首页 > 最新文献

Journal of Chromatography B最新文献

英文 中文
A sensitive method for confirming the clearance of caffeine and its three primary metabolites in sheep tissues destined for human consumption 一种确定供人食用的绵羊组织中咖啡因及其三种主要代谢物清除的灵敏方法
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-03 DOI: 10.1016/j.jchromb.2026.124912
Gregory S. Doran , Susan M. Robertson , Michael A. Friend , Scott H. Edwards
Caffeine is a naturally occurring stimulant that is consumed in coffee, tea, and energy and soft drinks, but may also be used therapeutically in humans and animals. Caffeine initially degrades rapidly via demethylation to theobromine, paraxanthine and theophylline, prior to further degradation to other metabolites. A method was developed to separate and quantify the four chemicals, which is generally complicated by co-elution of paraxanthine and theophylline, which are the two bioactive metabolites. Processes to extract and purify the four analytes from liver, kidney, muscle, brain, perirenal and abdominal fats, plasma and milk were also developed and provided limits of quantitation as low as 1 ng/g in solid tissues and 1 ng/mL in milk and plasma using a combination of solvent and solid phase extractions. Sample homogenisation considerations and recovery at different stages was investigated to identify the areas of greatest analyte loss. A study was conducted involving the daily oral caffeine dosing of pregnant merino ewes at a rate of 25 mg/kg for 52 days until the birth of lambs. Tissue samples and milk were collected from ewes and lambs up to 28 days after birth and analysed using the new methodologies. Caffeine and the three primary metabolites appeared in all tissues analysed at high concentrations at day 0 (birthing), but declined by 99.9 % in all tissues in ewes by day 7, and all tissues except plasma (98.5 %) in lambs by day 7. All analytes were only detected in milk from ewes at day 0, but provided a source of caffeine and its metabolites to suckling newborn lambs, which may be responsible for the slower clearance from lamb plasma. Theophylline was the dominant metabolite in both ewes and lambs, followed by paraxanthine, while theobromine was only detected at much lower concentrations. The results derived from this animal study using this new method indicate that accumulation of caffeine and its three primary metabolites does not occur in the milk or tissues of ewes or lambs.
咖啡因是一种天然存在的兴奋剂,存在于咖啡、茶、能量饮料和软饮料中,但也可用于人类和动物的治疗。咖啡因最初通过去甲基化迅速降解为可可碱、副黄嘌呤和茶碱,然后进一步降解为其他代谢物。建立了一种分离和定量四种化学物质的方法,该方法通常由副黄嘌呤和茶碱这两种生物活性代谢物共同洗脱而复杂。还开发了从肝脏、肾脏、肌肉、大脑、肾周和腹部脂肪、血浆和牛奶中提取和纯化这四种分析物的工艺,并提供了在固体组织中低至1ng /g,在牛奶和血浆中低至1ng /mL的定量限,使用溶剂和固相萃取的组合。样品均质考虑和回收在不同阶段进行了调查,以确定最大的分析物损失的区域。对怀孕的美利奴母羊进行了一项研究,在52天内每天口服咖啡因,剂量为25毫克/公斤,直到羔羊出生。从出生后28天的母羊和羔羊身上收集组织样本和牛奶,并使用新方法进行分析。在第0天(分娩),咖啡因和3种主要代谢物在所有组织中均以高浓度出现,但在第7天,母羊的所有组织中咖啡因和3种主要代谢物的浓度下降了99.9%,在第7天,羔羊除血浆外的所有组织中咖啡因和3种主要代谢物的浓度下降了98.5%。所有分析物仅在第0天母羊的奶中检测到,但为哺乳的新生羔羊提供了咖啡因及其代谢物的来源,这可能是羔羊血浆中咖啡因清除较慢的原因。母羊和羔羊的主要代谢物是茶碱,其次是副黄嘌呤,而可可碱仅在较低浓度下检测到。使用这种新方法的动物研究结果表明,咖啡因及其三种主要代谢物不会在母羊或羔羊的牛奶或组织中积累。
{"title":"A sensitive method for confirming the clearance of caffeine and its three primary metabolites in sheep tissues destined for human consumption","authors":"Gregory S. Doran ,&nbsp;Susan M. Robertson ,&nbsp;Michael A. Friend ,&nbsp;Scott H. Edwards","doi":"10.1016/j.jchromb.2026.124912","DOIUrl":"10.1016/j.jchromb.2026.124912","url":null,"abstract":"<div><div>Caffeine is a naturally occurring stimulant that is consumed in coffee, tea, and energy and soft drinks, but may also be used therapeutically in humans and animals. Caffeine initially degrades rapidly via demethylation to theobromine, paraxanthine and theophylline, prior to further degradation to other metabolites. A method was developed to separate and quantify the four chemicals, which is generally complicated by co-elution of paraxanthine and theophylline, which are the two bioactive metabolites. Processes to extract and purify the four analytes from liver, kidney, muscle, brain, perirenal and abdominal fats, plasma and milk were also developed and provided limits of quantitation as low as 1 ng/g in solid tissues and 1 ng/mL in milk and plasma using a combination of solvent and solid phase extractions. Sample homogenisation considerations and recovery at different stages was investigated to identify the areas of greatest analyte loss. A study was conducted involving the daily oral caffeine dosing of pregnant merino ewes at a rate of 25 mg/kg for 52 days until the birth of lambs. Tissue samples and milk were collected from ewes and lambs up to 28 days after birth and analysed using the new methodologies. Caffeine and the three primary metabolites appeared in all tissues analysed at high concentrations at day 0 (birthing), but declined by 99.9 % in all tissues in ewes by day 7, and all tissues except plasma (98.5 %) in lambs by day 7. All analytes were only detected in milk from ewes at day 0, but provided a source of caffeine and its metabolites to suckling newborn lambs, which may be responsible for the slower clearance from lamb plasma. Theophylline was the dominant metabolite in both ewes and lambs, followed by paraxanthine, while theobromine was only detected at much lower concentrations. The results derived from this animal study using this new method indicate that accumulation of caffeine and its three primary metabolites does not occur in the milk or tissues of ewes or lambs.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124912"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145895827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Removal of aspirin and tetracycline hydrochloride using UiO66/Polydopamine/bacterial cellulose composite: Phenomenological modeling and theoretical study UiO66/聚多巴胺/细菌纤维素复合材料去除阿司匹林和盐酸四环素:现象学模型和理论研究
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-08 DOI: 10.1016/j.jchromb.2025.124910
Nadia Bouaziz , Fatma Aouaini , Abdelmottaleb Ben Lamine
The effective removal of antibiotics and non-steroidal anti-inflammatory drugs and their combined pollutants is critical for both the aquatic environment and human health. This study evaluates the removal of pharmaceutical waste through the adsorption of aspirin and tetracycline hydrochloride (TC) onto the surface of a UiO-66/Polydopamine/Bacterial Cellulose composite (UiO-66/PDA/BC). Both theoretical and experimental approaches were considered to investigate the removal of aspirin and TC. The theoretical study, based on statistical physics theory, employs three analytical models to describe the adsorption phenomena of these drugs at the molecular level. According to the fitting results, the adsorption of both drugs onto the UiO-66/PDA/BC composite is an endothermic process and follows a monolayer adsorption mechanism with one type of site. Regarding the number of adsorbed molecules per site (n), the analysis indicates that both multi-docking (n < 1) and multi-molecular (n > 1) adsorption mechanisms are possible for aspirin and TC on UiO-66/PDA/BC. The adsorption capacities at saturation of the UiO-66/PDA/BC adsorbent deduced by the monolayer model are found to be 147.34–215.05 mg/g for aspirin and 193.72–246.66 mg/g for TC. Furthermore, adsorption energy calculations suggest that the removal of these pharmaceutical pollutants occurs primarily through physical interactions. The thermodynamic analysis confirms the spontaneous and feasible nature of the adsorption of both drugs onto the UiO-66/PDA/BC composite.
