Journal of pharmaceutical and biomedical analysis最新文献_第8页

Metabolite differences between stems and leaves of Sarcandrae herba: A basis for rational medicinal part selection 菝葜茎叶间代谢物差异：合理选择药用部位的依据。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-22 DOI: 10.1016/j.jpba.2025.117279

Yan-bo Xu , Wen-jing Gan , Xiao-kang Liu , Jia-wei Wang , Fei Huang , Zhen-wei Li , Dai-di Zhang , De-an Guo

The 2025 edition of the Chinese Pharmacopoeia designates the dried whole plant of Sarcandrae herba as the medicinal part; however, the frequent phenomenon of leaf detachment in practice raises concerns about its impact on the quality of the medicinal material. To address this, the present study systematically characterized the chemical basis of Sarcandrae herba using UPLC–Q–TOF–MS/MS technology. Combined with metabolomic approaches, we conducted chemical analyses on the stems and leaves of 30 batches of samples to identify and analyze differential metabolites and their distribution patterns. Additionally, the contents of two key marker components with significant differences in the whole plant, pure stems, and pure leaves were determined. The results demonstrated that a total of 103 chemical constituents were identified in the stems and leaves of Sarcandrae herba, encompassing organic acids, flavonoids, sesquiterpenoids, and coumarins, among which 24 were identified as significantly differential metabolites. Notably, isofraxidin exhibited specific enrichment in the stems, with its content in 15 batches of stems being five times higher than that in the leaves. Conversely, phenolic acids such as rosmarinic acid, chlorogenic acid, and neochlorogenic acid were predominantly distributed in the leaves, with the leaf content of rosmarinic acid being twice that of the stems. These findings reveal the characteristic distribution patterns of constituents in the stems and leaves of Sarcandrae herba, clarifying that leaf detachment may lead to the loss of bioactive phenolic acids and a relative increase in the proportion of isofraxidin, posing a quality risk for the medicinal material. This study provides a chemical foundation for understanding the scientific connotation of " whole plant for medicinal use" and offers important evidence for optimizing the quality control strategy of Sarcandrae herba medicinal materials.

2025年版《中国药典》将菝葜属植物的干燥全株指定为药用部位；然而，在实践中频繁出现的叶子脱离现象引起了人们对其对药材质量影响的关注。为了解决这一问题，本研究采用UPLC-Q-TOF-MS/MS技术系统地表征了菝葜属草本植物的化学基础。结合代谢组学方法，对30批样品的茎叶进行化学分析，鉴定和分析差异代谢物及其分布规律。此外，还测定了全株、纯茎和纯叶中两种差异显著的关键标记组分的含量。结果表明，在菝葜茎叶中共鉴定出103种化学成分，包括有机酸、黄酮类、倍半萜类和香豆素，其中24种化学成分被鉴定为显著差异代谢物。值得注意的是，异黄皮苷在茎中表现出特异性富集，15批茎中异黄皮苷的含量是叶中的5倍。相反，迷迭香酸、绿原酸和新绿原酸等酚酸主要分布在叶片中，迷迭香酸在叶片中的含量是茎中的两倍。这些发现揭示了菝葜茎叶中成分的特征分布模式，阐明了叶片脱落可能导致生物活性酚酸的损失和异黄皮苷的比例相对增加，对药材构成质量风险。本研究为理解“药用全植物”的科学内涵提供了化学基础，为优化菝葜属草本药材的质量控制策略提供了重要依据。

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引用次数: 0

Efficient separation of chemical constituents from shilajit by an inner-recycling counter-current chromatography and their molecular docking analysis for osteoporosis 内循环逆流色谱法分离石竹中的化学成分及其对骨质疏松的分子对接分析

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-21 DOI: 10.1016/j.jpba.2025.117274

Shahid Aziz , Iftikhar Ali , Manzoor Hussain , Sujjad Hyder , Ajmal Khan , Kashif Ali , Salma Batool , Xiao Wang

