Journal of Chromatography B最新文献_第9页

Bentonite modified with quaternary ammonium for enhanced human serum albumin adsorption 季铵改性膨润土增强人血清白蛋白吸附。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-11-10 DOI: 10.1016/j.jchromb.2025.124850

Saeed Barzegar , Mehran Javanbakht , Behrouz Akbari-Adergani

Human Serum Albumin (HSA) is an important protein that helps regulate oncotic pressure and transport of diverse molecules. A high-performance adsorbent for the separation of albumin is strategic to improve efficiency and selectivity, which can result in a low-cost and simplified process for obtaining high-purity albumin. The present study aimed to elucidate the adsorption of HSA on modified bentonite and represent the adsorption capacity. Various characterizations confirm the modification and structural changes of bentonite after surface modification with dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DTSACL). A central composite design (CCD) for the response surface methodology (RSM) was used to investigate and optimize the effect of the pH, concentration of HSA, and sorbent dosage on the adsorption. The findings demonstrated that electrostatic interaction and functionalization of the surface were implemented to enhance the adsorption capacity of the modified bentonite. Thermodynamic investigations indicate an exothermic adsorption process and preferred ambient temperatures, while isothermal examinations confirm that monolayer adsorption was a better fit for the Langmuir model. The maximum adsorption capacity (q_e) of 430 mg /g was obtained with a desirability of 0.98 at optimized pH = 6.2, initial HSA concentration = 418.9 mg/L, and DTSACL-bentonite dosage = 30.1 mg in aqueous solution of HSA. The adsorption of HSA from real human serum samples achieved an adsorption efficiency of 79.8 % and a recovery rate of 92.5 %. High-performance liquid chromatography (HPLC) and SDS-PAGE analyses confirmed that DTSACL-bentonite exhibits high selectivity and efficiency, highlighting its potential as a promising adsorbent for the separation and purification of HSA.

人血清白蛋白（HSA）是一种重要的蛋白，有助于调节肿瘤压力和多种分子的运输。高效的白蛋白吸附剂是提高白蛋白分离效率和选择性的重要手段，它可以使获得高纯度白蛋白的过程更简单、成本更低。本研究旨在阐明改性膨润土对HSA的吸附，并表示其吸附量。各种表征证实了二甲基十四烷基[3-（三甲氧基硅基）丙基]氯化铵（DTSACL）表面改性后膨润土的改性和结构变化。采用响应面法（RSM）的中心复合设计（CCD）研究并优化了pH、HSA浓度和吸附剂用量对吸附的影响。结果表明，改性膨润土通过静电相互作用和表面功能化来增强其吸附能力。热力学研究表明了放热吸附过程和首选的环境温度，而等温测试证实单层吸附更适合Langmuir模型。在pH = 6.2、HSA初始浓度= 418.9 mg/L、dtsacl -膨润土投加量= 30.1 mg的条件下，最大吸附量为430 mg/ g，最佳吸附量为0.98。对真实人血清样品的HSA吸附效率为79.8%，回收率为92.5%。高效液相色谱（HPLC）和SDS-PAGE分析证实了dtsacl -膨润土具有高选择性和高效率，显示了其作为分离纯化HSA吸附剂的潜力。

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引用次数: 0

Validation of a highly sensitive assay for the determination of rivastigmine in human plasma for pharmacokinetic studies. 用于药代动力学研究的人血浆中利瓦斯汀高灵敏度测定法的验证。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-10-26 DOI: 10.1016/j.jchromb.2025.124838

Maksim Dolov , Igor Rodin

A fast and highly sensitive LC–MS/MS method has been developed and validated for measuring rivastigmine levels in human plasma. The extraction of rivastigmine from plasma was performed using a simple and quick protein precipitation technique (PPT). The internal standard used was atazanavir-d₅. Chromatographic separation was achieved using a YMC-Triart C18 column, followed by detection with mass spectrometry. The mass transitions monitored were m/z 251.1 > 206.0 for rivastigmine, and m/z 710.5 > 168.1 for atazanavir-d₅. This method involves rapid plasma extraction, straightforward gradient chromatography, and mass spectrometric detection, allowing for the detection of rivastigmine at sub-nanogram per milliliter levels. The method was validated over a linear range of 25 to 5000 pg/ml, with a correlation coefficient of at least 0.9980. Both intra- and inter-day precision and accuracy were within 15 %. The overall recovery rates for rivastigmine and atazanavir-d₅ were close to 100 %. The total analysis time per sample was only 3 min. This method was successfully applied to determine the pharmacokinetic parameters of rivastigmine after a single oral dose of 1.5 mg (capsules) in 26 healthy volunteers.