有效去除抗生素和非甾体抗炎药及其复合污染物对水生环境和人类健康都至关重要。本研究评价了UiO-66/聚多巴胺/细菌纤维素复合材料(UiO-66/PDA/BC)表面吸附阿司匹林和盐酸四环素(TC)对制药废弃物的去除效果。我们考虑了理论和实验两种方法来研究阿司匹林和TC的去除。理论研究以统计物理理论为基础,采用三种分析模型来描述这些药物在分子水平上的吸附现象。拟合结果表明,两种药物在UiO-66/PDA/BC复合材料上的吸附均为吸热过程,且均为单位点的单层吸附机制。对于每个位点的吸附分子数(n),分析表明阿司匹林和TC在UiO-66/PDA/BC上可能存在多对接(n < 1)和多分子(n > 1)吸附机制。经单层模型计算,uiu -66/PDA/BC吸附剂对阿司匹林的饱和吸附量为147.34 ~ 215.05 mg/g,对TC的饱和吸附量为193.72 ~ 246.66 mg/g。此外,吸附能计算表明,这些药物污染物的去除主要是通过物理相互作用发生的。热力学分析证实了两种药物在UiO-66/PDA/BC复合材料上吸附的自发性和可行性。
{"title":"Removal of aspirin and tetracycline hydrochloride using UiO66/Polydopamine/bacterial cellulose composite: Phenomenological modeling and theoretical study","authors":"Nadia Bouaziz ,&nbsp;Fatma Aouaini ,&nbsp;Abdelmottaleb Ben Lamine","doi":"10.1016/j.jchromb.2025.124910","DOIUrl":"10.1016/j.jchromb.2025.124910","url":null,"abstract":"<div><div>The effective removal of antibiotics and non-steroidal anti-inflammatory drugs and their combined pollutants is critical for both the aquatic environment and human health. This study evaluates the removal of pharmaceutical waste through the adsorption of aspirin and tetracycline hydrochloride (TC) onto the surface of a UiO-66/Polydopamine/Bacterial Cellulose composite (UiO-66/PDA/BC). Both theoretical and experimental approaches were considered to investigate the removal of aspirin and TC. The theoretical study, based on statistical physics theory, employs three analytical models to describe the adsorption phenomena of these drugs at the molecular level. According to the fitting results, the adsorption of both drugs onto the UiO-66/PDA/BC composite is an endothermic process and follows a monolayer adsorption mechanism with one type of site. Regarding the number of adsorbed molecules per site (n), the analysis indicates that both multi-docking (<em>n</em> &lt; 1) and multi-molecular (<em>n</em> &gt; 1) adsorption mechanisms are possible for aspirin and TC on UiO-66/PDA/BC. The adsorption capacities at saturation of the UiO-66/PDA/BC adsorbent deduced by the monolayer model are found to be 147.34–215.05 mg/g for aspirin and 193.72–246.66 mg/g for TC. Furthermore, adsorption energy calculations suggest that the removal of these pharmaceutical pollutants occurs primarily through physical interactions. The thermodynamic analysis confirms the spontaneous and feasible nature of the adsorption of both drugs onto the UiO-66/PDA/BC composite.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124910"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Development and application of SPE-UHPLC-MS/MS methods for quantification of 29 steroids in rat serum SPE-UHPLC-MS/MS法测定大鼠血清中29种类固醇的含量及应用。
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-11 DOI: 10.1016/j.jchromb.2026.124924
Haiyang Cui , Dongliang Yang , Yonggang Zheng , Jing Li , Hairui Yan , Xiaoli Gao
Steroids and their metabolites play crucial roles in reproductive health, endocrine diseases, and tumor development. Comprehensive analysis of these compounds is essential for both mechanistic investigations and clinical evaluations. Although comprehensive analytical methods have been established for steroids in human clinical samples, their direct application to rat specimens remains limited, particularly with the absence of validated methods for detecting certain hydroxylated steroid metabolites in vivo. This study developed highly sensitive solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry methods for the quantitative analysis of 29 steroid metabolites. Through optimized derivatization of steroids and integration of two-dimensional liquid chromatography techniques, the method effectively reduces interference from non-volatile salts and overcomes trace-level detection challenges. Method validation demonstrated a wide linear range (0.002–250 ng/mL), low limits of quantification (2–100 pg/mL), high accuracy (88–114%), and precision (intra- and inter-batch CV ≤14%). The method exhibited acceptable extraction recovery, matrix effects, and stability, confirming compliance with regulatory standards. The validated method was successfully applied to characterize serum steroid metabolic profiles in both N-methyl-N-nitrosourea-induced breast cancer and ovariectomized rat models, revealing alterations in estrogen 2-hydroxylation and pregnenolone 21-hydroxylation pathways mediated by key steroidogenic enzymes, including CYP1A1/CYP1B1 and potentially CYP21A2-independent mechanisms, providing robust support for preclinical research on steroid-related diseases.