Shilajit (Asphaltum punjabianum), a herbomineral ethnomedicinal substance from layers of rocks in the Himalayas and Hindukush ranges, is reported to treat urinary bladder cancer, it reduces inflammation, oxidative stress and bone loss. To date, there are no such authentic reports on the detailed separation or isolation of the chemical constituents from shilajit. In the current study, the high-speed counter-current chromatography technique is used for the separation of chemical constituents from shilajit for the first time. Thus, the current investigation depicts an efficient separation of six components namely boscartin J (1), 3α-O-acetyl-11-keto-β-boswellic acid (2), boscartin K (3), hemerocallal A (4), 3α-hydroxytirucalla-8,24-dien-21-oic acid (5), and centrolobol (6) using HSCCC employing a solvent system MTBE/n-But/ACN/H2O (2:2:1:5, v/v). Using the molecular docking, compounds 2 and 5 exhibited a significant affinity for ERα (PDB 3OCB) and Cathepsin K (PDB 6WOK). It is worth mentioning that the simulated results reflect a profound alignment with established and renowned bone-protective pharmacological principles. 3α-O-Acetyl-11-keto-β-boswellic acid and 3α-hydroxytirucalla-8,24-dien-21-oic acid exhibit promising dual activity, modulation of ERα and inhibition of Cat. K for the treatment of osteoporosis.

Shilajit（旁遮普沥青）是一种来自喜马拉雅山脉和兴都库什山脉岩层的草本民族药材，据报道可以治疗膀胱癌，减少炎症、氧化应激和骨质流失。迄今为止，没有关于从shilajit中详细分离或分离化学成分的这种真实报告。本研究首次采用高速逆流色谱技术对石竹中的化学成分进行了分离。因此，本研究描述了在溶剂体系MTBE/n-But/ACN/H2O （2:2:1:5, v/v）下，HSCCC高效分离六种成分，即boscartin J(1)、3α- o-乙酰-11-酮-β-乳香酸(2)、boscartin K(3)、hememerocallal A(4)、3α-羟基tirucalla-8,24-二烯-21-oic酸(5)和centrolobol(6)。通过分子对接，化合物2和5对ERα (PDB 3OCB)和Cathepsin K （PDB 6WOK）表现出显著的亲和力。值得一提的是，模拟结果反映了与建立和著名的骨骼保护药理学原理的深刻对齐。3α- o-乙酰-11-酮-β-乳香酸和3α-羟基提鲁卡拉-8,24-二烯-21-酸表现出良好的双重活性，既能调节ERα，又能抑制Cat。K用于治疗骨质疏松症。

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引用次数: 0

Conformational dynamics and substrate selectivity in KPC-33: Molecular mechanisms of resistance to ceftazidime-avibactam revealed by multiscale molecular dynamics simulations KPC-33的构象动力学和底物选择性：多尺度分子动力学模拟揭示了对头孢他啶-阿维巴坦耐药的分子机制

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-21 DOI: 10.1016/j.jpba.2025.117272

Zuguo Zhao , Caihong Ye , Wei Zhang , Xianghao Yuan , Han Gao , Ruolin Zhu , Luguang Liang , Rui Yang , Jie Zeng , Xiaoxin Tian , Shengjun Feng , Qiujia Wen , Jiafan Guo , Tingting Zhi

The emergence of Klebsiella pneumoniae carbapenemase (KPC) −33, a D179Y variant of KPC-2, confers resistance to the combination of ceftazidime-avibactam (CAZ-AVI), but its atomic-level resistance mechanism remains unclear. To clarify conformational dynamics and substrate selectivity, we conducted extensive molecular dynamics simulations: 6μs equilibrium molecular dynamics simulations (MDs) for apo-enzymes of KPC-2 and KPC-33, 40μs free-binding MDs for enzymes and its substrates, and adaptive steered MD (ASMD), together with the interaction fingerprint and binding free energy analyses. KPC-33 exhibited greater conformational heterogeneity, especially in the Ω-loop region. Free-binding MD simulations showed that the thiazole ring of CAZ wedges into the interface between the Ω-loop and H6 of KPC-33, producing an induced-fit pre-catalytic binding configuration 4 (PCBC-4) that is absent in the simulations for CAZ-KPC-2 and is not reported previously. Binding free energy analysis showed that KPC-33 has higher affinity for CAZ but lower affinity for AVI due to residue-specific energy re-distributions. ASMD suggested that a lower barrier for Ω-loop-H6 separation in KPC-33 than in KPC-2, facilitating PCBC-4 formation. Dynamic network analysis further revealed tighter β-sheet/α-helix coupling in KPC-33, decreased modularity of the SXXK motif and Ω-loop communities. Together, these findings explain how D179Y substitution reshapes KPC-33's conformational landscape, favoring CAZ accommodation while evading avibactam inhibition. This study highlights the Ω-loop-H6 interface as a potential target to restore CAZ-AVI efficacy against resistant β-lactamases.

