Journal of Chromatography A最新文献_第3页

Advances in LC–MS strategies for comprehensive characterization and quantification of antibody-drug conjugates in preclinical and clinical settings 临床前和临床环境中抗体-药物偶联物的LC-MS综合表征和定量研究进展

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-04 DOI: 10.1016/j.chroma.2025.466606

Devendra Kumar , Neerja Trivedi

Antibody-Drug Conjugates (ADCs) represent a rapidly growing class of targeted therapeutics, combining the specificity of monoclonal antibodies with the potency of cytotoxic payloads. The structural complexity and heterogeneity of ADCs arising from variations in drug to antibody ratio (DAR), conjugation sites, and post translational modifications demand advanced analytical strategies for comprehensive characterization and quantification throughout development. Liquid chromatography coupled with mass spectrometry (LC–MS) has emerged as an indispensable platform for ADC analysis, offering high sensitivity, selectivity, and structural resolution across multiple levels. This review highlights recent advances in LC–MS workflows, including intact mass analysis, subunit/middle-down profiling, peptide mapping, and bioanalytical assays for free payloads and catabolites. We discuss emerging technologies such as multi-attribute methods (MAM), native MS, ion mobility, and hybrid ligand-binding assay (LBA)–LC–MS platforms that enhance throughput and analytical depth. Special focus is given to quantification strategies in biological matrices and regulatory expectations, including International Council for Harmonization (ICH) M10 and Food and Drug Administration (FDA) guidance on method validation. As ADC pipelines expand into new therapeutic areas, the integration of automation and AI-driven data processing is poised to transform LC–MS into a high throughput, intelligent tool for both product characterization and clinical monitoring. These innovations collectively support safer, more effective ADC development from discovery through approval.

抗体-药物偶联物（adc）结合了单克隆抗体的特异性和细胞毒性有效载荷的效力，代表了一种快速增长的靶向治疗方法。adc的结构复杂性和异质性是由药抗比（DAR）、偶联位点和翻译后修饰的变化引起的，需要先进的分析策略来在整个开发过程中进行全面的表征和定量。液相色谱联用质谱（LC-MS）已成为ADC分析不可或缺的平台，具有高灵敏度、选择性和跨多个水平的结构分辨率。本文重点介绍了LC-MS工作流程的最新进展，包括完整质量分析、亚基/中向下分析、肽图谱以及游离有效载荷和分解代谢物的生物分析分析。我们讨论了新兴技术，如多属性方法（MAM）、原生质谱、离子迁移率和混合配体结合测定(LBA)-LC-MS平台，这些技术提高了通量和分析深度。特别关注生物基质的定量策略和监管期望，包括国际协调委员会（ICH） M10和食品和药物管理局（FDA）关于方法验证的指导。随着ADC产品线扩展到新的治疗领域，自动化和人工智能驱动的数据处理的集成将使LC-MS转变为高通量、智能的产品表征和临床监测工具。这些创新共同支持从发现到批准的更安全、更有效的ADC开发。

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引用次数: 0

Quantitative profiling of aromatic amino acids and their host–microbial co-metabolites in human urine via UPLC–MS/MS 人尿中芳香氨基酸及其宿主-微生物共同代谢产物的UPLC-MS /MS定量分析

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-03 DOI: 10.1016/j.chroma.2025.466604

Domniki Gallou , Olga Begou , Helen Gika , Vasiliki Sarli , Ian D. Wilson , Georgios Theodoridis

The aromatic amino acids tryptophan, tyrosine and phenylalanine are involved in many biochemical pathways and their metabolism and co-metabolism by the human and the gut microbiota results in the production of a number of metabolites. Many of these are phenols which are excreted in the urine after either sulfation or glucuronidation by the host. These metabolic processes can be dysregulated due to factors such as inflammation, disease, dietary and/or pharmaceutical interventions. Validated, quantitative methods for the analysis of aromatic amino acids and their metabolites may therefore provide insights into host-gut microbiota symbiosis and its association with pathological conditions. As sulfation is often the favored form of conjugation for phenolic compounds, the development of analytical methods would benefit from access to the sulfate conjugates as reference standards, which unfortunately are scarce. To overcome this limitation nine sulfate conjugates were synthesized using standard chemical routes and were subsequently purified by preparative HPLC. Following structure confirmation (¹H NMR and MS/MS analysis) the standards were used along with 24 other analytes for the development and validation of a quantitative LC-MS/MS-based assay. The method used a CSH Phenyl-Hexyl column to separate and analyze 33 aromatic amino acids and their metabolites in urine. The method was validated and subsequently applied to the analysis of urine obtained from 20 healthy individuals to obtain information on the relevant concentrations in human urine.