类固醇及其代谢产物在生殖健康、内分泌疾病和肿瘤发展中起着至关重要的作用。这些化合物的综合分析是必不可少的机制调查和临床评价。尽管已经建立了针对人类临床样本中的类固醇的综合分析方法,但它们在大鼠样本中的直接应用仍然有限,特别是缺乏检测体内某些羟基化类固醇代谢物的有效方法。本研究建立了高灵敏度固相萃取-超高效液相色谱-串联质谱联用方法对29种类固醇代谢物进行定量分析。通过优化类固醇衍生化和整合二维液相色谱技术,该方法有效地减少了非挥发性盐的干扰,克服了痕量水平检测的挑战。结果表明,该方法线性范围宽(0.002 ~ 250 ng/mL),定量下限低(2 ~ 100 pg/mL),准确度高(88 ~ 114%),精密度高(批内和批间CV≤14%)。该方法具有良好的萃取回收率、基质效应和稳定性,符合国家标准。该方法成功应用于n -甲基-n -亚硝基源诱导的乳腺癌和去卵巢大鼠模型的血清类固醇代谢谱,揭示了关键类固醇生成酶介导的雌激素2-羟基化和孕烯醇酮21-羟基化途径的改变,包括CYP1A1/CYP1B1和潜在的cyp21a2独立机制,为类固醇相关疾病的临床前研究提供了有力支持。
{"title":"Development and application of SPE-UHPLC-MS/MS methods for quantification of 29 steroids in rat serum","authors":"Haiyang Cui ,&nbsp;Dongliang Yang ,&nbsp;Yonggang Zheng ,&nbsp;Jing Li ,&nbsp;Hairui Yan ,&nbsp;Xiaoli Gao","doi":"10.1016/j.jchromb.2026.124924","DOIUrl":"10.1016/j.jchromb.2026.124924","url":null,"abstract":"<div><div>Steroids and their metabolites play crucial roles in reproductive health, endocrine diseases, and tumor development. Comprehensive analysis of these compounds is essential for both mechanistic investigations and clinical evaluations. Although comprehensive analytical methods have been established for steroids in human clinical samples, their direct application to rat specimens remains limited, particularly with the absence of validated methods for detecting certain hydroxylated steroid metabolites in vivo. This study developed highly sensitive solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry methods for the quantitative analysis of 29 steroid metabolites. Through optimized derivatization of steroids and integration of two-dimensional liquid chromatography techniques, the method effectively reduces interference from non-volatile salts and overcomes trace-level detection challenges. Method validation demonstrated a wide linear range (0.002–250 ng/mL), low limits of quantification (2–100 pg/mL), high accuracy (88–114%), and precision (intra- and inter-batch CV ≤14%). The method exhibited acceptable extraction recovery, matrix effects, and stability, confirming compliance with regulatory standards. The validated method was successfully applied to characterize serum steroid metabolic profiles in both <em>N</em>-methyl-N-nitrosourea-induced breast cancer and ovariectomized rat models, revealing alterations in estrogen 2-hydroxylation and pregnenolone 21-hydroxylation pathways mediated by key steroidogenic enzymes, including CYP1A1/CYP1B1 and potentially CYP21A2-independent mechanisms, providing robust support for preclinical research on steroid-related diseases.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124924"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146004851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Portable mass spectrometry systems for point-of-care testing: technologies, applications, and clinical implementation 用于即时检测的便携式质谱系统:技术、应用和临床实施
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-11 DOI: 10.1016/j.jchromb.2026.124923
Brooks B. Pond, Madison Hoskins, Haylie Hollis, Stacy Brown
Portable mass spectrometry systems have shown great potential in point-of-care testing and field-based diagnostics, as they fill important gaps in decentralized clinical analysis. Conventional centralized laboratory testing may delay clinical decision making due to transport and analysis times. For conditions where transportation is limited, these delays disproportionately affect resource-poor settings. Advances in technology allow the design and assembly of lightweight, portable MS systems. Fundamental issues such as size reduction, power consumption, and vacuum requirements have been addressed as the MS miniaturization field has developed. These advances have generated commercially available devices and home-built instruments capable of rapid analysis, often without extensive sample preparation. Studies on applicability of portable MS have already shown utility in multiple fronts, such as therapeutic drug monitoring for HIV, psychiatric drugs, and cardiac interventions; drug profiling in forensic medicine; disease diagnosis including malaria detection; and cancer tissue analysis. Nevertheless, the tradeoff between miniaturization and sensitivity, small mass resolution relative to benchtop instruments, and challenges in reproducibility represent ongoing issues with miniaturized mass spectrometry. Despite these disadvantages, significant clinical potential can be concluded from current published evidence. With the development of complementary sample preparation methods and miniaturization technology, portable MS systems could see implementation in emergency departments, mobile clinics, and resource scarce clinic environments.
便携式质谱分析系统在护理点检测和现场诊断方面显示出巨大潜力,因为它们填补了分散临床分析的重要空白。由于运输和分析时间,传统的集中实验室检测可能会延迟临床决策。在交通有限的情况下,这些延误不成比例地影响资源贫乏的环境。技术的进步使得设计和组装轻便、便携的质谱系统成为可能。随着MS小型化领域的发展,诸如尺寸减小、功耗和真空要求等基本问题已经得到解决。这些进步产生了能够快速分析的商用设备和自制仪器,通常不需要大量的样品制备。便携式质谱的适用性研究已经在多个方面显示出实用性,例如艾滋病毒治疗药物监测,精神药物和心脏干预;法医学中的药物分析;疾病诊断,包括疟疾检测;还有癌症组织分析。然而,小型化和灵敏度之间的权衡,相对于台式仪器的小质量分辨率,以及再现性方面的挑战是小型化质谱法持续存在的问题。尽管存在这些缺点,但从目前发表的证据可以得出显著的临床潜力。随着补充制样方法和小型化技术的发展,便携式质谱系统可以在急诊科、流动诊所和资源稀缺的诊所环境中实施。
{"title":"Portable mass spectrometry systems for point-of-care testing: technologies, applications, and clinical implementation","authors":"Brooks B. Pond,&nbsp;Madison Hoskins,&nbsp;Haylie Hollis,&nbsp;Stacy Brown","doi":"10.1016/j.jchromb.2026.124923","DOIUrl":"10.1016/j.jchromb.2026.124923","url":null,"abstract":"<div><div>Portable mass spectrometry systems have shown great potential in point-of-care testing and field-based diagnostics, as they fill important gaps in decentralized clinical analysis. Conventional centralized laboratory testing may delay clinical decision making due to transport and analysis times. For conditions where transportation is limited, these delays disproportionately affect resource-poor settings. Advances in technology allow the design and assembly of lightweight, portable MS systems. Fundamental issues such as size reduction, power consumption, and vacuum requirements have been addressed as the MS miniaturization field has developed. These advances have generated commercially available devices and home-built instruments capable of rapid analysis, often without extensive sample preparation. Studies on applicability of portable MS have already shown utility in multiple fronts, such as therapeutic drug monitoring for HIV, psychiatric drugs, and cardiac interventions; drug profiling in forensic medicine; disease diagnosis including malaria detection; and cancer tissue analysis. Nevertheless, the tradeoff between miniaturization and sensitivity, small mass resolution relative to benchtop instruments, and challenges in reproducibility represent ongoing issues with miniaturized mass spectrometry. Despite these disadvantages, significant clinical potential can be concluded from current published evidence. With the development of complementary sample preparation methods and miniaturization technology, portable MS systems could see implementation in emergency departments, mobile clinics, and resource scarce clinic environments.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124923"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Simultaneous determination of short-chain fatty acids and tryptophan metabolites by a propyl chloroformate- derivatized GC–MS approach 氯甲酸丙酯衍生气相色谱-质谱法同时测定短链脂肪酸和色氨酸代谢物
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-12 DOI: 10.1016/j.jchromb.2026.124925
Yefeng Han , Xiaofang He , Yueling Gong , Mingxiao Li , Lili Sheng , Ningning Zheng , Jianbo Wan , Houkai Li , Yuanyuan Li
Short-chain fatty acids (SCFAs) and tryptophan metabolites, serve as key mediators of host-microbiota crosstalk, influencing physiological and pathological processes. Their interconnected roles necessitate simultaneous quantification to fully elucidate the potential mechanism of gut microbiota in metabolic diseases. However, they are difficult to be detected simultaneously due to differences in content or different polarity which contain specific carboxylic group, amino group or phenolic hydroxyl groups. In current study, our primary goal is to establish a rapid and sensitive quantitative measurement for SCFAs and tryptophan metabolites by using propyl chloroformate-(PCF) derivatization based on gas chromatography-triple quadrupole mass spectrometry (GC–MS/MS) analysis. Then, we applied the established method in measurement of serum samples from healthy subjects and metabolic associated fatty liver disease (MAFLD) patients. First, we optimized the reaction conditions including PCF volume, reaction time, extraction reagent ratio, and alkaline reagent concentration, enabling the simultaneous detection of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, 2-methylvaleric acid, hexanoic acid, indole, succinic acid, quinolinic acid, indole acetic acid, glutamine, indole butyric acid, 3-hydroxyanthranilic acid, melatonin, kynurenine, tyrosine, and tryptophan. 2-chloro-L-phenylalanine was set as the internal standard. The optimized condition showed good linearity, and the intra/inter-day precision achieved in the range from 0.81% to 14.88%. The recovery ranged from 85.68% to 114.50% and the matrix effect ranged from 85.81% to 113.42%. In addition, the influence of different storage conditions on sample stability was also acceptable. Finally, the quantitation result of serum samples indicated that isovaleric acid, quinolineic acid, indoleacetic acid, glutamine, melatonin, tyrosine, and tryptophan had significant difference between healthy subjects and MAFLD patients. The levels of isovaleric acid, indoleacetic acid, glutamine, tyrosine, and tryptophan had significant correlations with clinical indicators such as ALT, AST, Cap, Cr and TG, supporting their potential for further translational studies. In summary, this improved method is applicable for quantitative measurement of SCFAs and tryptophan metabolites, as well as those endogenous metabolites containing carboxylic, amino and phenolic hydroxyl groups.