肺炎克雷伯菌碳青霉烯酶(KPC) - 33， KPC-2的D179Y变体的出现，使其对头孢他啶-阿维巴坦（CAZ-AVI）联合耐药，但其原子水平的耐药机制尚不清楚。为了明确分子动力学和底物选择性，我们进行了广泛的分子动力学模拟：KPC-2和KPC-33载子酶的6μs平衡分子动力学模拟（MDs），酶及其底物的40μs自由结合分子动力学模拟（MDs），自适应导向分子动力学（ASMD），以及相互作用指纹图谱和结合自由能分析。KPC-33表现出更大的构象异质性，特别是在Ω-loop区域。自由结合的MD模拟表明，CAZ的噻唑环楔形进入KPC-33的Ω-loop和H6之间的界面，产生诱导拟合的预催化结合构型4 (PCBC-4)，这在CAZ- kpc -2的模拟中是不存在的，以前也没有报道过。结合自由能分析表明，KPC-33对CAZ具有较高的亲和力，而对AVI具有较低的亲和力。ASMD表明，与KPC-2相比，KPC-33的Ω-loop-H6分离屏障较低，有利于PCBC-4的形成。动态网络分析进一步发现KPC-33中β-sheet/α-helix耦合更紧密，SXXK基序和Ω-loop群落的模块化降低。总之，这些发现解释了D179Y取代如何重塑KPC-33的构象景观，有利于CAZ调节，同时逃避阿维巴坦抑制。这项研究强调Ω-loop-H6界面是恢复CAZ-AVI对耐药β-内酰胺酶疗效的潜在靶点。

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引用次数: 0

Characterizing the in vitro metabolic features of nine alicyclic fentanyl-type new psychoactive substances 九种脂环芬太尼型新型精神活性物质体外代谢特征的研究。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-20 DOI: 10.1016/j.jpba.2025.117276

Xuan Luo , Wenting Liu , Kejian Huang , Xiaofeng Liu , Ning Yang , Qiulian Luo

New psychoactive substances (NPS) represent a third-generation class of drugs characterized by high addictive potential, enhanced potency, low lethal doses, rapid in vivo metabolic rates, and fast structural diversification. Among these, fentanyl-type NPS (F-NPS), which are derived from the opioid analgesic “fentanyl”, present a category of NPS that simultaneously exhibit all five characteristics (in addition to nitazenes). Consequently, global F-NPS use poses a critical challenge, as metabolic data from F-NPS have often proven insufficient to support law enforcement agencies in combating emerging analogs. In this study, an in vitro human liver microsome model was used to incubate nine alicyclic F-NPS derivatives and the metabolites was analyzed by using liquid chromatography-ion trap tandem time-of-flight mass spectrometry. We systematically characterized the metabolic profiles of the alicyclic F-NPS and elucidated the structural determinants that influence metabolism. Our findings revealed that sp³-carbon monohydroxylation occurring at the alicyclic ring, a distinctive metabolic pathway in this subclass, is governed by the C–H Laplacian bond order, intermediate radical stability, alicyclic ring rigidity, and steric hindrance, while also competing with analogous metabolic pathways at other sites. Notably, alicyclic dehydrogenation, a previously unreported metabolic pathway, correlated with the stability of alicyclic alkenes and conjugative stabilization through adjacent carbonyl groups. N-oxidation metabolism was significantly modulated by N-dealkylation metabolism at adjacent positions and substituents on the phenyl group of the core. Furthermore, >N–CH₂–CH₂– serves as the primary metabolic hotspot within the F-NPS skeleton structure, with the total relative contents of corresponding metabolites accounting for > 80 % (and up to 98 %). The monohydroxylation of the phenyl group adhered to an electrophilic substitution mechanism governed by the electron density and both the theoretical calculation results and mass spectrometric data consistently indicated that sp²-carbon monohydroxylation occurred exclusively at the phenethyl group in the tail. The findings provide data and analytical strategies for the development of a universal F-NPS metabolic prediction model.

