2025.124813

Liwen Liang , Yunlong Guo , Xu Yao , Guangzhi Cai , Xiaokang Liu , Jiyu Gong

Diabetes Mellitus (DM) represents a significant global health challenge, necessitating the development of novel therapeutic agents with reduced adverse effects for effective management. This study investigated the hypoglycemic potential of total saponins derived from American ginseng (TSAG), focusing on its inhibitory activity against α-amylase and its modulatory effects on insulin resistance in HepG2 cells. Results from the α-amylase inhibition assay demonstrated that TSAG elicited significant, concentration-dependent inhibition. Enzyme kinetic analysis revealed that TSAG functions as a reversible, mixed-type inhibitor of α-amylase. In insulin-resistant HepG2 cells, TSAG significantly enhanced glucose consumption and promoted glycogen synthesis, indicating its capacity to ameliorate insulin sensitivity. Ultra-high-performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry (UHPLC-Q-Orbitrap/MS) analysis identified 40 ginsenoside constituents within TSAG. Molecular docking studies demonstrated high-affinity binding interactions between these key ginsenosides and α-amylase, corroborating their contribution to the observed inhibitory effects. Collectively, these findings highlight the therapeutic potential of American ginseng saponins in diabetes management, particularly in improving postprandial glycemic control and enhancing insulin sensitivity.

糖尿病（DM）是一个重大的全球健康挑战，需要开发新的治疗药物，减少不良反应的有效管理。本研究探讨了西洋参总皂苷（TSAG）的降糖潜能，重点研究了其对α-淀粉酶的抑制作用及其对HepG2细胞胰岛素抵抗的调节作用。α-淀粉酶抑制实验结果表明，TSAG具有显著的浓度依赖性抑制作用。酶动力学分析表明，TSAG是一种可逆的混合型α-淀粉酶抑制剂。在胰岛素抵抗的HepG2细胞中，TSAG显著增加葡萄糖消耗，促进糖原合成，表明其改善胰岛素敏感性的能力。超高效液相色谱-四极杆轨道阱质谱联用（UHPLC-Q-Orbitrap/MS）分析鉴定出人参皂苷的40种成分。分子对接研究表明，这些关键人参皂苷与α-淀粉酶之间存在高亲和力的结合作用，证实了它们对观察到的抑制作用的贡献。总的来说，这些发现强调了西洋参皂苷在糖尿病治疗中的治疗潜力，特别是在改善餐后血糖控制和提高胰岛素敏感性方面。

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引用次数: 0

Secretory expression and optimization of type II L-asparaginase from Pseudomonas sp. PCH199 假单胞菌PCH199型l -天冬酰胺酶的分泌表达及优化

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-08 DOI: 10.1016/j.jchromb.2025.124814

Subhash Kumar , Virender Kumar , Dharam Singh

L-asparaginase (L-ASNase) is used to treat acute lymphoblastic leukemia and acrylamide reduction in food. Presently, recombinant L-ASNase formulations are produced in the cytoplasmic fractions of E. coli. Here, we describe an efficient approach for secretory expression of recombinant L-ASNase II (Pg-ASNase II) with pelB signal peptide in the E. coli expression host. Pg-asn II was successfully cloned and expressed in pET-26b(+) having pelB leader sequence for periplasmic expression. It led to the successful secretion of Pg-ASNase II in the supernatant. However, a significant amount of Pg-ASNase II was also left in the cytoplasmic space. The protein secretion increased from 0.33 to 0.77 mg mL^-1 with 0.2 % Tween 80 in the medium. Pg-ASNase II was purified in a single chromatographic step using a Q-sepharose column with 8.0 mg L⁻¹ purified protein production and 60.0 U mg^-1 Pg-ASNase II activity. It demonstrated a 75 % yield and a 15-fold increase in purification. The current study reported an increased extracellular L-ASNase expression compared to the wild organism and helped reduce the cost of the downstream process. Hence, this research can contribute to upcoming investigations of periplasmic Pg-ASNase II for therapeutic applications.