芳香氨基酸色氨酸、酪氨酸和苯丙氨酸参与许多生化途径，它们在人体和肠道微生物群中的代谢和协同代谢导致许多代谢物的产生。其中许多是酚类物质，经宿主磺化或葡萄糖醛酸化后随尿液排出。这些代谢过程可能由于炎症、疾病、饮食和/或药物干预等因素而失调。经过验证的芳香氨基酸及其代谢物的定量分析方法可能因此为宿主-肠道微生物群共生及其与病理状况的关联提供见解。由于硫酸化通常是酚类化合物的首选偶联形式，因此获得硫酸盐偶联物作为参考标准将有利于分析方法的发展，不幸的是，这些参考标准很少。为了克服这一限制，采用标准化学路线合成了9种硫酸盐缀合物，随后采用制备高效液相色谱法纯化。在结构确认（1H NMR和MS/MS分析）之后，将标准品与其他24种分析物一起用于开发和验证定量LC-MS/MS-based分析。该方法采用CSH苯基-己基柱分离分析尿液中的33种芳香氨基酸及其代谢物。对该方法进行了验证，并随后将其应用于20名健康个体的尿液分析，以获得人类尿液中相关浓度的信息。

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引用次数: 0

Analytical characterization and stability evaluation of high-purity melittin isolated from crude bee venom via one-step, scalable reversed-phase liquid chromatography 用一步可扩展反相液相色谱法从粗蜂毒中分离高纯度蜂毒素的分析表征和稳定性评价

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-03 DOI: 10.1016/j.chroma.2025.466605

Farnaz Fatahian , Hassan Rezadoost , Seyed Mohammad Jafar Seyed Golestan , Hossein Behboudi , Martina Catani , Alberto Cavazzini , Alireza Ghassempour

Melittin (MEL), the principal peptide component of honeybee venom (Apis mellifera), is of considerable interest owing to its broad biological activities. However, the isolation of MEL in high purity remains analytically challenging, primarily due to the chemical complexity of crude venom and the amphipathic nature of the peptide. Here, we describe a one-step, scalable reversed-phase liquid chromatography (RP-HPLC) approach that affords MEL with 99.05 % recovery and 98.44 % purity, markedly exceeding the quality of available commercial standards. The identity and structural integrity of the purified peptide were confirmed through complementary analytical techniques, including RP-HPLC, electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and circular dichroism (CD) spectroscopy. Accelerated stability testing under ICH conditions (40°C, 75 % RH) over six months demonstrated a substantially higher chemical and conformational stability of purified MEL compared with crude venom, with degradation following first-order kinetics. Taken together, these findings establish an efficient and reproducible chromatographic workflow for the preparative isolation and analytical characterization of MEL from a complex natural source, while stability profiling highlights its suitability for downstream pharmaceutical applications.

蜂毒素（MEL）是蜂毒（Apis mellifera）的主要肽成分，由于其广泛的生物活性而引起了相当大的兴趣。然而，高纯度MEL的分离在分析上仍然具有挑战性，主要是由于粗毒液的化学复杂性和肽的两性性质。在这里，我们描述了一个一步，可扩展的反相液相色谱（RP-HPLC）方法，提供MEL 99.05%的回收率和98.44%的纯度，明显超过现有的商业标准的质量。通过RP-HPLC、电喷雾质谱（ESI-MS）、基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和圆二色（CD）光谱等互补分析技术确认了纯化肽的身份和结构完整性。在ICH条件下（40°C, 75% RH）超过6个月的加速稳定性测试表明，与粗毒液相比，纯化MEL的化学和构象稳定性显著提高，降解遵循一级动力学。综上所述，这些发现为复杂天然来源MEL的制备分离和分析表征建立了一个高效、可重复的色谱工作流程，而稳定性分析强调了其对下游制药应用的适用性。

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引用次数: 0

On the intrinsic effect of the particle size distribution on the permeability of particulate liquid chromatography columns. A theoretical overview 粒径分布对颗粒液相色谱柱渗透性的内在影响。理论概述。

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-02 DOI: 10.1016/j.chroma.2025.466601