新型精神活性物质（NPS）是第三代药物，具有高成瘾性、强效性、低致死剂量、快速体内代谢率和快速结构多样化等特点。其中，源自阿片类镇痛药“芬太尼”的芬太尼型NPS （F-NPS）是一类同时表现出所有五种特征的NPS（除了nitazene）。因此，全球使用F-NPS带来了严峻的挑战，因为来自F-NPS的代谢数据经常被证明不足以支持执法机构打击新兴类似物。本研究采用体外人肝微粒体模型孵育9种脂环类F-NPS衍生物，并采用液相色谱-离子阱串联飞行时间质谱法分析其代谢产物。我们系统地表征了脂环F-NPS的代谢谱，并阐明了影响代谢的结构决定因素。我们的研究结果表明，sp3-碳单羟基化发生在脂环上，这是该亚类中一个独特的代谢途径，受C-H拉普拉斯键顺序、中间自由基稳定性、脂环刚性和位阻的控制，同时也与其他类似的代谢途径竞争。值得注意的是，脂环脱氢是一种以前未报道的代谢途径，与脂环烯烃的稳定性和相邻羰基的共轭稳定性相关。邻近位置的n -脱烷基代谢和核心苯基上的取代基显著调节了n -氧化代谢。此外，>N-CH₂- ch₂-是F-NPS骨架结构内的主要代谢热点，其代谢产物的总相对含量占> 80 %（最高可达98 %）。苯基的单羟基化遵循由电子密度控制的亲电取代机制，理论计算结果和质谱数据一致表明sp2-碳单羟基化只发生在尾部的苯基上。这些发现为开发通用的F-NPS代谢预测模型提供了数据和分析策略。

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引用次数: 0

Voltammetric analysis of entacapone, levodopa, and carbidopa in tablets: A chemometric approach 恩他卡彭、左旋多巴和卡比多巴片剂的伏安分析：化学计量学方法

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-20 DOI: 10.1016/j.jpba.2025.117275

Azam Safarnejad , Fatma Budak , Ahmet Cetinkaya , Burcu Dogan-Topal , Sibel A. Ozkan

The objective of the present work is to develop a voltammetric method for the determination of entacapone (ENT) in the presence of levodopa (LEV) and carbidopa (CAR) in synthetic mixtures and pharmaceutical formulations, using a multivariate approach. ENT is an inhibitor of Catechol-O-methyltransferase (COMT), used in the treatment of Parkinson’s disease as an adjunct for the combination therapy with LEV and CAR. With the optimized parameters in a pH 3.0 Britton-Robinson buffer, the quantitative analyses were assessed by the root mean square error in prediction (RMSEP), as well as the root mean square error of calibration (RMSEC) and the relative error of prediction (REP). The Radial Basis Function Artificial Neural Network (RBF-ANN) and Partial Least Squares regression (PLS) were applied for the determination of ENT in the presence of LEV and CAR. In the RBF-ANN model, the REP of 0.87 and RMSEP of 2.14

\times

10⁻⁷ are obtained for the test data when quantifying the ENT. The RBF model, as a nonlinear calibration method, was successful for predicting ENT concentration. To evaluate the validity of the recommended method, a commercial pharmaceutical tablet (Stalevo ® 200 mg) was analyzed. RBF model has the ability to overcome difficulties such as non-linearity and overlaps in the voltammograms.

本研究的目的是建立一种伏安法测定合成混合物和药物制剂中左旋多巴（LEV）和卡比多巴（CAR）存在时恩他卡彭（ENT）的含量。ENT是一种儿茶酚- o -甲基转移酶（COMT）抑制剂，用于治疗帕金森病，作为LEV和CAR联合治疗的辅助疗法。在pH为3.0的布里顿-罗宾逊缓冲液中，采用预测均方根误差（RMSEP）、校准均方根误差（RMSEC）和相对预测误差（REP）对优化后的参数进行定量分析。采用径向基函数人工神经网络（RBF-ANN）和偏最小二乘回归（PLS）测定LEV和CAR存在时的ENT。在RBF-ANN模型中，在量化ENT时，测试数据的REP为0.87，RMSEP为2.14×10−7。RBF模型作为一种非线性定标方法，在预测ENT浓度方面取得了成功。为评价所推荐方法的有效性，对市售片剂（Stalevo®200 mg）进行了分析。RBF模型具有克服伏安图非线性和重叠等困难的能力。

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引用次数: 0

Point-of-care fentanyl detection: Combining smartphone imaging and chemometrics for reliable quantitative analysis 即时芬太尼检测：结合智能手机成像和化学计量学进行可靠的定量分析

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-20 DOI: 10.1016/j.jpba.2025.117277