建立了一种快速、高灵敏度的LC-MS /MS方法，用于测定人血浆中伐斯汀的含量。采用简单快速蛋白沉淀技术（PPT）从血浆中提取利瓦斯汀。内标为atazanvir -d5。采用YMC-Triart C18色谱柱进行色谱分离，然后采用质谱法进行检测。监测到的质量转移为：瑞瓦斯汀为m/z 251.1 > 206.0，阿扎那韦-d5为m/z 710.5 > 168.1。该方法包括快速血浆提取，直接梯度色谱和质谱检测，允许检测亚纳克/毫升水平的利瓦斯汀。在25 ~ 5000 pg/ml的线性范围内，相关系数至少为0.9980。日内、日间的精密度和准确度均在15%以内。瑞瓦斯汀和阿扎那韦-d5的总回收率接近100%。每个样品的总分析时间仅为3分钟。该方法成功地测定了26名健康志愿者单次口服1.5 mg（胶囊）利瓦斯汀的药动学参数。

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引用次数: 0

Molecular imprinted solid-phase extraction and analysis of Entecavir in presence of its induced degradation products and co-administered drug(s) in spiked human plasma, environmental three-color assessment and sustainability profiling 恩替卡韦在人血浆中诱导降解产物和共给药的分子印迹固相萃取和分析，环境三色评估和可持续性分析

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-11-04 DOI: 10.1016/j.jchromb.2025.124843

Sarah S. Saleh, Heba T. Elbalkiny

Entecavir (ETV) is an antiviral drug that acts by blocking the active viral replication process due to its chemical similarity to guanine. Through this study, the extraction of ETV samples was described, for the first time, using a synthesized molecular imprinted polymer solid phase extraction (MISPE). The MISPE was characterized using TEM and FTIR analysis. A green RP- HPLC method was developed for the quantitation of ETV using C18 column and a mobile phase consisting of 0.1 % phosphoric acid in water and methanol in a gradient mode delivered at a rate of 1 ml/min at room temperature. The UV detection was carried out at 245 nm. The analytical method was validated according to ICH guidelines with a linear range of (5–250 μg/mL). The specific extraction of ETV was carried out successfully using MISPE in presence of its induced acidic, and basic degradation products. MISPE was also used for the extraction of ETV from spiked human samples containing the co-administered drug lamivudine. The MISPE showed excellent selectivity, reusability and high recovery percentages (>90 %) when compared to Oasis HLB cartridges. The analytical procedure was compared to the reported methods in terms of environmental three-color assessment (ETCA): greenness (using AGREE, AGREEMIP and ComplexMoGAPI), blueness (using BAGI), and whiteness (using RGB-12 algorithms). The proposed method transcended in saving energy, efficiency and applicability. The sustainability profile for the proposed method was established using the efficient-valid-green (EVG) framework displayed via its radar chart, that showed balance between the three pillars, and the NQS index that displays the excellent alignment of the method with the UN-17 SDGs.

恩替卡韦（ETV）是一种抗病毒药物，由于其化学成分与鸟嘌呤相似，通过阻断活跃的病毒复制过程起作用。通过本研究，首次描述了利用合成分子印迹聚合物固相萃取（MISPE）提取ETV样品的方法。用TEM和FTIR对MISPE进行了表征。采用C18色谱柱，流动相为0.1%磷酸-水-甲醇，在室温下以1 ml/min的速度梯度输送，建立了绿色反相高效液相色谱法定量ETV。紫外检测波长为245 nm。按照ICH指南，在5 ~ 250 μg/mL的线性范围内对方法进行验证。在其诱导的酸性和碱性降解产物存在的情况下，利用MISPE对ETV进行了特异性萃取。MISPE也用于从含有共同给药拉米夫定的加标人样品中提取ETV。与Oasis HLB墨盒相比，MISPE具有良好的选择性、可重复使用性和高回收率（90%）。在环境三色评估（ETCA）方面，将分析程序与报告的方法进行比较：绿色（使用AGREE， AGREEMIP和ComplexMoGAPI），蓝色（使用BAGI）和白色（使用RGB-12算法）。该方法在节能、效率和适用性方面均有超越。通过雷达图显示的有效绿色（EVG）框架和NQS指数建立了该方法的可持续性特征，该框架显示了三个支柱之间的平衡，而NQS指数显示了该方法与联合国-17可持续发展目标的良好一致性。