l -天冬酰胺酶（L-ASNase）用于治疗急性淋巴细胞白血病和减少食物中的丙烯酰胺。目前，重组L-ASNase制剂是在大肠杆菌的细胞质部分中生产的。在此，我们描述了一种在大肠杆菌表达宿主中分泌表达含有pelB信号肽的重组L-ASNase II （Pg-ASNase II）的有效方法。成功克隆出Pg-asn II，并在pET-26b（+）中表达，具有pelB领导序列，可在质周表达。这导致Pg-ASNase II在上清液中成功分泌。然而，大量的Pg-ASNase II也留在细胞质间隙中。当培养基中添加0.2%的Tween 80时，蛋白质分泌量从0.33 mg mL-1增加到0.77 mg mL-1。Pg-ASNase II采用Q-sepharose柱纯化，纯化蛋白产量为8.0 mg L-1，活性为60.0 U mg- 1pg - asnase II。它证明了75%的产量和15倍的净化。目前的研究报告了与野生生物相比，细胞外L-ASNase表达增加，有助于降低下游过程的成本。因此，本研究可以为即将开展的质周Pg-ASNase II的治疗应用研究做出贡献。

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引用次数: 0

Innovative multifunctional tag system for protein purification and analytical characterization 用于蛋白质纯化和分析表征的创新多功能标签系统。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-08 DOI: 10.1016/j.jchromb.2025.124811

María Jesús Leopold, Verónica Ferrando , Ricardo Kratje, Marcos Oggero, Natalia Ceaglio

Epitope tag–monoclonal antibody (mAb) systems are essential tools in biotechnology. We developed a novel system based on the mGMOP peptide—derived from GM-CSF with six O-glycosylation sites and an N-terminal APARSPS epitope—and the in-house mAb CC1H7, which binds this epitope, in single or multiple copies, with enhanced affinity under high ionic strength. The mGMOP–mAb CC1H7 system was applied to immunoaffinity chromatography, Western blot, and competitive ELISA. Optimization used two engineered interferon variants: one with a single tag (mGMOP–IFN) and another with four tags (mGMOP3–IFN–mGMOP). A third variant, mGMOP3–EPO–mGMOP, was constructed using erythropoietin for further validation. Immunoaffinity purification was optimized via factorial design, testing different salts. The final protocol used 1 M Na₂SO₄ for loading and 50 mM sodium phosphate at pH 11 for elution, achieving 80–92 % recovery and 100 % purity. Competitive ELISA was optimized using a Box-Behnken design, yielding low LOD and LOQ. Western blotting confirmed detection of both IFN variants, also with low LOD and LOQ. All methods were successfully applied to tagged EPO. The platform enables standardized workflows for characterizing recombinant proteins without requiring protein-specific antibodies, and offers a versatile approach for protein bioprocessing.

表位标签-单克隆抗体（mAb）系统是生物技术中必不可少的工具。我们开发了一种基于mGMOP肽（源自GM-CSF，具有6个o糖基化位点和一个n端APARSPS表位）和内部单克隆抗体CC1H7的新系统，该单克隆或多拷贝结合该表位，在高离子强度下具有增强的亲和力。mgop - mab CC1H7系统应用于免疫亲和层析、Western blot和竞争性ELISA。优化使用了两种工程干扰素变体：一种带有单个标签（mgmopp - ifn），另一种带有四个标签（mGMOP3-IFN-mGMOP）。第三个变体mGMOP3-EPO-mGMOP使用促红细胞生成素构建以进一步验证。通过析因设计优化免疫亲和纯化，检测不同盐类。最终方案采用1 M硫酸钠（Na₂SO₄）和50 mM磷酸钠（pH 11）洗脱，回收率为80- 92%，纯度为100%。竞争性ELISA采用Box-Behnken设计优化，LOD和LOQ均较低。Western blotting证实检测到两种IFN变体，同样具有低LOD和LOQ。所有方法均成功应用于标记EPO。该平台实现了标准化的工作流程，无需蛋白质特异性抗体即可表征重组蛋白，并为蛋白质生物处理提供了一种通用的方法。

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引用次数: 0

Accurate determination of fluoxetine and its metabolites in Mediterranean mussel organs based on solid phase extraction clean-up and HPLC-HRMS 固相萃取净化- HPLC-HRMS法精确测定地中海贻贝器官中氟西汀及其代谢物

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-08 DOI: 10.1016/j.jchromb.2025.124808