Yasaman Sefid-Sefidehkhan , Mohammad Hasanzadeh , Abolghasem Jouyban , Sedigheh Mohammadzadeh , Mohamadbahger Hosseini , Vahid Jouyban-Gharamaleki , Elaheh Rahimpour

In this work, the possibility of using smartphone image analysis in combination with the spot test as a quick and simple method of fentanyl analysis was investigated. A probe composed of silver nanoprism-graphene quantum dots (AgNprs-GQD) has been used to induce color variations at different fentanyl concentrations. The data for these variations were recorded by spectrophotometry, spectrofluorimetry, and the digital image colorimetry methodology for comparison. Image analysis was achieved using the PhotoMetrix smartphone App, which uses univariate calibration of the collected images. For further investigation of image analysis, multivariate calibration was performed using the parallel factor analysis (PARAFAC) method in the MATLAB environment. The method was validated by comparing the results obtained from the image analysis methods and spectrophotometry as a standard method. Under optimized conditions, linear ranges and limits of detection (LOD) were 0.001– 0.3 µg/mL and 0.0007 µg/mL for the spectrophotometry method, 0.01– 0.5 µg/mL and 0.009 µg/mL for the spectrofluorimetry method, 0.001– 3.0 µg/mL and 0.0008 µg/mL for the smartphone colorimetry method, and 0.001– 0.5 µg/mL and 0.0007 µg/mL for PARAFAC. Intra-day and inter-day precisions were obtained at ≤ 6.4 % and ≤ 10.8 %. The method was successfully applied to exhaled breath condensate samples from ventilated neonates, demonstrating a non-invasive, rapid, and field-deployable approach for point-of-care fentanyl quantification.

在这项工作中，研究了使用智能手机图像分析与现场测试相结合作为芬太尼分析快速简便方法的可能性。一种由银纳米棱镜-石墨烯量子点（AgNprs-GQD）组成的探针被用来诱导不同芬太尼浓度下的颜色变化。通过分光光度法、荧光光谱法和数字图像比色法记录这些变化的数据进行比较。使用PhotoMetrix智能手机应用程序实现图像分析，该应用程序对收集的图像进行单变量校准。为了进一步研究图像分析，在MATLAB环境下使用平行因子分析（PARAFAC）方法进行多变量校准。通过比较图像分析方法和分光光度法作为标准方法的结果，验证了该方法的有效性。在优化条件下，分光光度法的线性范围和检出限分别为0.001 ~ 0.3 µg/mL和0.0007 µg/mL，分光光度法的线性范围和检出限分别为0.01 ~ 0.5 µg/mL和0.009 µg/mL，智能手机比色法的线性范围和检出限分别为0.001 ~ 3.0 µg/mL和0.0008 µg/mL， PARAFAC的线性范围和检出限分别为0.001 ~ 0.5 µg/mL和0.0007 µg/mL。日内和日内精密度分别为≤ 6.4 %和≤ 10.8 %。该方法已成功应用于通气新生儿呼出冷凝水样本，展示了一种无创、快速、可现场部署的芬太尼即时定量方法。

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引用次数: 0

Facile synthesis of ternary magnetic chitosan-graphene oxide composites for efficient extraction of urinary modified nucleosides prior to HPLC-MS/MS analysis 三元磁性壳聚糖-氧化石墨烯复合材料的简单合成，用于高效提取尿液修饰核苷，用于HPLC-MS/MS分析

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-19 DOI: 10.1016/j.jpba.2025.117271

Qiao Deng , Jianliang Li , Jingjing Xu , Xiaojing Zhao , Dan Wei , Xu Wang

The accurate quantification of urinary modified nucleosides, promising non-invasive cancer biomarkers, is hindered by their trace concentrations and complex matrix interference. To overcome this challenge, we developed the facile synthesis of a novel ternary magnetic composite (MCS-100GO) by integrating chitosan (CS) and graphene oxide (GO) onto the magnetic core, utilized as adsorbent for magnetic solid-phase extraction (MSPE). The adsorbent leverages the combined effects of hydrogen bonding (from CS and GO) and π-π interactions (from GO) for highly efficient capture, while the magnetic core enables rapid and simple separation. Systematic optimization demonstrated exceptional performance for 3-methylcytidine (m³C) and 7-methylguanosine (m⁷G). The MCS-100GO composite achieved rapid adsorption equilibrium (3 min), high maximum adsorption capacities (0.7730 mg/g for m³C and 0.7561 mg/g for m⁷G). The developed MCS-100GO based MSPE-HPLC-MS/MS method, validated according to FDA guidelines, exhibited excellent sensitivity (LODs: 0.015–0.030 μg/L), a wide linear range (0.050–50 μg/L; R² > 0.9995), and high precision (RSD < 6.4 %). Furthermore, parallelism experiments with a coefficient of variation (CV) of 9.7 % confirmed no significant matrix effects. Compared to existing methods, this approach offers a superior balance of low adsorbent dosage, sensitivity, speed, presenting a promising and efficient strategy for high-throughput analysis of urinary nucleoside biomarkers.