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引用次数: 0

An LC-MS/MS method for simultaneous quantification of pyrotinib, docetaxel and paclitaxel in human plasma and its application to therapeutic drug monitoring LC-MS/MS同时定量人血浆中吡罗替尼、多西他赛和紫杉醇的方法及其在治疗药物监测中的应用。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-10-29 DOI: 10.1016/j.jchromb.2025.124840

Lingxiao Zhang , Jie Qu , Xinyan Sun , Haiyan Dong , Hongping Yao , Xiaoliang Cheng

Pyrotinib which is a novel irreversible tyrosine kinase inhibitor plus docetaxel or paclitaxel is effective for patients with Her2 positive early or advanced breast cancer including those who failed in first-line treatment. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and verified for simultaneous quantification of pyrotinib, docetaxel and paclitaxel in human plasma, and applied to therapeutic drug monitoring. A reversed-phase Hypersil GOLD aQ column eluted by a gradient mobile phase composed of water and acetonitrile both containing 0.1 % formic acid under flow rate of 0.3 mL min⁻¹ was used for chromatographic separation. The mass spectrometry was operated in positive electrospray ionization mode, and selective reaction monitoring was applied for quantitative analysis. With imatinib as internal standard, one-step deproteinization approach with acetonitrile was applied to extract analytes and purify specimens. This method was adequately validated according to guidelines in terms of specificity and selectivity, sensitivity, linearity, extraction recovery, matrix effect, precision and accuracy, dilution integration and stability. The validated method was applied to therapeutic drug monitoring for breast cancer patients receiving pyrotinib and taxanes based chemotherapy. The therapeutic drug monitoring results showed that the plasma concentration of pyrotinib, docetaxel and paclitaxel varied significantly among individuals. Therapeutic drug monitoring for pyrotinib, docetaxel and paclitaxel is essential for individualized treatment to ensure efficacy and safety.

Pyrotinib是一种新型的不可逆酪氨酸激酶抑制剂，与多西紫杉醇或紫杉醇联合使用对Her2阳性的早期或晚期乳腺癌患者有效，包括那些在一线治疗失败的患者。建立并验证了同时定量测定人血浆中吡罗替尼、多西他赛和紫杉醇的液相色谱-串联质谱（LC-MS/MS）方法，并将其应用于治疗药物监测。采用反相Hypersil GOLD aQ色谱柱，以含有0.1%甲酸的水和乙腈组成的梯度流动相洗脱，流速为0.3 mL min-1。质谱分析采用正电喷雾电离模式，定量分析采用选择性反应监测。以伊马替尼为内标，采用乙腈一步脱蛋白法提取分析物，纯化样品。本方法在特异性和选择性、灵敏度、线性度、提取回收率、基质效应、精密度和准确度、稀释积分和稳定性等方面均按照指南进行了充分验证。将验证的方法应用于以吡罗替尼和紫杉烷为基础的化疗的乳腺癌患者的治疗药物监测。治疗药物监测结果显示，吡罗替尼、多西他赛和紫杉醇的血药浓度在个体间差异显著。吡罗替尼、多西紫杉醇和紫杉醇的治疗药物监测对于个体化治疗至关重要，以确保疗效和安全性。

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引用次数: 0

Identification of byakangelicin metabolites in rats via the UHPLC-Q-exactive orbitrap mass spectrometer uhplc - q -高纯度轨道阱质谱联用法鉴定大鼠黄姜黄素代谢物。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-11-07 DOI: 10.1016/j.jchromb.2025.124841