短链脂肪酸(SCFAs)和色氨酸代谢物是宿主-微生物群串扰的关键介质,影响生理和病理过程。它们相互关联的作用需要同时量化,以充分阐明肠道微生物群在代谢性疾病中的潜在机制。但由于它们含有特定的羧基、氨基或酚羟基,含量不同或极性不同,难以同时检测。在本研究中,我们的主要目标是建立一种基于气相色谱-三重四极杆质谱(GC-MS /MS)分析的氯甲酸丙酯-(PCF)衍生化快速、灵敏的SCFAs和色氨酸代谢物定量测定方法。然后,我们将建立的方法应用于健康受试者和代谢性脂肪性肝病(MAFLD)患者的血清样本测量。首先,对PCF体积、反应时间、提取试剂配比、碱性试剂浓度等条件进行优化,实现了乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、2-甲基戊酸、己酸、吲哚、琥珀酸、喹啉酸、吲哚乙酸、谷氨酰胺、吲哚丁酸、3-羟基苯甲酸、褪黑素、犬尿氨酸、酪氨酸、色氨酸的同时检测。内标为2-氯- l -苯丙氨酸。优化条件线性良好,日内/日间精密度范围为0.81% ~ 14.88%。加样回收率为85.68% ~ 114.50%,基质效应为85.81% ~ 113.42%。此外,不同的储存条件对样品稳定性的影响也是可以接受的。最后,血清样品的定量结果显示,健康受试者与MAFLD患者的异戊酸、喹啉酸、吲哚乙酸、谷氨酰胺、褪黑素、酪氨酸和色氨酸存在显著差异。异戊酸、吲哚乙酸、谷氨酰胺、酪氨酸和色氨酸的水平与ALT、AST、Cap、Cr和TG等临床指标有显著相关性,支持其进一步转化研究的潜力。综上所述,该改进方法适用于SCFAs和色氨酸代谢物的定量测定,以及含有羧基、氨基和酚羟基的内源性代谢物的定量测定。
{"title":"Simultaneous determination of short-chain fatty acids and tryptophan metabolites by a propyl chloroformate- derivatized GC–MS approach","authors":"Yefeng Han ,&nbsp;Xiaofang He ,&nbsp;Yueling Gong ,&nbsp;Mingxiao Li ,&nbsp;Lili Sheng ,&nbsp;Ningning Zheng ,&nbsp;Jianbo Wan ,&nbsp;Houkai Li ,&nbsp;Yuanyuan Li","doi":"10.1016/j.jchromb.2026.124925","DOIUrl":"10.1016/j.jchromb.2026.124925","url":null,"abstract":"<div><div>Short-chain fatty acids (SCFAs) and tryptophan metabolites, serve as key mediators of host-microbiota crosstalk, influencing physiological and pathological processes. Their interconnected roles necessitate simultaneous quantification to fully elucidate the potential mechanism of gut microbiota in metabolic diseases. However, they are difficult to be detected simultaneously due to differences in content or different polarity which contain specific carboxylic group, amino group or phenolic hydroxyl groups. In current study, our primary goal is to establish a rapid and sensitive quantitative measurement for SCFAs and tryptophan metabolites by using propyl chloroformate-(PCF) derivatization based on gas chromatography-triple quadrupole mass spectrometry (GC–MS/MS) analysis. Then, we applied the established method in measurement of serum samples from healthy subjects and metabolic associated fatty liver disease (MAFLD) patients. First, we optimized the reaction conditions including PCF volume, reaction time, extraction reagent ratio, and alkaline reagent concentration, enabling the simultaneous detection of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, 2-methylvaleric acid, hexanoic acid, indole, succinic acid, quinolinic acid, indole acetic acid, glutamine, indole butyric acid, 3-hydroxyanthranilic acid, melatonin, kynurenine, tyrosine, and tryptophan. 2-chloro-<em>L</em>-phenylalanine was set as the internal standard. The optimized condition showed good linearity, and the intra/inter-day precision achieved in the range from 0.81% to 14.88%. The recovery ranged from 85.68% to 114.50% and the matrix effect ranged from 85.81% to 113.42%. In addition, the influence of different storage conditions on sample stability was also acceptable. Finally, the quantitation result of serum samples indicated that isovaleric acid, quinolineic acid, indoleacetic acid, glutamine, melatonin, tyrosine, and tryptophan had significant difference between healthy subjects and MAFLD patients. The levels of isovaleric acid, indoleacetic acid, glutamine, tyrosine, and tryptophan had significant correlations with clinical indicators such as ALT, AST, Cap, Cr and TG, supporting their potential for further translational studies. In summary, this improved method is applicable for quantitative measurement of SCFAs and tryptophan metabolites, as well as those endogenous metabolites containing carboxylic, amino and phenolic hydroxyl groups.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124925"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Library-assisted UHPLC-Q-TOF-MS/MS bioanalytical profiling of Qiling Hushen granules: In-vivo metabolic mapping and kidney-biased distribution 库辅助UHPLC-Q-TOF-MS/MS:七龄护肾颗粒体内代谢图谱和肾脏偏倚分布
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-05 DOI: 10.1016/j.jchromb.2026.124913
Pei Sheng , Mingjin Zhang , Shuqi Wang , Shuyi Peng , Jiakun Li , Yi Liu , Kaixuan Xu , Xiaofei An
Qiling Hushen Granules (QLHSG), a multi-herb prescription widely used for diabetic kidney disease (DKD), lacks system-level evidence linking chemical features with in-vivo disposition. A library-assisted UHPLC–Q-TOF-MS/MS workflow was developed to integrate in-vitro phytochemical profiling with in-vivo prototype/metabolite elucidation and organ-level biodistribution. An in-house spectral library was constructed using reference standards, open databases and diagnostic-ion rules, enabling high-confidence annotation based on retention time, accurate mass and MS/MS fragmentation. In total, 166 in-vitro constituents were cataloged. In vivo, 78 prototype constituents were characterized. Metabolites were assigned by matrix (bile 46, feces 44, plasma 27, urine 156), delineating phase I/II biotransformation and elimination routes. Tissue mapping demonstrated kidney-biased accumulation of multiple prototypes and metabolites, indicating that renal targeting is consistent with the therapeutic context. Dominant exposure classes included iridoids and flavonoids. Representative bioactives were astragaloside IV, calycosin, paeoniflorin, secologanoside and sweroside. Network analysis combined with molecular docking connected absorbed constituents to DKD-relevant targets (e.g., TP53, EGFR, AKT1, STAT3, HIF1α), providing plausible mechanistic insights. Overall, this workflow establishes a reproducible pathway, from chemical profiling to systemic exposure and organ targeting in complex herbal formulations. For the first time, it provides system-level chemical evidence supporting the renoprotective effects of QLHSG.