尿修饰核苷是一种有前途的非侵入性癌症生物标志物，但其痕量浓度和复杂基质干扰阻碍了其准确定量。为了克服这一挑战，我们通过将壳聚糖（CS）和氧化石墨烯（GO）整合到磁芯上，开发了一种新型的三重磁性复合材料（MCS-100GO），作为磁性固相萃取（MSPE）的吸附剂。吸附剂利用氢键（来自CS和GO）和π-π相互作用（来自GO）的综合效应进行高效捕获，而磁芯则实现快速简单的分离。系统优化表明，3-甲基胞苷（m3C）和7-甲基鸟苷（m7G）具有优异的性能。MCS-100GO复合材料实现了快速吸附平衡（3 min），最大吸附量高（m3C为0.7730 mg/g， m7G为0.7561 mg/g）。所建立的基于MCS-100GO的MSPE-HPLC-MS/MS方法具有良好的灵敏度（lod: 0.015 ~ 0.030 μg/L）、较宽的线性范围（0.050 ~ 50 μg/L; R2 > 0.9995）和较高的精密度（RSD < 6.4 %）。此外，在变异系数（CV）为9.7 %时，平行度实验证实没有显著的矩阵效应。与现有的方法相比，该方法在低吸附剂用量、灵敏度和速度方面取得了良好的平衡，为尿核苷生物标志物的高通量分析提供了一种有前途的有效策略。

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引用次数: 0

Leveraging digestion-assisted capillary electrophoresis sodium dodecyl sulfate (CE-SDS) to monitor galactosylation in monoclonal antibodies 利用消化辅助毛细管电泳十二烷基硫酸钠（CE-SDS）监测单克隆抗体的半乳糖基化。

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-19 DOI: 10.1016/j.jpba.2025.117273

Ramya Rao, Dylan A. Howie, Deanna Di Grandi, Lloydine Clarke, Emily Westin, Vasilis Drainas, Michael P. Rosconi, Erica A. Pyles, Jennifer B. Nguyen

CE-SDS and MCE-SDS have become industry-standard methods for characterizing product-related size variants and identifying potential product-related impurities of biologics. These methods are used as analytical tools to guide manufacturing process parameters, as well as to establish comparability among sample lots for release. An asymmetric heavy chain doublet was recently observed among multiple monospecific monoclonal antibody samples while performing routine lot-to-lot comparability assessments under reduced CE-SDS conditions. Notably, this splitting pattern was not observed under MCE-SDS. Using a combination of enzymatic digestions and oligosaccharide analysis, we confirm the specific identity of the slower-migrating heavy chain peak as galactosylation variants. Leveraging this method for extended product characterization, it was revealed that galactosylation variants are consistently elevated in enriched acidic charge variant fractions of several mAbs. This digestion-assisted CE-SDS method can also be applied successfully to Fc fusion proteins to enable domain-specific characterization of galactose content. Overall, we demonstrate that, in the absence of more complex mass-spectrometry based glycan profiling techniques, IdeS pre-treatment of mAbs and Fc fusion proteins prior to reduced CE-SDS analysis can readily detect differences in galactose content across various samples while simultaneously informing on N-glycan occupancy. Finally, we propose that the analytical strategy outlined in this study provides CE-SDS peak identification, specifically for galactosylation variants, and can support extended characterization.

