Journal of Chromatography B最新文献

{"title":"Estimation of protein aggregates in Darbepoetin alfa formulations by developing a single validated analytical method","authors":"Nupur Garg,&nbsp;Sanjay Mendiratta,&nbsp;Gurminder Bindra,&nbsp;Sakshi Gehlot,&nbsp;Krutika Goswami,&nbsp;Charu Mehra Kamal","doi":"10.1016/j.jchromb.2025.124518","DOIUrl":"10.1016/j.jchromb.2025.124518","url":null,"abstract":"<div><div>Darbepoetin alfa is a new generation recombinant human erythropoiesis stimulating agent having extended half-life as compared to recombinant human erythropoietin. It is given as a life-saving drug for anemic disorders caused by chronic kidney diseases, cancer, etc. With the presence of commercial formulations of darbepoetin biosimilars in the market and non-availability of any established method in literature or testing guidelines in any compendia for their purity and quality, this research aims to develop a sensitive and robust method to determine the aggregate impurities by using size exclusion chromatographic technique. The method was optimized for sharp and high-resolution monomer peak of darbepoetin and other interferences which was further validated according to ICH Q2(R2) guidelines. The developed method was further tested on the commercial samples to verify the same. Additionally, stress testing of samples was done to simulate the after-production exposure and stability assessment. The results demonstrated that the method for analysing aggregates in darbepoetin is complying the criteria of being sensitive, specific, accurate, robust and reliable.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1254 ","pages":"Article 124518"},"PeriodicalIF":2.8,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143394585","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}

{"title":"Salting-out assisted liquid-liquid extraction for UPLC-MS/MS determination of bile acids and kynurenine-, indole- and serotonin-pathway metabolites of tryptophan in human serum of healthy probands","authors":"Celine Oanes ,&nbsp;Marina Alexeeva ,&nbsp;Kjetil Søreide ,&nbsp;Cato Brede","doi":"10.1016/j.jchromb.2025.124519","DOIUrl":"10.1016/j.jchromb.2025.124519","url":null,"abstract":"<div><div>The bacterial composition of the gut has been found to affect many diseases, including several gastrointestinal cancers. The microbiome appears central in the production of certain metabolites that enter circulation, especially those from bile acids and the essential amino acid tryptophan. The tumor-microenvironment may also produce changes in metabolites, such as those from the tryptophan-kynurenine pathway, of which several compounds may be measured in the blood. As data emerges from large scale metabolomics studies, there will be a need to validate metabolomic biomarkers to confirm their clinical utility. This task also requires knowledge about biological variation of the same metabolites in a healthy population. For this purpose, a novel method was developed for quantification of bile acids and tryptophan metabolites in samples of human serum by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Salting-out assisted liquid-liquid extraction was optimized with the ion-pairing reagent trifluoroacetic acid. In this way, both polar tryptophan metabolites and non-polar bile acids could be extracted with a high recovery, favorable matrix effects, and improved chromatographic focusing, by using straightforward robot pipetting. The instrumental analysis was fast (4 min and 32 s) and with sample injections done directly from the extraction microplate. The method was applied to quantify metabolites in serum from healthy probands, and for investigating inter- and intraindividual variations over six hours.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1255 ","pages":"Article 124519"},"PeriodicalIF":2.8,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143419679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Uncovering the antidepressant active ingredients and related molecular mechanisms of Xiaoyao Pill using integrated pharmacological strategy","authors":"Siqian Zhou ,&nbsp;Yajing Wang ,&nbsp;Mingxia Xie ,&nbsp;Jing Li ,&nbsp;Pan Meng ,&nbsp;Juan Wu ,&nbsp;Lin Jiang ,&nbsp;Hongping Long","doi":"10.1016/j.jchromb.2025.124502","DOIUrl":"10.1016/j.jchromb.2025.124502","url":null,"abstract":"<div><h3>Purpose</h3><div>To investigate antidepressant active ingredients of XYP (Xiaoyao Pill), while predicting its primary pharmacodynamic material basis and underlying mechanisms of action.