七龄护肾颗粒(QLHSG)是一种广泛用于糖尿病肾病(DKD)的多药处方,缺乏系统水平的证据将化学特征与体内处置联系起来。开发了一个库辅助UHPLC-Q-TOF-MS /MS工作流程,将体外植物化学分析与体内原型/代谢物阐明和器官水平的生物分布结合起来。利用参考标准、开放数据库和诊断规则构建了内部光谱库,实现了基于保留时间、精确质量和MS/MS碎片的高置信度标注。共鉴定出166种体外成分。在体内,78个原型成分被表征。代谢产物按基质分配(胆汁46,粪便44,血浆27,尿液156),描绘I/II期生物转化和消除途径。组织制图显示多种原型和代谢物的肾脏偏向性积累,表明肾脏靶向与治疗背景一致。主要暴露种类包括环烯醚萜和类黄酮。具有代表性的生物活性成分有黄芪甲苷、毛蕊花苷、芍药苷、龙葵苷和苦参苷。网络分析结合分子对接将吸收成分与dkd相关靶点(如TP53、EGFR、AKT1、STAT3、HIF1α)连接起来,提供了合理的机制见解。总的来说,这个工作流程建立了一个可重复的途径,从化学分析到系统暴露和复杂草药配方的器官靶向。该研究首次提供了支持QLHSG肾保护作用的系统级化学证据。
{"title":"Library-assisted UHPLC-Q-TOF-MS/MS bioanalytical profiling of Qiling Hushen granules: In-vivo metabolic mapping and kidney-biased distribution","authors":"Pei Sheng ,&nbsp;Mingjin Zhang ,&nbsp;Shuqi Wang ,&nbsp;Shuyi Peng ,&nbsp;Jiakun Li ,&nbsp;Yi Liu ,&nbsp;Kaixuan Xu ,&nbsp;Xiaofei An","doi":"10.1016/j.jchromb.2026.124913","DOIUrl":"10.1016/j.jchromb.2026.124913","url":null,"abstract":"<div><div>Qiling Hushen Granules (QLHSG), a multi-herb prescription widely used for diabetic kidney disease (DKD), lacks system-level evidence linking chemical features with <em>in-vivo</em> disposition. A library-assisted UHPLC–Q-TOF-MS/MS workflow was developed to integrate <em>in-vitro</em> phytochemical profiling with <em>in-vivo</em> prototype/metabolite elucidation and organ-level biodistribution. An in-house spectral library was constructed using reference standards, open databases and diagnostic-ion rules, enabling high-confidence annotation based on retention time, accurate mass and MS/MS fragmentation. In total, 166 <em>in-vitro</em> constituents were cataloged. <em>In vivo</em>, 78 prototype constituents were characterized. Metabolites were assigned by matrix (bile 46, feces 44, plasma 27, urine 156), delineating phase I/II biotransformation and elimination routes. Tissue mapping demonstrated kidney-biased accumulation of multiple prototypes and metabolites, indicating that renal targeting is consistent with the therapeutic context. Dominant exposure classes included iridoids and flavonoids. Representative bioactives were astragaloside IV, calycosin, paeoniflorin, secologanoside and sweroside. Network analysis combined with molecular docking connected absorbed constituents to DKD-relevant targets (<em>e.g.</em>, TP53, EGFR, AKT1, STAT3, HIF1α), providing plausible mechanistic insights. Overall, this workflow establishes a reproducible pathway, from chemical profiling to systemic exposure and organ targeting in complex herbal formulations. For the first time, it provides system-level chemical evidence supporting the renoprotective effects of QLHSG.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124913"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145923079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Toward needle-free lamotrigine monitoring: Proof-of-concept for saliva and dried saliva spot analysis by LC-MS/MS - a short communication 迈向无针拉莫三嗪监测:LC-MS/MS用于唾液和干唾液斑点分析的概念验证-简短交流。
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-16 DOI: 10.1016/j.jchromb.2026.124929
Viktória Ďurčová , Marta Pelcová , Pavel Šmak , Ondřej Strýček , Jana Gregorová , Ondřej Peš , Zdeněk Glatz , Pavel Šištík , Jan Juřica
Recent advancements in micro-sampling methods provide non-invasive options for collecting biological samples used in therapeutic drug monitoring (TDM). Lately, alternative matrices such as capillary blood and saliva/oral fluid in the form of dried saliva spots, have attracted considerable interest. These matrices represent a promising approach for TDM, as they could improve accessibility while preserving analytical precision.
The study developed and validated an LC-MS method for lamotrigine quantitation in saliva and Dried Saliva Spots (DSS). Samples were prepared via protein precipitation (methanol: acetonitrile: water, 7:2:1) and dilution, followed by separation on a Luna Omega C18 Polar column and detection in ESI+ mode. An isotopically labelled internal standard was employed, ensuring compliance with EMA guidelines. The linearity was demonstrated for lamotrigine concentrations ranging from 0.20 to 25 mg/L in both dried saliva spot and saliva samples.
Saliva and DSS samples from patients were used to compare lamotrigine concentrations with plasma levels determined by a hospital-accredited laboratory. Findings demonstrated a strong correlation between evaluated matrices and plasma (R = 0.847 for saliva and plasma, and R = 0.839 for DSS and plasma, both p < 0.0001), underscoring the promise of saliva and DSS as substitutes for TDM. This technique improves patient comfort and accessibility through a non-invasive sampling method while ensuring accuracy.
These findings provide a foundation for broader clinical implementation of alternative-matrix TDM, representing a significant step toward patient-centred, accessible pharmacotherapy and enabling improved LTG monitoring in ambulatory and remote settings.