Ruijun Cai , Zhongnan Mao , Tao An , Yunbo Zhang , Li Zhou

Byakangelicin is a furanocoumarin derived from the roots of Angelica dahurica. It exhibits pharmacological properties, including anti-tumor activity, as well as against liver injury and fibrosis. In this study, Ultra-High-Performance Liquid Chromatography with Q-Exactive Orbitrap Mass Spectrometry technology was employed to investigate the metabolic profile of byakangelicin in rats. Following intragastric administration the suspension of byakangelicin, plasma and tissue specimens were harvested. According to high-resolution extracted ion chromatograms, the metabolites of byakangelicin were identified by comparing accurate mass, diagnostic fragment ions, and chromatographic retention times. Data collection was performed in positive ion mode to facilitate metabolite characterization of byakangelicin. Overall, 46 metabolites were successfully characterised. The primary metabolic pathways included hydroxylation, dehydroxylation, methylation, demethylation, reduction, glucuronidation, glycination, and cysteinylation. This systematic investigation of the metabolic characteristics and pathways of byakangelicin in rats provides a valuable reference for future pharmacodynamic evaluations, pharmacological research, and drug development.

白芷素是一种呋喃香豆素，从白芷的根中提取。它具有药理特性，包括抗肿瘤活性，以及抗肝损伤和纤维化。本研究采用超高效液相色谱- Q-Exactive Orbitrap质谱联用技术对大鼠体内黄姜白素的代谢谱进行了研究。经灌胃给药后，收集血浆和组织标本。根据高分辨率提取离子色谱图，通过比较准确的质量、诊断片段离子和色谱保留时间，鉴定了黄姜白素的代谢产物。在正离子模式下进行数据收集，以方便黄姜白素代谢产物的表征。总体而言，46种代谢物被成功表征。主要代谢途径包括羟基化、去羟基化、甲基化、去甲基化、还原、葡萄糖醛酸化、甘氨酸化和半胱氨酸化。本研究对黄姜白素在大鼠体内的代谢特性和代谢途径进行了系统的研究，为今后的药效学评价、药理研究和药物开发提供了有价值的参考。

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引用次数: 0

Establishment of a LC-MS/MS method for the simultaneous quantitative determination of trimethylamine N-oxide and four precursors in human saliva 建立同时定量测定人唾液中三甲胺n -氧化物及四种前体的LC-MS/MS方法

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-10-22 DOI: 10.1016/j.jchromb.2025.124829

Jianyue Li , Bin Hu , Jie Wu , Dazhen Wang , Junyan Xia , Yanan Li , Yonghong Gao , Baorong Chen , Jianfeng Zhou , Zenghe Li

In this work, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantitatively detect the choline (CHL), trimethylamine N-oxide (TMAO), γ-butyrobetaine (GBB), betaine (BET), and ʟ-carnitine (CAR) in human saliva for the first time. Saliva samples collected from healthy volunteers were treated with acetonitrile for protein precipitation, followed by direct injection into the LC-MS/MS system for analysis. The method validation was performed in accordance with the CLSI C62-A standard guidelines. The linearity was in the range of 0.300–300.000 μmol/L for CHL, 0.005–5.000 μmol/L for TMAO, 0.050–50.000 μmol/L for GBB, 0.020–20.000 μmol/L for BET, and 0.100–100.000 μmol/L for CAR. The intra-day and inter-day precision ranged from 1.13 % to 8.34 % and from 1.63 % to 10.65 %, respectively. The recoveries and matrix effects of these analytes ranged from 94.31 % to 108.00 % and from 90.59 % to 111.37 %, respectively. The performance of the method met the requirements of the guidelines. The validated method was subsequently applied to analyze test saliva samples from 43 healthy volunteers and 4 coronary heart disease (CHD) inpatients, to evaluate the distributions of five analytes in healthy people and CHD inpatients. This study provides a reliable and accurate analytical approach for the clinical detection of five analytes in saliva.

本文首次建立了液相色谱-串联质谱（LC-MS/MS）同时定量检测人唾液中胆碱（CHL）、三甲胺n -氧化物（TMAO）、γ-丁甜菜碱（GBB）、甜菜碱（BET）和肉碱（CAR）的方法。健康志愿者唾液样品经乙腈沉淀蛋白后，直接注入LC-MS/MS系统分析。方法验证按照CLSI C62-A标准指南进行。CHL的线性范围为0.300 ~ 300.000 μmol/L， TMAO为0.005 ~ 5.000 μmol/L， GBB为0.050 ~ 50.000 μmol/L， BET为0.020 ~ 20.000 μmol/L， CAR为0.100 ~ 100.000 μmol/L。日内精度为1.13% ~ 8.34%，日内精度为1.63% ~ 10.65%。加样回收率为94.31% ~ 108.00%，基质效应为90.59% ~ 111.37%。该方法的性能满足指南的要求。随后将验证的方法应用于43名健康志愿者和4名冠心病住院患者的唾液样本分析，评估5种分析物在健康人群和冠心病住院患者中的分布。本研究为临床检测唾液中5种分析物提供了可靠、准确的分析方法。