</div></div><div><h3>Methods</h3><div>UPLC-Q-TOF-MS/MS was used to identify the active ingredients of XYP. In addition, based on the analysis of components, network pharmacology and molecular docking were used to investigate potential therapeutic targets and possible signaling pathways of XYP in the treatment of depression.</div></div><div><h3>Results</h3><div>A total of 102 chemical components, 10 prototype components and 16 metabolites absorbed in the brain were identified in XYP. Network pharmacology analysis showed that these compounds shared 420 common targets with depression, TP53, EGFR, PTGS2, ESR1, PPARG and other 68 targets were considered as core targets, mainly enriched in PI3K-Akt and MAPK signaling pathways. GO analysis unveiled associated apoptosis and inflammatory response. Molecular docking revealed that paeoniflorin, liquiritin, and atractylenolide III were found to have the highest binding energy to TP53, ESR1 and PPARG.</div></div><div><h3>Conclusion</h3><div>These findings suggested that XYP may exert antidepressant effects through atractylenolide III, paeoniflorin, saikosaponin D, liquiritin, formononetin, affecting the PIK3/AKT signaling pathway. This lays the foundation for the research on the quality standards and clinical rational application of traditional Chinese medicine formulas.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1255 ","pages":"Article 124502"},"PeriodicalIF":2.8,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143479326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An exploratory SWATH plasma proteomics analysis of phyllodes tumor- a type of female breast tumor","authors":"M.D. Quasid Akhter ,&nbsp;Munish Dwivedi ,&nbsp;Shivani Chitkara ,&nbsp;Navneet Kaur ,&nbsp;Swarnendu Bag","doi":"10.1016/j.jchromb.2025.124508","DOIUrl":"10.1016/j.jchromb.2025.124508","url":null,"abstract":"<div><h3>Objective</h3><div>Phyllodes tumors (PT) are rare fibroepithelial breast tumors with poorly understood molecular pathology. This study intent to identify the potential plasma markers of phyllodes tumors compared to controls using Sequential window acquisition of all theoretical fragment ion spectra-mass spectrometry (SWATH-LC-MS) based proteomics approach.</div></div><div><h3>Method</h3><div>Plasma samples from phyllodes tumor cases and controls underwent SWATH-LC-MS/MS based untargeted proteomics analysis. Proteins with 1.5 fold changes &amp; <em>p</em> &lt; 0.05 in PT cases compared to control were considered for further analysis. Statistical analysis was done by using R 4.3.1 software and proteomics analysis was performed by using Spectronaut Software</div></div><div><h3>Result &amp; conclusion</h3><div>Three hundred and nineteen proteins were identified. Amongst them 30 proteins were significantly altered in PT case compared to control. 26 were upregulated and 4 were downregulated. Again Protein-Protein network analysis revealed that 21 proteins were matched with STRING data base and out of 21 proteins 19 were highly connected in the interaction analysis. As per our knowledge, this is the first exploratory study on LC-MS/MS-SWATH based phyllodes tumor proteomics. Different proteins like APMAP, HGFAC,TTR, PNO3 etc., and associated pathways like FCGR3A-mediated IL10 synthesis, arylesterase activity, EMT related Wnt/β-catenin Pathway etc. were significantly altered in Phyllodes tumor cases. Hence. this study will help to find the plausible theranostic markers and druggable targets for the phyllodes tumors in future.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1254 ","pages":"Article 124508"},"PeriodicalIF":2.8,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143377759","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}

{"title":"Residual and pharmacokinetic behavior of berberine in Carassius auratus under intraperitoneal injection conditions by HPLC-Q-TOF/MS","authors":"Shuai Zhang ,&nbsp;Le Yang ,&nbsp;Hao Jin ,&nbsp;Yuxiang Wang ,&nbsp;Qiaoqiao Teng ,&nbsp;Qi Meng ,&nbsp;Zhiqiang Cai","doi":"10.1016/j.jchromb.2025.124507","DOIUrl":"10.1016/j.jchromb.2025.124507","url":null,"abstract":"<div><div>The present study investigated the distribution characteristics of Berberine (BBR) in <em>Carassius auratus</em> under artificial environmental conditions (temperature: 26 ± 1.0 °C; continuous air pumping) following a single administration via intraperitoneal injection of an appropriate dosage of 2.0 mg/Kg per body weight. Additionally, studies have investigated the residual distribution of BBR in eight different tissues using high performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-Q-TOF/MS) technology. A total of 36 <em>Carassius auratus</em> (3 parallel samples per group) were in the drug-exposed group and 3 were in the control group. <em>Carassius auratus</em> were sampled at the indicated times (sampling interval: 24–96 h), lasting for approximately one month. The residual metabolic drug concentration-time curves were plotted according to the concentration of BBR in different tissues.