微采样方法的最新进展为治疗药物监测(TDM)中使用的生物样品的收集提供了非侵入性选择。最近,替代的基质,如毛细管血和唾液/口服液在干燥的唾液斑点的形式,已经引起了相当大的兴趣。这些矩阵代表了一种很有前途的TDM方法,因为它们可以在保持分析精度的同时提高可访问性。本研究建立并验证了一种用于唾液和干唾液斑(DSS)中拉莫三嗪定量的LC-MS方法。样品经蛋白质沉淀(甲醇:乙腈:水,7:2:1)和稀释制备,在Luna Omega C18 Polar柱上分离,ESI+模式下检测。采用同位素标记的内部标准,确保符合EMA指南。拉莫三嗪浓度在0.20 ~ 25mg /L范围内呈线性关系。患者唾液和DSS样本用于比较拉莫三嗪浓度与医院认可的实验室测定的血浆水平。结果显示,评估基质与血浆之间存在很强的相关性(唾液和血浆的R = 0.847, DSS和血浆的R = 0.839,均为p
{"title":"Toward needle-free lamotrigine monitoring: Proof-of-concept for saliva and dried saliva spot analysis by LC-MS/MS - a short communication","authors":"Viktória Ďurčová ,&nbsp;Marta Pelcová ,&nbsp;Pavel Šmak ,&nbsp;Ondřej Strýček ,&nbsp;Jana Gregorová ,&nbsp;Ondřej Peš ,&nbsp;Zdeněk Glatz ,&nbsp;Pavel Šištík ,&nbsp;Jan Juřica","doi":"10.1016/j.jchromb.2026.124929","DOIUrl":"10.1016/j.jchromb.2026.124929","url":null,"abstract":"<div><div>Recent advancements in micro-sampling methods provide non-invasive options for collecting biological samples used in therapeutic drug monitoring (TDM). Lately, alternative matrices such as capillary blood and saliva/oral fluid in the form of dried saliva spots, have attracted considerable interest. These matrices represent a promising approach for TDM, as they could improve accessibility while preserving analytical precision.</div><div>The study developed and validated an LC-MS method for lamotrigine quantitation in saliva and Dried Saliva Spots (DSS). Samples were prepared via protein precipitation (methanol: acetonitrile: water, 7:2:1) and dilution, followed by separation on a Luna Omega C18 Polar column and detection in ESI+ mode. An isotopically labelled internal standard was employed, ensuring compliance with EMA guidelines. The linearity was demonstrated for lamotrigine concentrations ranging from 0.20 to 25 mg/L in both dried saliva spot and saliva samples.</div><div>Saliva and DSS samples from patients were used to compare lamotrigine concentrations with plasma levels determined by a hospital-accredited laboratory. Findings demonstrated a strong correlation between evaluated matrices and plasma (<em>R</em> = 0.847 for saliva and plasma, and <em>R</em> = 0.839 for DSS and plasma, both <em>p</em> &lt; 0.0001), underscoring the promise of saliva and DSS as substitutes for TDM. This technique improves patient comfort and accessibility through a non-invasive sampling method while ensuring accuracy.</div><div>These findings provide a foundation for broader clinical implementation of alternative-matrix TDM, representing a significant step toward patient-centred, accessible pharmacotherapy and enabling improved LTG monitoring in ambulatory and remote settings.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124929"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146014196","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Applying a vacuum ultraviolet detector for liquid chromatography: Simultaneous analysis of four organic acids and four carbohydrates in a fermentation process 应用真空紫外检测器进行液相色谱分析:发酵过程中四种有机酸和四种碳水化合物的同时分析
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-08 DOI: 10.1016/j.jchromb.2026.124921
Mayur Bansal , Annika Dombrowski , Chinoso Nwanebi , Dale Harrison , Hal S. Alper
The initial application of a prototype vacuum ultraviolet (VUV) absorption detector system for high performance liquid chromatography (HPLC) was validated and characterized. This detector enabled simultaneous detection and quantification of four organic acids—lactic, acetic, itaconic, and citric along with four carbohydrates—glucose, fructose, galactose, and lactose. This collection of analytes was selected based on their prevalence in fermentations, metabolic production interest, and occurrence in food processes/chemistry. Full spectrum VUV data, collected across 7 discrete wavelength bands centered at 177.3, 190.3, 203.1, 215.8, 228.3, 240.7, and 252.9 nm, yielded unique fingerprints that allowed for separation and quantification of strongly co-eluting peaks, most notably the hexose sugars. Separations were performed on an Aminex HPX-87H ion exclusion column using 5 mM H₂SO₄ as the isocratic mobile phase at 0.6 mL/min, with samples preheated to 40 °C. Across the eight analytes, Hydra limits of detection spanned 0.0228–3.59 g/L, with calibration linearity of R2 = 0.941–0.998. The VUV prototype maintained linear response up to 100 g/L for all analytes (60 g/L for itaconic acid), whereas refractive index detection saturated between ∼10–60 g/L depending on the analyte. Spectral deconvolution recovered co-eluting organic acids with ∼1–4% error and the co-eluting hexose cluster within ∼11–19% error, demonstrating practical spectral selectivity for overlapped peaks. This extended dynamic range can eliminate the need for sample dilution thus greatly streamlining workflow processes and minimizing the dilution of trace components. The combination of quantitative accuracy and spectral selectivity was demonstrated though the monitoring of glucose utilization and lactic acid production during a seven-day Saccharomyces cerevisiae fermentation with minimal sample preparation. These results support the potential of LC-VUV detection as a robust, high-throughput method for metabolite analysis in high titer bioprocessing applications. However, a current limitation of the prototype is reduced sensitivity at low concentrations relative to conventional detectors, indicating that present performance is best suited to higher titer samples.