Fabrice Gritti

This study presents a theoretical investigation into the intrinsic influence of particle size distribution (PSD) on the permeability of packed beds in liquid chromatography columns. Using a log-normal PSD derived from experimental data on

2 μ m

BEH 66

Å

Particles, the work explores how different definitions of average particle size, number-, volume-, and surface-area-weighted (Sauter mean), affect the comparison between monodisperse and polydisperse particle packings when estimating the solid-to-fluid surface area in various models of column permeability. By normalizing permeability to a constant void fraction and average particle size, it is shown that the calculation of the permeability of columns packed with polydisperse particles is found to be either higher or lower than that of monodisperse packings, depending on the chosen reference particle size. Notably, for the same void fraction and Sauter mean diameter, the width of the particle size distribution (PSD) of any chromatographic packing material has no intrinsic impact on the column permeability. This confirms that, in addition to void fraction, the Sauter mean diameter or the specific surface area is the second most critical parameter for fair and meaningful comparison. These findings are supported by analytical derivations and validated against published fluid dynamics simulations across a broad range of microstructures composed of both highly polydisperse and strictly monodisperse non-overlapping sphere packings as well as few simulated and measured permeability data of liquid chromatography columns. Overall, the results offer a robust framework for interpreting column permeability and guiding particle design strategies aimed at maximizing chromatographic speed and performance.

本文从理论上探讨了粒径分布对液相色谱柱填充层渗透率的内在影响。利用2μm BEH 66Å颗粒实验数据得出的对数正态PSD，研究了在各种柱渗透率模型中，平均粒径、数量、体积和表面积加权（Sauter平均值）的不同定义如何影响单分散和多分散颗粒填料在估计固液比表面积时的比较。通过将渗透率归一化为恒定的孔隙率和平均粒径，可以发现，根据所选择的参考粒径，多分散颗粒填充柱的渗透率计算值高于或低于单分散填料的渗透率。值得注意的是，对于相同的孔隙率和Sauter平均直径，任何色谱填料的粒径分布宽度（PSD）对柱渗透率没有内在影响。这证实，除了孔隙率之外，Sauter平均直径或比表面积是进行公平和有意义比较的第二个最关键参数。这些发现得到了分析推导的支持，并通过广泛的流体动力学模拟得到了验证，这些模拟包括高度多分散和严格单分散的非重叠球体填料，以及很少的液相色谱柱的模拟和测量渗透率数据。总的来说，结果为解释柱渗透率和指导颗粒设计策略提供了一个强大的框架，旨在最大限度地提高色谱速度和性能。

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引用次数: 0

Recent advances in modern extracellular vesicle isolation and separation techniques 现代细胞外囊泡分离技术的最新进展。

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-02 DOI: 10.1016/j.chroma.2025.466602

Thanaporn Liangsupree , Evgen Multia , Marja-Liisa Riekkola

Since slightly sluggish beginning some twenty years ago, heterogeneous nanosized extracellular vesicles (EVs), secreted by almost all mammalian cells, all Gram-negative and Gram-positive bacteria and several plant tissues, have grabbed an enormous attention especially during the last years due to their functional role in intercellular and interorgan communications, offering possibilities for great innovations in diagnostics and therapeutic monitoring and prognosis. The availability of their reliable isolation and separation techniques that can conquer most of the challenges related to purity, yield, scalability, integrity, nanoscale contaminants, operation time, and complex sample matrices is a crucial prerequisite for the exploitation of EVs and their subpopulations. The utmost goal of this review, that is a continuation of our overview published in 2021, is to introduce the most recent modern and emerging isolation and separation approaches and trends, with their specific properties and applications. The focus is again on size-, charge-, and affinity-based techniques and ultracentrifugation and precipitation-based techniques are included only for comparison purposes. The isolation techniques will be briefly compared with each other in terms of different important parameters. Furthermore, a few industrial promising applications are introduced. Although in the newly developed isolation and separation techniques and methods especially the presence of interfering nanocomponents, causing lower purity, has been much better minimized or even eliminated, and although even in vivo studies have more frequently been carried out, it is obvious that only combined and hyphenated techniques can overcome at least most of the challenges.