</div><div>The maximum BBR concentrations (<em>C</em>_<em>max</em>) in all tissues were achieved 24 h after the administration of the intraperitoneal injection. The order of the <em>C</em>_<em>max</em> was: muscle &lt; eye &lt; gill &lt; brain &lt; kidney &lt; intestine&lt; liver &lt; bile. According to the distribution characteristics of BBR between tissues, the drug concentrations in muscle, brain, eye, and gill tissues were relatively low (∼100 to &gt;400 ng/g). Their metabolism was rapid, and BBR residue was significantly reduced to tens of ng/g from 24 to 200 h. Meanwhile, the bile, kidney, intestine, and liver contained significantly higher concentrations of BBR (maintained at 1200 to 6000 ng/g). The concentration shows a fluctuating and decreasing characteristic, with the drug remaining for a longer period. Following the 31 days BBR withdrawal period, the pharmacokinetic parameters of maximum observed concentration(<em>C</em>_<em>max</em>), terminal half-life (<em>T</em>_<em>1/2</em>), elimination rate constant during terminal phase (<em>λ</em>_<em>z</em>), the volume of distribution (Vd/F), and total body clearance (Cl/F) were calculated employing non-compartmental analysis (NCA) using the PK Solver software. The research results of the article explain the retention of BBR in <em>Carassius auratus</em> until excretion and can be used to determine residual levels over time.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1254 ","pages":"Article 124507"},"PeriodicalIF":2.8,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143372031","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}

{"title":"Identification and characterization of the absorbed components and metabolites of Gandouling tablets in rats' plasma, liver, and urine by UPLC-Q-TOF-MSE","authors":"Tiantian Wang ,&nbsp;Qing Zuo ,&nbsp;Ting Dong ,&nbsp;Peng Wu ,&nbsp;Shijian Cao ,&nbsp;Hongfei Wu ,&nbsp;An Zhou","doi":"10.1016/j.jchromb.2025.124503","DOIUrl":"10.1016/j.jchromb.2025.124503","url":null,"abstract":"<div><div>Gandouling (GDL), a famous proprietary Chinese medicine, has been utilized in China's clinics for decades to treat Wilson's disease. However, its metabolism in vivo needs to be clarified. In this study, Ultra Performance Liquid Chromatography-quadrupole time-of-flight mass spectrometry-tandem (UPLC-Q-TOF-MS^E) was employed to analyze the metabolic pathways of these critical components in the rat after identifying the prototypes and metabolites of GDL in the plasma, liver, and urine of both normal and copper-loaded rats. As a result, 49 components were detected in the plasma of normally administered rats, including 29 prototype compounds and 20 metabolites; and 26 components were detected in the liver of normally administered rats, including 16 prototype compounds and 10 metabolites. 43 components were detected in the plasma of copper-laden administered rats, including 25 prototype compounds and 18 metabolites; and 23 components were detected in the liver of copper-laden administered rats, including 15 prototype compounds and 8 metabolites. A total of 73 GDL-related substances were detected in the urine of rats. The study results showed that the compositions in rats' plasma, liver, and urine were similar, mainly alkaloids and anthraquinones. The alkaloid components are mainly metabolized by phase I metabolism in vivo and the metabolic pathways are methylation, demethylation, etc. The anthraquinone components are mainly metabolized by phase II metabolism in vivo, and the metabolism modes are mainly glucuronidation and sulfation. The present study comprehensively analyses the metabolic properties of GDL and sets an essential basis for further investigations on the pharmacokinetics, in vivo bioactive components, and mechanism of action of GDL.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1254 ","pages":"Article 124503"},"PeriodicalIF":2.8,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143372517","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}