E. Lemaire , E. Gomez , D. Rosain , F. Courant

Fluoxetine, a widely prescribed antidepressant, is frequently detected in marine environments, exposing marine invertebrates to this emerging contaminant. Due to its potential to bioaccumulate and undergo biotransformation, it is essential to quantify both the parent compound and its metabolites for accurate risk assessment. Furthermore, tissue-specific measurements are crucial for identifying organ-specific accumulation that may cause localized toxic effects. An analytical method was then developed to quantify fluoxetine and three of its metabolites in individual mussel tissues (soft tissues, gills, digestive glands) using a minimal sample size (40 or 400 mg). Four solid-phase extraction sorbents were evaluated: MCX, HLB, C18, and Phree phospholipid removal. The Phree sorbent was selected as the optimal cleanup method. Further optimization involved testing two extraction solvents (acetonitrile and methanol) and two extraction techniques: ultrasonic-assisted extraction and bead-based extraction. Identification and quantification were performed using HPLC-Orbitrap-MS, relying on accurate mass measurements of selected MS/MS fragments. The method demonstrated satisfactory Method Quantification Limits (MQL) for fluoxetine (1.2, 10.5, and 10.4 μg/kg dw) and its main metabolite, norfluoxetine (5.1, 25.6, and 24.3 μg/kg dw) in soft tissues, digestive glands, and gills, respectively. These MQL values align with literature data, especially given the low sample size and the expression of concentrations on a dry weight basis. The final method allows the sensitive quantification of fluoxetine and its metabolites from small volumes, facilitating the assessment of their distribution across three target organs. These advancements support the development of toxicokinetic/toxicodynamic (TK/TD) models, contributing to environmental risk assessment of emerging contaminants.

氟西汀是一种广泛使用的抗抑郁药，经常在海洋环境中检测到，使海洋无脊椎动物暴露于这种新出现的污染物中。由于其具有生物积累和生物转化的潜力，因此对母体化合物及其代谢物进行量化以进行准确的风险评估至关重要。此外，组织特异性测量对于识别可能导致局部毒性作用的器官特异性积累至关重要。然后开发了一种分析方法，使用最小样本量（40或400毫克）对单个贻贝组织（软组织、鳃、消化腺）中的氟西汀及其三种代谢物进行定量。评估了四种固相萃取吸附剂：MCX， HLB， C18和Phree磷脂去除。选择Phree吸附剂作为最佳的清除方法。进一步的优化包括测试两种提取溶剂（乙腈和甲醇）和两种提取技术：超声波辅助提取和珠状提取。使用HPLC-Orbitrap-MS进行鉴定和定量，依赖于所选MS/MS片段的精确质量测量。该方法在软组织、消化腺和鳃中分别检测到氟西汀（1.2、10.5和10.4 μg/kg dw）及其主要代谢物去甲氟西汀（5.1、25.6和24.3 μg/kg dw）的定量限（MQL）满意。这些MQL值与文献数据一致，特别是考虑到低样本量和以干重为基础的浓度表达。最后一种方法允许小体积的氟西汀及其代谢物的敏感定量，便于评估它们在三个目标器官中的分布。这些进展支持毒物动力学/毒物动力学（TK/TD）模型的发展，有助于对新出现的污染物进行环境风险评估。

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引用次数: 0

A fast GC–MS/MS method for the simultaneous measurement of key metabolites of peroxisomal beta-oxidation and ether lipid biosynthesis in human fibroblasts 同时测定人成纤维细胞过氧化物酶体β -氧化和醚类脂质生物合成关键代谢物的快速GC-MS /MS方法

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-06 DOI: 10.1016/j.jchromb.2025.124806

Karin Preindl , Thomas Stimpfl , Gunda Koellensperger , Fabian Dorninger , Johannes Berger , Christoph Wiesinger

Peroxisomes are subcellular compartments that host a variety of metabolic pathways, including the chain shortening of fatty acids (FAs) by beta-oxidation and certain steps in the formation of ether lipids. Here, we describe the development of a GC–MS/MS-based method for the simultaneous and reproducible determination of key metabolites of these pathways, also including less common FA species related to peroxisomal metabolism that are typically not part of standard analytical methods.

We for the first time utilize 1-chlorobutane for the extraction of FAs as an effective alternative to commonly used extraction solvents. 1-Chlorobutane offers a broader polarity range than hexane and lower toxicity relative to chloroform with solvent consumption of less than one mL per sample.