自20年前开始，几乎所有哺乳动物细胞、所有革兰氏阴性和革兰氏阳性细菌以及几种植物组织都分泌异质纳米胞外囊泡（ev），由于其在细胞间和器官间通讯中的功能作用，特别是在最近几年引起了极大的关注，为诊断、治疗监测和预后的巨大创新提供了可能性。可靠的分离技术可以克服纯度、产量、可扩展性、完整性、纳米级污染物、操作时间和复杂样品基质等方面的挑战，这是开发电动汽车及其亚群的关键先决条件。这篇综述的最大目标是介绍最新的现代和新兴的隔离和分离方法和趋势，以及它们的具体特性和应用，这是我们在2021年发表的综述的延续。重点再次放在基于尺寸、电荷和亲和力的技术上，而基于超离心和沉淀的技术仅用于比较目的。本文将根据不同的重要参数对这些隔离技术进行简要比较。此外，还介绍了几种具有工业前景的应用。尽管在新开发的分离技术和方法中，特别是干扰纳米成分的存在，导致纯度降低，已经更好地最小化甚至消除，尽管甚至更频繁地进行体内研究，但很明显，只有结合和连接技术才能克服至少大部分的挑战。

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引用次数: 0

The use of multiple liquid chromatography methods augmented by phosphorus-31 nuclear magnetic resonance to characterize the diastereomer composition in synthetic oligonucleotides 利用磷-31核磁共振增强的多种液相色谱方法表征合成寡核苷酸的非对映体组成

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-02 DOI: 10.1016/j.chroma.2025.466600

Mohsin Ali , Akanksha Manghrani , Mirandia Szramowski , Ahmed M. Abdel-Megied , Likan Liang , Nils F. Aberg , Kui Yang , Kang Chen , Yan Wang , Deyi Zhang , Steven Fletcher , Robert G. Brinson , Jace W. Jones

Synthetic oligonucleotide therapeutics represent a rapidly advancing class of drugs that target RNA to modulate gene expression. The phosphorothioate modification to the nucleic acid backbone is routinely used to increase resistance to enzymatic degradation and improve the pharmacological profile. Each phosphorothioate modification creates a stereogenic center, leading to the formation of diastereomers at every modified linkage. This results in a final drug product containing a complex and heterogenous mixture of diastereomers. With the increasing regulatory approval of synthetic oligonucleotide drug products, there is a pressing need to develop analytical methods that can characterize their diastereomer composition. We detail the use of four liquid chromatography methods complemented by ³¹P NMR to characterize the diastereomer composition of fully phosphorothioated short (2- and 5-mers) and full-length (20-mers) oligonucleotide sequences. We also assessed the effect of how specific chemical activators influenced the diastereomer composition in these synthetic sequences. Our data showed that the use of multiple liquid chromatography methods augmented by ³¹P NMR provided complementary and additional insight into the diastereomer composition of phosphorothioate-linked oligonucleotides. Further, our data also indicated that the chemical activator substantially influenced the diastereomer content but other important factors including the overall chemical synthesis process and sequence specific nucleobases and modifications play an important role as well.

合成寡核苷酸疗法代表了一类快速发展的药物，靶向RNA来调节基因表达。对核酸主干的硫代磷酸酯修饰通常用于增加对酶降解的抵抗力和改善药理学特征。每一个硫代磷酸酯修饰都会产生一个立体中心，导致在每一个修饰的键上形成非对映体。这导致最终的药物产品含有复杂的非对映体的异质混合物。随着越来越多的监管机构批准合成寡核苷酸药物产品，迫切需要开发能够表征其非对映体组成的分析方法。我们详细地使用了四种液相色谱方法，辅以31P核磁共振来表征完全磷酸化的短（2和5米）和全长（20米）寡核苷酸序列的非对映体组成。我们还评估了特定化学活化剂如何影响这些合成序列中的非对映体组成。我们的数据表明，使用多种液相色谱方法增强31P核磁共振提供了补充和额外的洞察非对映体组成的硫代连接寡核苷酸。此外，我们的数据还表明，化学活化剂在很大程度上影响了非对映体的含量，但其他重要因素，包括整个化学合成过程和序列特异性核碱基和修饰也起着重要作用。

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引用次数: 0

Rational design of a mesoporous COF with tailored porosity for synergistic extraction and analysis of organic ultraviolet stabilizers 为有机紫外稳定剂的协同萃取和分析，合理设计具有定制孔隙率的介孔COF。

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-02 DOI: 10.1016/j.chroma.2025.466603

Shuang Li , Yiling Zhu , Zhen Ren , Chunying Zheng , Jinhua Li , Xiaoyan Wang , Jiping Ma , Lingxin Chen