CE-SDS和MCE-SDS已成为表征产品相关尺寸变化和识别生物制剂潜在产品相关杂质的行业标准方法。这些方法被用作指导生产工艺参数的分析工具，以及建立放行样品批次之间的可比性。最近，在降低CE-SDS条件下进行常规批对批可比性评估时，在多个单特异性单克隆抗体样品中观察到不对称重链双链。值得注意的是，MCE-SDS没有观察到这种分裂模式。利用酶解和低聚糖分析的结合，我们确认了迁移较慢的重链峰作为半乳糖基化变体的具体身份。利用这种方法扩展产品表征，揭示了半乳糖基化变体在几种单克隆抗体的富集酸性电荷变体部分中持续升高。这种消化辅助CE-SDS方法也可以成功地应用于Fc融合蛋白，以实现半乳糖含量的结构域特异性表征。总的来说，我们证明，在缺乏更复杂的基于质谱的聚糖分析技术的情况下，在减少CE-SDS分析之前，对单克隆抗体和Fc融合蛋白进行IdeS预处理可以很容易地检测到不同样品中半乳糖含量的差异，同时提供n -聚糖占用的信息。最后，我们提出本研究中概述的分析策略提供CE-SDS峰识别，特别是半乳糖基化变体，并且可以支持扩展表征。

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引用次数: 0

A quantitative, broad-panel LC-MS/MS method for the analysis of intact steroid conjugates: A novel approach to steroid profiling for biomarker research in corticoid-dependent diseases 一种用于完整类固醇偶联物分析的定量、宽面板LC-MS/MS方法：一种用于皮质激素依赖性疾病生物标志物研究的类固醇谱分析的新方法

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-18 DOI: 10.1016/j.jpba.2025.117258

Eleanor North , Arne Gessner , Max Kurlbaum , Martin Fassnacht , Matthias Kroiss , Martin F. Fromm , Nora Bartels

In endocrine oncology changes in hormone synthesis and metabolism play a major role in disease formation and progression. Accordingly, steroid metabolism is a possible pathophysiological factor in adrenal tumours and metabolic conjugates of steroids are promising biomarker candidates for the clinically challenging differential diagnosis of highly prevalent, benign adrenocortical adenomas versus rare, aggressive adrenocortical carcinomas. The analysis of steroidal compounds in human biological samples is most accurately achieved by mass spectrometry-based approaches. Commonly, metabolic end products of steroids, such as sulfate and glucuronide conjugates, are hydrolysed before measurements to simplify the procedure. Therefore, data on actual steroid conjugate concentrations in humans is scarce. In this work we present a compact LC-MS/MS method for the absolute quantification of 22 intact steroid conjugates in urine and plasma with an instrument run time of 11 min and a straightforward sample preparation procedure. The method was successfully validated according to established international guidelines and applied to samples of patients with adrenal tumours to demonstrate the method’s suitability regarding sample preparation, analyte selection and quantifiable concentration ranges. Furthermore, the exploratory comparison of steroid abundance in the patient samples support the previously proposed potential of steroid conjugates as differentiating biomarkers between adrenocortical carcinoma and adenoma (e.g., 17-OH-pregnenolone sulfate, median plasma concentration in carcinoma (114 ng/ml) versus adenoma (3.76 ng/ml), p < 0.001). The newly developed method enables absolute quantification of multiple steroid conjugates in humans which provides the basis for profound biomarker research for the early detection of adrenocortical carcinomas and valuable data for related research questions.

在内分泌肿瘤学中，激素合成和代谢的变化在疾病的形成和发展中起着重要作用。因此，类固醇代谢可能是肾上腺肿瘤的病理生理因素，类固醇代谢偶联物是临床上具有挑战性的高发病率、良性肾上腺皮质腺瘤与罕见的侵袭性肾上腺皮质癌鉴别诊断的有希望的生物标志物候选物。人类生物样品中甾体化合物的分析最准确地实现了基于质谱的方法。通常，类固醇的代谢终产物，如硫酸盐和葡萄糖醛酸缀合物，在测量前水解以简化程序。因此，关于人体内实际类固醇偶联物浓度的数据很少。在这项工作中，我们提出了一种紧凑的LC-MS/MS方法，用于尿液和血浆中22种完整的类固醇偶联物的绝对定量，仪器运行时间为11 min，样品制备过程简单。根据既定的国际指南成功验证了该方法，并将其应用于肾上腺肿瘤患者的样本，以证明该方法在样品制备，分析物选择和可量化浓度范围方面的适用性。此外，对患者样本中类固醇丰度的探索性比较支持了先前提出的类固醇偶联物作为肾上腺皮质癌和腺瘤之间区分生物标志物的潜力（例如，17- oh -孕烯醇酮硫酸酯，癌（114 ng/ml）与腺瘤（3.76 ng/ml）的中位血浆浓度，p <； 0.001）。新开发的方法可以对人体多种类固醇偶联物进行绝对定量，为肾上腺皮质癌的早期检测提供深入的生物标志物研究基础，并为相关研究问题提供有价值的数据。