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引用次数: 0

LC-HRMS- and TLC-based metabolomics for the identification and authentication of Sida rhombifolia 以LC-HRMS和tlc为基础的代谢组学方法鉴别和鉴定白桦。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-10-31 DOI: 10.1016/j.jchromb.2025.124834

Uswatun Hasanah , Eti Rohaeti , Irmanida Batubara , Utami Dyah Syafitri , Rudi Heryanto , Taopik Ridwan , Nancy Dewi Yuliana , Mohamad Rafi

Ensuring the authenticity and quality of Sida rhombifolia raw materials is crucial for its herbal medicinal product's consistent efficacy, quality, and safety. In this study, we developed an identification and authentication method for identifying and authenticating S. rhombifolia from Turnera subulata. T. subulata has the same leaf morphology as S. rhombifolia, so that it could be used as an adulteration raw material for S. rhombifolia. We employed liquid chromatography-high resolution mass spectrometry (LC-HRMS)- and thin-layer chromatography (TLC)-based metabolomics for that purpose. Distinct chemical fingerprints of S. rhombifolia from T. subulata were obtained using LC-HRMS and TLC fingerprint analysis. Mixtures of S. rhombifolia and T. subulata powdered samples at varying concentrations (5 %, 25 %, and 50 % w/w) were analyzed using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to see differences of each group. The PCA score plot from the TLC analysis explained over 70 % of the total variance, while LC-HRMS data provided the highest classification accuracy at 97.05 %. This integrated approach enhances the reliability of S. rhombifolia authentication by combining TLC's rapid profiling capability with LC-HRMS's analytical precision. This study provides a robust analytical framework for the quality control of herbal medicines, specifically addressing challenges related to adulteration.

确保思达连翘原料的真实性和质量对其草药产品的一致疗效、质量和安全性至关重要。在本研究中，我们建立了一种鉴别和鉴定地下土白桦的鉴定方法。毛茛叶形态与黄双歧花相同，可作为黄双歧花的掺假原料。为此，我们采用了液相色谱-高分辨率质谱（LC-HRMS）和基于薄层色谱（TLC）的代谢组学。采用LC-HRMS和TLC指纹图谱分析方法，获得了不同种类的金缕兰化学指纹图谱。采用主成分分析（PCA）和正交偏最小二乘判别分析（OPLS-DA）对不同浓度（5%、25%和50% w/w）的白桦和毛茛混合粉末样品进行分析，比较各组间的差异。来自TLC分析的PCA评分图解释了总方差的70%以上，而LC-HRMS数据提供了最高的分类准确率，为97.05%。这种综合方法结合了薄层色谱的快速分析能力和LC-HRMS的分析精度，提高了白桦鉴别的可靠性。本研究为草药质量控制提供了一个强有力的分析框架，特别是针对掺假相关的挑战。

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引用次数: 0

Development and validation of an LC-MS/MS method for Tirzepatide, a dual GIP/GLP-1 receptor agonist, in rat plasma for application to a pharmacokinetic study 大鼠血浆中双GIP/GLP-1受体激动剂tizepatide的LC-MS/MS方法的开发和验证，用于药代动力学研究。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-10-26 DOI: 10.1016/j.jchromb.2025.124836

Hae-In Choi , Hyeon-Cheol Jeong , Jong-Woo Jeong , Jaeyoung Lee , Da Hae Kim , Kyong-Cheol Ko , Yoon-Jee Chae , Kyeong-Ryoon Lee