Six saturated long to very long-chain FAs, nine polyunsaturated FAs (PUFAs), two dicarboxylic FAs and 1-O-octadecyl glycerol (ODG, batyl alcohol) were extracted simultaneously. The method was validated using fibroblasts and for the majority of FA species accuracies ranged from 80 to 110 % with precision values (CV %) from 6 to 20 %. The measurement of ODG is for the first time described as marker for the estimation of the cellular ether lipid synthesis rate. The suitability of the method was demonstrated by the analysis of primary human fibroblasts from controls and individuals with peroxisomal disorders. This cell type represents a widely used model system for the investigation of peroxisomal metabolism and disease, thus rendering our protocol a valuable addition to the toolkit for studying peroxisomal pathways.

过氧化物酶体是承载多种代谢途径的亚细胞室，包括通过β -氧化缩短脂肪酸（FAs）链和形成醚类脂质的某些步骤。在这里，我们描述了一种基于GC-MS / ms的方法的发展，用于同时和可重复性地测定这些途径的关键代谢物，也包括与过氧化物酶体代谢相关的不太常见的FA物种，这些物种通常不是标准分析方法的一部分。我们首次利用1-氯丁烷作为一种有效的萃取溶剂来萃取脂肪酸。1-氯丁烷的极性范围比己烷宽，毒性比氯仿低，每个样品的溶剂消耗量小于1 mL。同时提取了6个饱和长链至甚长链脂肪酸，9个多不饱和脂肪酸（PUFAs）， 2个二羧基脂肪酸和1- o -十八烷基甘油（ODG, batyl alcohol）。用成纤维细胞验证了该方法，对大多数FA物种的准确度在80%到110%之间，精度值（CV %）在6%到20%之间。ODG的测定首次被描述为估计细胞醚脂质合成速率的标记物。通过分析来自对照组和过氧化物酶体疾病患者的原代人成纤维细胞，证明了该方法的适用性。这种细胞类型代表了研究过氧化物酶体代谢和疾病的广泛使用的模型系统，因此使我们的方案成为研究过氧化物酶体途径的工具包的有价值的补充。

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引用次数: 0

Recommendation of a milder cleaning-in-place method for Cytiva's second-generation protein L resin MabSelect VL 推荐Cytiva第二代蛋白L树脂MabSelect VL的温和就地清洗方法。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-10-04 DOI: 10.1016/j.jchromb.2025.124810

Ju Qu, Yifeng Li, Yan Wan

Protein L-based media are a type of affinity resin whose application in antibody purification is only secondary to Protein A resins. As Protein L resins exhibit stronger byproduct separation capabilities than Protein A resins, they are particularly preferred when purifying complex molecules such as bispecific antibodies (bsAbs). Despite the growing use of Protein L resins, comprehensive evaluations of their lifetime and cleaning-in-place (CIP) methods are generally lacking. In the current study, we examined MabSelect VL, Cytiva's second-generation Protein L resin, for its alkaline stability. In specific, the resin's performance was evaluated after 80 cycles of sanitization with 0.1 M sodium hydroxide (NaOH). In addition, the binding behavior of monomer and aggregates to aged resin was studied to understand the change in resin ligands during use. According to the data, 0.1 M NaOH is not well tolerated by MabSelect VL, and its repeated treatment causes performance degradation. Therefore, we recommend a milder sanitization method, 0.05 M NaOH, which can be combined with 1 % benzyl alcohol to improve microbial control.

蛋白l基介质是一种亲和树脂，其在抗体纯化中的应用仅次于蛋白a树脂。由于蛋白L树脂比蛋白A树脂具有更强的副产物分离能力，因此它们在纯化复杂分子（如双特异性抗体（bsab））时特别受欢迎。尽管蛋白L树脂的使用越来越多，但通常缺乏对其使用寿命和就地清洗（CIP）方法的全面评估。在目前的研究中，我们检测了MabSelect VL， Cytiva的第二代蛋白L树脂，其碱性稳定性。以0.1 M氢氧化钠（NaOH）消毒80次后，对树脂的性能进行了评价。此外，研究了单体和聚集体与老化树脂的结合行为，以了解树脂配体在使用过程中的变化。数据显示，MabSelect VL对0.1 M NaOH的耐受性不佳，反复处理会导致性能下降。因此，我们推荐一种较温和的消毒方法，0.05 M NaOH，可与1%苄醇结合使用，以改善微生物控制。

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引用次数: 0