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引用次数: 0

Solid phase extraction of palbociclib from palbociclib-loaded therapeutic micelles: Novel synthesis of hybrid reduced graphene oxide–modified magnetic iron oxide (rGO@SiO2@Fe3O4)nanosorbent-based plasma extraction and pharmacokinetics study in rats 从负载palbociclib的治疗胶束中固相提取palbociclib：基于混合还原氧化石墨烯修饰磁性氧化铁（rGO@SiO2@Fe3O4）纳米吸附剂的新型合成及其大鼠药代动力学研究

IF 3.1 3区医学 Q2 CHEMISTRY, ANALYTICAL

Journal of pharmaceutical and biomedical analysis

Pub Date : 2025-11-17 DOI: 10.1016/j.jpba.2025.117270

Medapati Nikitha Lakshmi Suseela , Patharaj Gokul , Rajnish Kumar , Matte Kasi Viswanadh , Jyotsana Pandey , M. Sterlin Leo Hudson , Joseph Selvin , Madaswamy.S. Muthu

Accurate and sensitive quantification of anticancer agents in biological matrices is challenging yet crucial for pharmacokinetic evaluation and therapeutic monitoring. In this study, a reduced graphene oxide–modified magnetic iron oxide nanosorbent (

{rGO@SiO}_{2} {@Fe}_{3} O_{4}

) was synthesized via in-situ co-precipitation and the same was applied for the selective extraction and enrichment of palbociclib (PLB), using bosutinib (BSTB) as an internal standard. The nanosorbent was comprehensively characterized by electron microscopy with elemental mapping (FE-SEM/EDX), surface vibrational spectroscopy (ATR-FTIR), powder X-ray diffraction (PXRD), and superconducting quantum interference device (MPMS) magnetometer. The synthesized hybrid nanosorbent demonstrated favourable morphological, structural, and magnetic properties, enabling rapid magnetic separation and enhanced surface interactions. The established chromatographic analysis showed excellent linearity across 100–10,000 ng/mL, alongside a quantification limit of 100 ng/mL and a minimum detectable concentration of 50 ng/mL. Application of the developed method to rat plasma samples yielded good recovery and reproducibility, validating its performance in real biological matrices. In addition, PLB-loaded TPGS micelles (100, 125, and 150 mg) were successfully formulated, exhibiting particle sizes of 13.3–15.8 nm, supporting their potential for drug delivery applications. The integration of magnetic separability with enhanced sorption capacity makes

{rGO@SiO}_{2} {@Fe}_{3} O_{4}

a reusable and environmentally sustainable platform for plasma extraction. This work offers a promising strategy for sensitive, reliable, and environmental friendly quantification of PLB.

准确和敏感地定量生物基质中的抗癌药物是具有挑战性的，但对于药代动力学评估和治疗监测至关重要。本研究通过原位共沉淀法合成了还原氧化石墨烯修饰的磁性氧化铁纳米吸附剂rGO@SiO2@Fe3O4，并以博舒替尼（BSTB）为内标，将其用于选择性提取富集帕博西尼（PLB）。采用元素映射电镜（FE-SEM/EDX）、表面振动光谱（ATR-FTIR）、粉末x射线衍射（PXRD）和超导量子干涉装置（MPMS）磁强计对纳米吸附剂进行了全面表征。合成的杂化纳米吸附剂表现出良好的形态、结构和磁性能，能够实现快速磁分离和增强表面相互作用。所建立的色谱分析在100 - 10,000 ng/mL范围内显示出良好的线性，定量限为100 ng/mL，最低检测浓度为50 ng/mL。将该方法应用于大鼠血浆样品，回收率和重现性良好，验证了其在真实生物基质中的性能。此外，成功制备了负载plb的TPGS胶束（100、125和150 mg），其粒径为13.3-15.8 nm，具有潜在的药物递送应用前景。磁性可分离性与增强的吸附能力的集成使rGO@SiO2@Fe3O4成为可重复使用且环境可持续的等离子体提取平台。这项工作为敏感、可靠和环保的PLB定量提供了一个有前途的策略。

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引用次数: 0