对真空紫外(VUV)吸收检测器系统在高效液相色谱(HPLC)中的初步应用进行了验证和表征。该检测器能够同时检测和定量四种有机酸——乳酸、乙酸、衣康酸和柠檬酸,以及四种碳水化合物——葡萄糖、果糖、半乳糖和乳糖。这组分析物的选择是基于它们在发酵、代谢生产兴趣和食品加工/化学中的发生率。在以177.3、190.3、203.1、215.8、228.3、240.7和252.9 nm为中心的7个独立波长波段收集的全光谱VUV数据,产生了独特的指纹图谱,可以分离和定量强共洗脱峰,最明显的是己糖。在Aminex HPX-87H离子隔离柱上进行分离,以5 mM H₂SO₄为等密度流动相,流速为0.6 mL/min,样品预热至40℃。8种分析物的Hydra检出限范围为0.0228 ~ 3.59 g/L,线性R2 = 0.941 ~ 0.998。VUV原型对所有分析物保持高达100 g/L的线性响应(衣康酸为60 g/L),而折射率检测在~ 10-60 g/L之间饱和,具体取决于分析物。光谱反褶积恢复共洗脱有机酸的误差为~ 1-4%,共洗脱己糖簇的误差为~ 11-19%,证明了对重叠峰的实际光谱选择性。这种扩展的动态范围可以消除对样品稀释的需要,从而大大简化了工作流程并最大限度地减少了痕量成分的稀释。通过对7天酿酒酵母发酵过程中葡萄糖利用和乳酸产量的监测,证明了定量准确性和光谱选择性的结合。这些结果支持LC-VUV检测作为高滴度生物处理应用中代谢物分析的稳健、高通量方法的潜力。然而,目前原型的一个限制是相对于传统检测器在低浓度下的灵敏度降低,这表明目前的性能最适合于高滴度的样品。
{"title":"Applying a vacuum ultraviolet detector for liquid chromatography: Simultaneous analysis of four organic acids and four carbohydrates in a fermentation process","authors":"Mayur Bansal ,&nbsp;Annika Dombrowski ,&nbsp;Chinoso Nwanebi ,&nbsp;Dale Harrison ,&nbsp;Hal S. Alper","doi":"10.1016/j.jchromb.2026.124921","DOIUrl":"10.1016/j.jchromb.2026.124921","url":null,"abstract":"<div><div>The initial application of a prototype vacuum ultraviolet (VUV) absorption detector system for high performance liquid chromatography (HPLC) was validated and characterized. This detector enabled simultaneous detection and quantification of four organic acids—lactic, acetic, itaconic, and citric along with four carbohydrates—glucose, fructose, galactose, and lactose. This collection of analytes was selected based on their prevalence in fermentations, metabolic production interest, and occurrence in food processes/chemistry. Full spectrum VUV data, collected across 7 discrete wavelength bands centered at 177.3, 190.3, 203.1, 215.8, 228.3, 240.7, and 252.9 nm, yielded unique fingerprints that allowed for separation and quantification of strongly co-eluting peaks, most notably the hexose sugars. Separations were performed on an Aminex HPX-87H ion exclusion column using 5 mM H₂SO₄ as the isocratic mobile phase at 0.6 mL/min, with samples preheated to 40 °C. Across the eight analytes, Hydra limits of detection spanned 0.0228–3.59 g/L, with calibration linearity of R<sup>2</sup> = 0.941–0.998. The VUV prototype maintained linear response up to 100 g/L for all analytes (60 g/L for itaconic acid), whereas refractive index detection saturated between ∼10–60 g/L depending on the analyte. Spectral deconvolution recovered co-eluting organic acids with ∼1–4% error and the co-eluting hexose cluster within ∼11–19% error, demonstrating practical spectral selectivity for overlapped peaks. This extended dynamic range can eliminate the need for sample dilution thus greatly streamlining workflow processes and minimizing the dilution of trace components. The combination of quantitative accuracy and spectral selectivity was demonstrated though the monitoring of glucose utilization and lactic acid production during a seven-day <em>Saccharomyces cerevisiae</em> fermentation with minimal sample preparation. These results support the potential of LC-VUV detection as a robust, high-throughput method for metabolite analysis in high titer bioprocessing applications. However, a current limitation of the prototype is reduced sensitivity at low concentrations relative to conventional detectors, indicating that present performance is best suited to higher titer samples.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124921"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145923080","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Simple ion-pairing reversed phase LC-UV method for the relative quantification of capping species in mRNA-based modalities 简单离子配对反相LC-UV方法在mrna为基础的模式相对定量封顶物种
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-03-01 Epub Date: 2026-01-10 DOI: 10.1016/j.jchromb.2026.124922
Jonathan Maurer , Denis Bouchard , Agathe Bousquier , Camille Malburet , Jean-François Cotte , Davy Guillarme
Messenger RNA (mRNA)-based therapeutics and vaccines rely on proper 5′ capping to ensure translational efficiency, stability, and reduced reactogenicity. Current analytical approaches for capping evaluation often rely on mass spectrometry (MS) and fluorinated solvents, which are costly, technically demanding, and not always suitable for quality control (QC) laboratories. Here, we describe the development of a simple, robust, and QC-compatible ion-pairing reversed-phase liquid chromatography method coupled with UV detection (IP-RPLC-UV) for the relative quantification of key 5′ capping species in mRNA modalities, including uncapped, Cap 0, Cap 1, and Cap G transcripts, as well as non-templated additions (+G). The method employs targeted cleavage of the 5′ end using an oligo hybridization and RNase H digestion strategy, enabling baseline resolution of capping species within 20 min. A systematic screening of various ion-pairing reagents demonstrated that butylammonium acetate offered the optimal balance between retention and resolution, while a design of experiments approach identified critical parameters influencing performance and ensured method robustness. Method sensitivity was confirmed with a lower limit of quantification at 0.01 mg/mL for uncapped species, and repeatability testing showed consistent results across samples. This study introduces the first IP-RPLC-UV method tailored for routine relative quantification of cap structures, providing a practical alternative to MS-based workflows and facilitating the implementation of accessible, reliable analytical control strategies for mRNA drug development.
基于信使RNA (mRNA)的疗法和疫苗依赖于适当的5 '封顶来确保翻译效率、稳定性和降低反应原性。目前用于封盖评价的分析方法通常依赖于质谱法(MS)和氟化溶剂,这些方法成本高,技术要求高,而且并不总是适合质量控制(QC)实验室。在这里,我们描述了一种简单、可靠且与qc兼容的离子配对反相液相色谱法与紫外检测(ip - hplc -UV)的发展,用于相对定量mRNA模式中的关键5 '封顶物种,包括未封顶、Cap 0、Cap 1和Cap G转录本,以及非模板添加物(+G)。该方法使用寡核苷酸杂交和RNase H酶切策略对5 '端进行靶向切割,可在20分钟内实现封顶物种的基线分辨率。对各种离子配对试剂的系统筛选表明,乙酸丁胺在保留和分辨率之间提供了最佳平衡,而实验方法的设计确定了影响性能的关键参数,并确保了方法的稳健性。方法灵敏度为0.01 mg/mL,重复性测试结果一致。本研究引入了首个专为常规帽结构相对定量而设计的ip - hplc - uv方法,为基于ms的工作流程提供了一种实用的替代方案,并促进了mRNA药物开发中易于获取、可靠的分析控制策略的实施。
{"title":"Simple ion-pairing reversed phase LC-UV method for the relative quantification of capping species in mRNA-based modalities","authors":"Jonathan Maurer ,&nbsp;Denis Bouchard ,&nbsp;Agathe Bousquier ,&nbsp;Camille Malburet ,&nbsp;Jean-François Cotte ,&nbsp;Davy Guillarme","doi":"10.1016/j.jchromb.2026.124922","DOIUrl":"10.1016/j.jchromb.2026.124922","url":null,"abstract":"<div><div>Messenger RNA (mRNA)-based therapeutics and vaccines rely on proper 5′ capping to ensure translational efficiency, stability, and reduced reactogenicity. Current analytical approaches for capping evaluation often rely on mass spectrometry (MS) and fluorinated solvents, which are costly, technically demanding, and not always suitable for quality control (QC) laboratories. Here, we describe the development of a simple, robust, and QC-compatible ion-pairing reversed-phase liquid chromatography method coupled with UV detection (IP-RPLC-UV) for the relative quantification of key 5′ capping species in mRNA modalities, including uncapped, Cap 0, Cap 1, and Cap G transcripts, as well as non-templated additions (+G). The method employs targeted cleavage of the 5′ end using an oligo hybridization and RNase H digestion strategy, enabling baseline resolution of capping species within 20 min. A systematic screening of various ion-pairing reagents demonstrated that butylammonium acetate offered the optimal balance between retention and resolution, while a design of experiments approach identified critical parameters influencing performance and ensured method robustness. Method sensitivity was confirmed with a lower limit of quantification at 0.01 mg/mL for uncapped species, and repeatability testing showed consistent results across samples. This study introduces the first IP-RPLC-UV method tailored for routine relative quantification of cap structures, providing a practical alternative to MS-based workflows and facilitating the implementation of accessible, reliable analytical control strategies for mRNA drug development.