A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of tirzepatide, a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors in rat plasma. Tirzepatide was extracted from rat plasma by protein precipitation using methanol. Chromatographic separation was achieved using a peptide C18 column with gradient elution of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed in positive electrospray ionization mode using multiple reaction monitoring with transitions of m/z 1204.4 → 1473.6 for tirzepatide and m/z 1029.4 → 1238.4 for the internal standard, semaglutide. The developed method exhibited good linearity over a concentration range of 1–1000 ng/mL (r² > 0.99). Intra- and inter-day accuracy (−4.324–5.057 %) and precision (5.250–9.000 %) met the regulatory criteria at all quality control levels, and were stable under various plasma handling and storage conditions. The validated method was successfully applied to a pharmacokinetic study in rats following the intravenous and subcutaneous injection of tirzepatide at 0.3 mg/kg. The terminal half-lives were 10.04 h and 9.803 h after intravenous and subcutaneous administration, respectively, indicating comparable elimination profiles. The bioavailability following subcutaneous dosing was estimated to be approximately 62.38 %. These findings highlight the robustness and applicability of the developed method, suggesting its potential utility for the quantitative analysis of other peptide therapeutics with structures or mechanisms of action similar to those of tirzepatide.

建立了一种灵敏、快速的液相色谱-串联质谱（LC-MS/MS）方法，用于定量大鼠血浆中葡萄糖依赖性胰岛素性多肽（GIP）和胰高血糖素样肽-1 （GLP-1）受体双激动剂替西肽。采用甲醇蛋白沉淀法从大鼠血浆中提取替西帕肽。色谱分离采用肽C18柱，梯度洗脱水和含有0.1%甲酸的乙腈。质谱检测采用正电喷雾电离模式，采用多重反应监测，替西肽为m/z 1204.4→1473.6，内标semaglutide为m/z 1029.4→1238.4。该方法在1 ~ 1000 ng/mL （r2 > 0.99）的浓度范围内线性良好。日内和日间准确度（-4.324- 5.057%）和精密度（5.250-9.000 %）均符合所有质量控制水平的监管标准，且在各种血浆处理和储存条件下均稳定。该方法成功应用于0.3 mg/kg替西帕肽静脉注射和皮下注射后大鼠体内的药代动力学研究。静脉和皮下给药后的终末半衰期分别为10.04 h和9.803 h，表明消除情况相当。皮下给药后的生物利用度估计约为62.38%。这些发现突出了所开发方法的稳健性和适用性，表明其对其他结构或作用机制类似于替西肽的肽疗法的定量分析的潜在效用。

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引用次数: 0

Aptamer enrichment strategy for the detection of erythropoietin-receptor agonists in blood samples: A potential alternative to antibody-based assays 适体富集策略检测血液样本中的促红细胞生成素受体激动剂：一个潜在的替代抗体为基础的分析。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-11-07 DOI: 10.1016/j.jchromb.2025.124846

Yeşim Somay Selbes

Optimal detection of Erythropoietin receptor agonists (ERAs) in anti-doping analyses relies heavily on efficient immunopurification (IP) strategies. While antibody-based IP is routinely applied, aptamers have emerged as promising alternatives. This proof-of-principle study investigated the applicability of an anti-EPO DNA aptamer for ERA enrichment in serum, followed by Sarkosyl-Polyacrylamide Gel Electrophoresis (SAR-PAGE) analysis.

The aptamer-based method successfully captured recombinant ERA variants (rEPO, dEPO, EPO-Fc, CERA) with defined limits of detection (LOD) in serum: 10 mIU/mL for rEPO, 10 pg/mL for dEPO, and 25 pg/mL for EPO-Fc and CERA. In comparison, the antibody-based method achieved lower serum LODs of 5 mIU/mL for rEPO, 5 pg/mL for dEPO, and 12.5 pg/mL for EPO-Fc and CERA. Both methods demonstrated good selectivity, as no non-specific bands were detected in blank serum matrices. However, endogenous blood EPO (bEPO) was not enriched by the aptamer, likely reflecting glycosylation-related structural differences between endogenous and recombinant isoforms.

These findings highlight that aptamers can provide highly specific recognition of recombinant ERAs without cross-reactivity to bEPO, which may simplify interpretation in doping control analyses. Nonetheless, further optimization—such as refining immobilization chemistries, incorporating differently glycosylated EPO forms during SELEX, and sequence improvements—is required to enhance capture of native isoforms. Overall, this study establishes a foundation for aptamer-based enrichment as a complementary alternative to antibody-based methods in anti-doping applications.