Organic ultraviolet stabilizers (OUVSs) have raised significant concerns due to their widespread occurrence and potential endocrine-disrupting effects. Herein, a kind of novel mesoporous covalent organic frameworks (COF), i.e., Fe₃O₄@TFPB-BD, was rationally designed and fabricated for the vortex-assisted magnetic solid-phase extraction (MSPE) of seven kinds of OUVSs from water samples prior to ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) analysis. The pore size (42.1 Å) of the material was pre-designed using density functional theory (DFT) to match the molecular dimensions of the OUVSs (10.6–12.5 Å), facilitating rapid mass transfer. The adsorption mechanism was elucidated to involve synergistic π-π conjugation and hydrogen bonding interactions. Under optimized MSPE conditions, the developed method demonstrated exceptional performance with a remarkably short pretreatment time of only 8 min, excellent precision (intra-day RSDs of 4.2–14.4 %; inter-day RSDs of 1.8–14.8 %), and ultra-low detection limits between 0.23 and 0.35 ng/L. The method was successfully applied to the analysis of coastal seawater and swimming pool water, achieving satisfactory spiked recoveries ranging from 83.66 % to 118.48 %. This study presents a rapid, sensitive, and robust approach for monitoring trace-level OUVSs in complex aquatic environments.

有机紫外线稳定剂（OUVSs）由于其广泛存在和潜在的内分泌干扰作用而引起了人们的极大关注。本文合理设计并制备了一种新型介孔共价有机骨架（COF） Fe3O4@TFPB-BD，用于涡旋辅助磁固相萃取（MSPE）提取水样中7种ouvs，并进行超高效液相色谱-串联质谱（UHPLC-MS/MS）分析。利用密度泛函理论（DFT）预先设计了材料的孔径（42.1 Å），以匹配OUVSs的分子尺寸（10.6-12.5 Å），从而促进快速传质。吸附机理涉及协同π-π共轭和氢键相互作用。在优化的MSPE条件下，该方法预处理时间短，仅为8 min，精密度高（日内rsd为4.2 ~ 14.4%，日内rsd为1.8 ~ 14.8%），检出限在0.23 ~ 0.35 ng/L之间。该方法成功地应用于沿海海水和游泳池水的分析，加标回收率为83.66% ~ 118.48%。本研究提出了一种快速、灵敏、可靠的方法来监测复杂水生环境中痕量OUVSs。

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引用次数: 0

Analysis of N-methyl-2-pyrrolidone and its hydroxy metabolite at low concentration level in water samples using liquid chromatography tandem mass spectrometry 液相色谱串联质谱法分析水样中低浓度n -甲基-2-吡咯烷酮及其羟基代谢物。

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-01 DOI: 10.1016/j.chroma.2025.466599

Ádám Tölgyesi , Carlos Gonçalves , Eszter Benes , Andrea Simon , Virender K. Sharma

N-methyl-2-pyrrolidone (NMP) is a synthetic organic compound used as a solvent in several industrial processes such as battery and cosmetics production. There are growing concerns regarding the toxicity of NMP in the environment. We present for the first time a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the determination of NMP and its hydroxy metabolite (5‑hydroxy-N-methyl-2-pyrrolidone, 5-OHNMP) in water at trace concentration (< 1.0 ng/mL). The HPLC separation was carried out on an aqueous mixed-mode column packed with C18 and anion exchange particles that enabled appropriate retention for both compounds. Quick sample preparation was performed by mixing isotopically labelled internal standards with the samples, followed by extraction with ethyl acetate in the presence of a QuEChERS salt mixture. After eliminating the use of plasticware from the entire sample treatment process, the target analytes could be detected at the 0.1 ng/mL level. The further reducing of the limit of quantification (LOQ) in real samples was limited by the cross contamination of NMP originating from the equipment used for the analysis. The method was validated between the 0.1 ng/mL and 10 ng/mL levels and the recovery ranged from 101 % to 109 % with high precision (RSD = 1.69 % - 7.34 %), with the exception for NMP at 0.10 ng/mL (RSD% = 24.7 %). The method was applied to the analysis of NMP in thirty-five surface and groundwater samples.