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124922"},"PeriodicalIF":2.8,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Bupleurum polysaccharide improves CUMS-induced depressive behavior in rats by regulating the “microbiota-gut-brain Axis”: a mechanism study based on metabolomics and metagenomics 柴胡多糖通过调节“微生物-肠-脑轴”改善cms诱导大鼠抑郁行为:基于代谢组学和宏基因组学的机制研究
IF 2.8 3区 医学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2026-02-01 Epub Date: 2025-12-23 DOI: 10.1016/j.jchromb.2025.124905
Hongcai Zhang, Shuxiang Zhang , Xuan Li, Wenran Wang, Haixue Kuang
This study aimed to comprehensively investigate the antidepressant mechanisms of Bupleurum polysaccharide (BP) through the microbiota-gut-brain axis, employing an integrated multi-omics approach. Using a chronic unpredictable mild stress (CUMS) rat model of depression, we evaluated BP's effects on depressive-like behaviors and analyzed its regulatory mechanisms on metabolites and gut microbiota through combined metabolomics and metagenomics. Structural characterization revealed that Bupleurum polysaccharide SPAP-1 is an acidic homogeneous polysaccharide with a molecular weight of approximately 100 kDa, primarily composed of glucose, mannose, rhamnose, and other monosaccharides. Pharmacodynamic assessments demonstrated that BP significantly ameliorated CUMS-induced depressive behaviors, including weight loss, reduced food intake, anhedonia, and behavioral despair (P < 0.05). Metabolomic analysis identified 19 differential metabolites, with BP reversing 11 of them, primarily involved in phenylalanine and tryptophan metabolism pathways. Western blot analysis confirmed BP's regulatory effects on key enzymes Got1 and Lta4h. Metagenomic results showed that BP remarkably reshaped gut microbiota structure, restored microbial diversity, optimized the Firmicutes/Bacteroidetes ratio, enriched beneficial genera (Agathobacter, Phocaeicola), and inhibited pathogenic genera (Ruminococcus). Crucially, integrated multi-omics analysis revealed significant microbiota-metabolite correlations, demonstrating that BP-promoted beneficial bacteria positively correlated with neurotransmitter precursors, while BP-inhibited pathogenic bacteria associated with pro-inflammatory mediators. Mediation analysis further established the “microbiota → metabolite → behavior” causal chain, with Ruminococcus → LTB4 → despair behavior accounting for 42.3 % of the mediation effect. In conclusion, Bupleurum polysaccharide ameliorates depressive-like behaviors through multi-target regulation of the metabolite-microbiota interaction network, highlighting its potential as an antidepressant agent or functional food and providing a novel research paradigm for understanding the multi-target characteristics of traditional Chinese medicine polysaccharides.
本研究旨在采用综合多组学方法,通过微生物-肠-脑轴,全面探讨柴胡多糖(BP)抗抑郁机制。采用慢性不可预测轻度应激(CUMS)大鼠抑郁模型,通过代谢组学和宏基因组学联合分析BP对抑郁样行为的影响,并分析其对代谢物和肠道微生物群的调节机制。结构表征表明柴胡多糖SPAP-1是一种酸性均质多糖,分子量约为100 kDa,主要由葡萄糖、甘露糖、鼠李糖等单糖组成。药效学评估表明,BP可显著改善cms诱导的抑郁行为,包括体重减轻、食物摄入减少、快感缺乏和行为绝望(P < 0.05)。代谢组学分析确定了19种差异代谢物,BP逆转了其中的11种,主要涉及苯丙氨酸和色氨酸的代谢途径。Western blot分析证实了BP对关键酶Got1和Lta4h的调控作用。宏基因组学结果显示,BP显著重塑了肠道菌群结构,恢复了微生物多样性,优化了厚壁菌门/拟杆菌门的比例,丰富了有益菌属(Agathobacter, Phocaeicola),抑制了致病性菌属(Ruminococcus)。至关重要的是,综合多组学分析揭示了显著的微生物群代谢物相关性,表明bp促进有益细菌与神经递质前体正相关,而bp抑制与促炎介质相关的致病菌。中介分析进一步建立了“微生物群→代谢物→行为”的因果链,其中Ruminococcus→LTB4→绝望行为占中介效应的42.3%。综上所述,柴胡多糖通过多靶点调节代谢-微生物相互作用网络改善抑郁样行为,突出了其作为抗抑郁剂或功能食品的潜力,并为了解中药多糖的多靶点特性提供了新的研究范式。
{"title":"Bupleurum polysaccharide improves CUMS-induced depressive behavior in rats by regulating the “microbiota-gut-brain Axis”: a mechanism study based on metabolomics and metagenomics","authors":"Hongcai Zhang,&nbsp;Shuxiang Zhang ,&nbsp;Xuan Li,&nbsp;Wenran Wang,&nbsp;Haixue Kuang","doi":"10.1016/j.jchromb.2025.124905","DOIUrl":"10.1016/j.jchromb.2025.124905","url":null,"abstract":"<div><div>This study aimed to comprehensively investigate the antidepressant mechanisms of Bupleurum polysaccharide (BP) through the microbiota-gut-brain axis, employing an integrated multi-omics approach. Using a chronic unpredictable mild stress (CUMS) rat model of depression, we evaluated BP's effects on depressive-like behaviors and analyzed its regulatory mechanisms on metabolites and gut microbiota through combined metabolomics and metagenomics. Structural characterization revealed that Bupleurum polysaccharide SPAP-1 is an acidic homogeneous polysaccharide with a molecular weight of approximately 100 kDa, primarily composed of glucose, mannose, rhamnose, and other monosaccharides. Pharmacodynamic assessments demonstrated that BP significantly ameliorated CUMS-induced depressive behaviors, including weight loss, reduced food intake, anhedonia, and behavioral despair (<em>P</em> &lt; 0.05). Metabolomic analysis identified 19 differential metabolites, with BP reversing 11 of them, primarily involved in phenylalanine and tryptophan metabolism pathways. Western blot analysis confirmed BP's regulatory effects on key enzymes Got1 and Lta4h. Metagenomic results showed that BP remarkably reshaped gut microbiota structure, restored microbial diversity, optimized the Firmicutes/Bacteroidetes ratio, enriched beneficial genera (Agathobacter, Phocaeicola), and inhibited pathogenic genera (Ruminococcus). Crucially, integrated multi-omics analysis revealed significant microbiota-metabolite correlations, demonstrating that BP-promoted beneficial bacteria positively correlated with neurotransmitter precursors, while BP-inhibited pathogenic bacteria associated with pro-inflammatory mediators. Mediation analysis further established the “microbiota → metabolite → behavior” causal chain, with Ruminococcus → LTB4 → despair behavior accounting for 42.3 % of the mediation effect. In conclusion, Bupleurum polysaccharide ameliorates depressive-like behaviors through multi-target regulation of the metabolite-microbiota interaction network, highlighting its potential as an antidepressant agent or functional food and providing a novel research paradigm for understanding the multi-target characteristics of traditional Chinese medicine polysaccharides.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124905"},"PeriodicalIF":2.8,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145880738","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
期刊
Journal of Chromatography B
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1