在反兴奋剂分析中，促红细胞生成素受体激动剂（ERAs）的最佳检测在很大程度上依赖于高效的免疫纯化（IP）策略。虽然基于抗体的IP是常规应用，但适体已成为有希望的替代方案。这项原理验证研究调查了抗epo DNA适体在血清中富集ERA的适用性，随后进行了萨科齐-聚丙烯酰胺凝胶电泳（SAR-PAGE）分析。基于适体的方法成功捕获了重组ERA变体（rEPO, dEPO, EPO-Fc， CERA），其血清检测限（LOD）为：rEPO为10 mIU/mL， dEPO为10 pg/mL， EPO-Fc和CERA为25 pg/mL。相比之下，基于抗体的方法获得了较低的血清lod， rEPO为5 mIU/mL， dEPO为5 pg/mL， EPO-Fc和CERA为12.5 pg/mL。两种方法均表现出良好的选择性，因为在空白血清基质中未检测到非特异性条带。然而，内源性血液EPO （bEPO）没有被适体富集，可能反映了内源性和重组亚型之间糖基化相关的结构差异。这些发现表明，适体可以在不与bEPO发生交叉反应的情况下对重组era进行高度特异性识别，这可能会简化兴奋剂检测分析的解释。尽管如此，需要进一步优化，如改进固定化学物质，在SELEX过程中加入不同的糖基化EPO形式，以及序列改进，以增强天然同工异构体的捕获。总的来说，本研究为基于适配体的富集作为基于抗体的方法在反兴奋剂应用中的补充替代方法奠定了基础。

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引用次数: 0

Biotransformation and its effects on anxiolytic activity and toxicity of Polygalae Radix saponins 聚总皂苷的生物转化及其对抗焦虑活性和毒性的影响。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2026-01-01 Epub Date: 2025-11-02 DOI: 10.1016/j.jchromb.2025.124842

Yuhan Sun , Yihong Li , Jiaqi Xie , Yulu Liang , Hongqian Kui , Huang Jianmei

Saponins, accounting for over 2.0% (w/w) in Polygalae Radix extracts, act as both active and toxic constituents, with their glycosyl structures hypothesized to modulate efficacy and toxicity. This study aimed to modify the glycosyl structures of Polygalae Radix (PR) saponins via snail enzyme biotransformation and evaluate changes in the activity of inhibiting anxiety-like behavior in the EPM and gastrointestinal toxicity. The biotransformation process was optimized using one-way experiments and response surface methodology, identifying optimal conditions as 46 °C, pH 6.4, 72-h reaction time, and an enzyme-substrate ratio of 25:1. UPLC-Q-Exactive/MS analysis revealed 37 differential saponin-like components with truncated glycosyl chains. In the elevated plus-maze (EPM) test, both untransformed and transformed PR saponins exhibited a significant effect of inhibiting anxiety-like behavior in the EPM, with transformed products showing enhanced activity. Gastrointestinal toxicity assessments, including weight monitoring and gastric tissue morphology observation, demonstrated reduced flatulence and weight loss in mice administered transformed saponins. These results confirm that glycosyl chain degradation in PR saponins reduces gastrointestinal toxicity while preserving the efficacy of inhibiting anxiety-like behavior in the EPM, providing a scientific basis for improving the safety and clinical application of PR.

总皂苷含量超过2.0% (w/w)，是有效成分和毒性成分，其糖基结构可能调节药效和毒性。本研究旨在通过蜗牛酶生物转化修饰聚galae Radix （PR）皂苷的糖基结构，并评价其在EPM和胃肠道毒性中抑制焦虑样行为活性的变化。采用单向实验和响应面法对生物转化过程进行优化，确定最佳条件为46°C、pH 6.4、反应时间72 h、酶底物比25:1。UPLC-Q-Exactive/MS分析显示37种糖基链截断的差异皂苷样成分。在升高+迷宫（EPM）实验中，未转化和转化的PR皂苷均表现出抑制EPM中焦虑样行为的显著作用，转化产物的活性增强。胃肠道毒性评估，包括体重监测和胃组织形态学观察，显示小鼠服用转化皂苷后肠胃胀气减少，体重减轻。这些结果证实了白藜芦醇皂苷的糖基链降解在降低胃肠道毒性的同时保留了抑制EPM中焦虑样行为的功效，为提高白藜芦醇的安全性和临床应用提供了科学依据。

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引用次数: 0