n -甲基-2-吡咯烷酮（NMP）是一种合成有机化合物，在电池和化妆品生产等多种工业过程中用作溶剂。人们越来越关注NMP在环境中的毒性。本文首次建立了液相色谱串联质谱（LC-MS/MS）测定水中微量浓度（< 1.0 ng/mL） NMP及其羟基代谢物（5 -羟基- n -甲基-2-吡罗烷酮，5- ohnmp）的方法。高效液相色谱分离是在水混合模式柱上进行的，柱上填充了C18和阴离子交换颗粒，使两种化合物都能适当保留。通过将同位素标记的内标物与样品混合进行快速样品制备，然后在QuEChERS盐混合物存在下用乙酸乙酯萃取。在整个样品处理过程中消除塑料制品的使用后，目标分析物可以在0.1 ng/mL的水平上检测到。进一步降低实际样品的定量限（LOQ）受到来自分析所用设备的NMP交叉污染的限制。该方法在0.1 ng/mL ~ 10 ng/mL范围内有效，加样回收率为101% ~ 109%，精密度（RSD = 1.69% ~ 7.34%），除NMP为0.10 ng/mL （RSD% = 24.7%）外。将该方法应用于35个地表水和地下水样品的NMP分析。

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引用次数: 0

Theoretical and experimental evaluation of biosolvents with different degrees of hydrophobicity for the extraction of safranal and crocins from saffron 不同疏水度的生物溶剂提取藏红花中的藏红花醛和藏红花素的理论和实验评价

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-01 DOI: 10.1016/j.chroma.2025.466597

Marta Rivas-Piña , Adal Mena-García , Ana Isabel Ruiz-Matute , Rosa Lebrón-Aguilar , María Luz , Jesús Eduardo Quintanilla-López

Saffron (Crocus sativus) is a rich source of safranal and crocins, compounds responsible for its organoleptic and bioactive properties. Currently, there is a growing interest in obtaining saffron-based bioactive ingredients using sustainable technologies to develop functional products.

This study explores the use of green biosolvents, including natural deep eutectic solvents (NADESs), for the extraction of safranal and crocins. Solvent selection was guided by COSMO-RS (COnductor-like Screening MOdel for Real Solvents) predictions, also considering solvent polarity, toxicity level, and commercial availability. These predictions were also validated through σ-profile analysis and molecular dynamics simulations. Selected solvents, namely verbenone, carvacrol, and NADESs based on thymol and choline chloride, were evaluated against conventional solvents (H₂O and MeOH:H₂O). Saffron extracts were analysed using HPLC-DAD-MS, with choline chloride:ethylene glycol emerging as the most effective extractant for safranal (2.55 mg g⁻¹), comparable to MeOH:H₂O (2.56 mg g⁻¹), and superior to H₂O (1.29 mg g⁻¹). These solvents also provided the highest crocin recoveries (114.76-117.16 mg g⁻¹). Among the terpenoid-based solvents, carvacrol exhibited a higher selectivity for safranal (1.04 mg g⁻¹), with only 0.91 mg g⁻¹ for crocins.

Overall, the hydrophilic solvents were more efficient than the hydrophobic ones, finding a clear relationship between polarity and extraction capacity. These findings suggest the potential application of choline chloride:ethylene glycol NADES in the food industry for the extraction of bioactives from saffron, providing ecological benefits with low health risks and environmental impact.

藏红花（藏红花）是藏红花醛和藏红花素的丰富来源，这些化合物负责其感官和生物活性特性。目前，人们对利用可持续技术获得藏红花基生物活性成分以开发功能性产品的兴趣日益浓厚。本研究探索了绿色生物溶剂的使用，包括天然深共晶溶剂（NADESs），用于提取藏红花和藏红花。溶剂选择以cosmos - rs（真实溶剂类导体筛选模型）预测为指导，同时考虑溶剂极性、毒性水平和商业可用性。这些预测也通过σ-剖面分析和分子动力学模拟得到了验证。选择溶剂，即马鞭草酮，香芹酚，以及基于百里酚和氯化胆碱的NADESs，对传统溶剂（h2o和MeOH: h2o）进行了评价。用高效液相色谱-双相质谱法分析藏红花提取物，氯化胆碱：乙二醇是藏红花最有效的萃取剂（2.55 mg g⁻¹），与MeOH:H₂O （2.56 mg g⁻¹）相当，优于H₂O （1.29 mg⁻¹）。这些溶剂也提供了最高的藏红花素回收率（114.76-117.16 mg g⁻）。在萜类溶剂中，香芹酚对沙弗兰（1.04 mg g⁻）有较高的选择性，而对藏红花（0.91 mg g⁻）有较高的选择性。总的来说，亲水溶剂比疏水溶剂更有效，极性和萃取量之间存在明确的关系。这些发现表明氯化胆碱：乙二醇NADES在食品工业中的潜在应用，用于从藏红花中提取生物活性物质，提供低健康风险和环境影响的生态效益。

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引用次数: 0

Purification-free immobilization of dopamine D2 receptor and glucocorticoid receptor by alkyne–azide cycloaddition for affinity chromatography-based drug screening 烷基叠氮环加成法无纯化固定多巴胺D2受体和糖皮质激素受体的亲和层析药物筛选

IF 4 2区化学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography A

Pub Date : 2025-12-01 DOI: 10.1016/j.chroma.2025.466596

Ruoxue Bai, Manling Li, Yingyi Zhang, Jiangwei He, Yujin Lin, Haoran Zhang, Ping Shu, Xiaojing Yan, Bo Wang, Tingting Huang, Xue Zhao, Xinfeng Zhao

Immobilized protein-based affinity chromatography offers a promising alternative for high-throughput drug screening, yet its effectiveness is often hampered by conventional immobilization techniques as they result in functional loss of the protein due to random orientation and multi-step purification. Herein, we developed a universal strategy for fabricating highly efficient chromatographic methods by integrating genetic code expansion with alkyne-azide click cycloaddition in the immobilization of dopamine D2 receptor (DRD2) and glucocorticoid receptor (GR) strain-promoted alkyne-azide cycloaddition (SPAAC). The site-specific incorporation of p-azidophenylalanine (pAzF) into the two receptors using an engineered E. coli C321.ΔA strain enabled direct, oriented, and covalent immobilization of them onto dibenzocyclooctyne (DBCO)-functionalized silica by a copper-free click reaction without the need for prior purification. The resulting immobilized DRD2_p_AzF and GR_p_AzF stationary phases were thoroughly characterized by XPS, SEM/EDS, and immunofluorescence, confirming successful immobilization and preserved receptor activity. These exhibited exceptional specificity, stability (>30 days), and reproducibility (RSD<1 %). Applications immobilized DRD2 and GR in analyzing Wendan Decoction identified liquiritigenin, neohesperidin, and naringin as active components of the prescription. Among them, liquiritigenin, neohesperidin were characterized as dual-target ligands. Determination of their binding affinities to the receptors by frontal analysis enables calculation of drug-like efficiency indices (SEI, BEI, LLE). Cellular assays in HT22 cells further confirmed their bioactivities and target specificity to the receptors. This work is possible to advance the discovery of potential leads from natural products as it has the properties of streamlining the construction of reliable receptor chromatography methods, integrating the screening with binding affinity measurement, and preliminary drug-likeness evaluation.

基于固定蛋白的亲和层析为高通量药物筛选提供了一种很有前途的选择，但其有效性经常受到传统固定技术的阻碍，因为它们会导致蛋白质因随机取向和多步骤纯化而失去功能。在此，我们开发了一种通用策略，通过整合遗传密码扩展和炔-叠氮化物点击环加成来固定多巴胺D2受体（DRD2）和糖皮质激素受体（GR）株促进的炔-叠氮化物环加成（SPAAC），以构建高效的色谱方法。利用工程化大肠杆菌C321将对叠氮苯丙氨酸（pAzF）特异地结合到这两种受体中。ΔA菌株通过无铜点击反应将它们直接、定向和共价固定在二苯并环环泰（DBCO）功能化二氧化硅上，而无需事先纯化。通过XPS、SEM/EDS和免疫荧光对固定化后的DRD2pAzF和GRpAzF固定相进行了全面的表征，证实了固定化成功并保留了受体活性。这些方法具有特殊的特异性、稳定性（30天）和重复性（rsd1 %）。应用固定化DRD2和GR对温胆汤进行分析，确定其有效成分为清肾素、新橙皮苷和柚皮苷。其中，利尿原素、新橙皮苷为双靶配体。通过正面分析确定它们与受体的结合亲和力，可以计算药物样效率指数（SEI， BEI， LLE）。HT22细胞实验进一步证实了它们的生物活性和对受体的靶向特异性。该方法简化了可靠的受体层析方法的构建，将筛选与结合亲和性测量相结合，并进行了初步的药物相似性评估，因此有可能促进从天然产物中发现潜在的线索。

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