{"title":"Development of an analytical method for the analysis of diflubenzuron in whole blood, plasma, and serum using liquid chromatography tandem mass spectrometry","authors":"Greg Jellick ,&nbsp;Kimberly Lohmeyer ,&nbsp;Stephan Bach","doi":"10.1016/j.jchromb.2025.124506","DOIUrl":"10.1016/j.jchromb.2025.124506","url":null,"abstract":"<div><div>The integration of free-range beef livestock into the human food chain has been continuously threatened by outbreaks of babesiosis, which is propagated by ticks that vector the protozoans that cause the disease. The protozoans are spread among livestock by certain species of ticks, known collectively as cattle fever ticks (CFT). Although eliminated from the US in the 1940s, CFT continue to infiltrate the US by crossing the US/Mexico border on stray livestock and wildlife, and infest cattle herds. Orally administered acaricides are part of the strategy to control CFT. Parallel dosing studies, utilizing oral administration of the benzoylphenyl urea (BPU) compound diflubenzuron (DFB), were conducted in cattle and white tailed deer (WTD). We developed and validated a sensitive (&lt;1 ppb) liquid chromatography/ tandem mass spectrometry (LC-MS/MS) method for DFB quantification in toxicology specimens. In fit-for-purpose testing, the method demonstrated accuracies within ±9 % of QC targets, and coefficient of variation percentages (%CV) of 14.8 % for whole blood, 9.0 % for plasma, and 8.7 % for serum. This work outlines the extraction and instrumental method for DFB analysis in these matrices. LC-MS/MS had not previously been used to detect and quantify DFB in whole blood, plasma, or serum. This is also the first method to employ carbon-13 labeled DFB (¹³C₆) as an internal standard for measuring DFB in blood matrices from bovine dosing studies<em>.</em></div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1256 ","pages":"Article 124506"},"PeriodicalIF":2.8,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143512227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery of peptide quality markers for quality control and identification of toad venom using LC-MS/MS and label-free methods","authors":"Juan Chen ,&nbsp;Fei Xue ,&nbsp;Yingying Xie ,&nbsp;Weiliang Cui ,&nbsp;Lejun Tan ,&nbsp;Yan Niu ,&nbsp;Li Shi ,&nbsp;Lin Wang ,&nbsp;Li Liu ,&nbsp;Bing Wang ,&nbsp;Yang Jiao ,&nbsp;Yongqiang Lin","doi":"10.1016/j.jchromb.2025.124504","DOIUrl":"10.1016/j.jchromb.2025.124504","url":null,"abstract":"<div><div>Toad venom, a secretion from the parotoid glands of toads such as <em>Bufo bufo gargarizans</em> Cantor, is a traditional Chinese medicine renowned for its cardiotonic, anti-inflammatory, and antitumor properties. However, due to the rising price of toad venom, its quality control faces significant challenges from widespread adulteration with substances like toad skin and dried toads, which compromise its authenticity and therapeutic efficacy. Currently, no comprehensive method exists for the simultaneous detection of these adulterants. In this study, dried toads were subdivided into skin, flesh, and bone and compared with toad venom from <em>Bufo bufo gargarizans</em> Cantor using nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) combined with multivariate statistical analysis. This is the first study to propose a peptide-based method for simultaneously identifying and quantifying adulterants such as toad skin, flesh, and bone in toad venom, offering a promising approach to improve quality control techniques for toad venom. Twenty-seven peptide quality markers were identified, including eight for toad venom, six for toad skin, five for toad flesh, and eight for toad bone. These markers were validated via bioinformatics analysis and HPLC-Triple Quadrupole MS, enabling the establishment of a novel and reliable quality control strategy. A method for determining toad venom content was established using the peptide quality marker NNAYDINEER. The method demonstrated high specificity, excellent linearity (R² &gt; 0.9977), a limit of quantification (LOQ) of 1 ng/mL, and outstanding repeatability, precision, and stability. Analysis of five batches of toad venom samples revealed their content ranged from 0.0387 % to 0.0963 %. This study offers an innovative approach for both qualitative identification and quantitative determination, providing critical technical and theoretical support for ensuring the quality and authenticity of toad venom-based traditional medicines and related formulations.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1254 ","pages":"Article 124504"},"PeriodicalIF":2.8,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143350624","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}

{"title":"Automatic identification of oligonucleotide metabolites in complex biological samples using ultra high-performance liquid chromatography high-resolution mass spectrometry combined with the molecule profiler software","authors":"Xinyuan Ye ,&nbsp;Lili Jiang ,&nbsp;Ying Xiong ,&nbsp;Cui Zhang ,&nbsp;Chenxi Wang ,&nbsp;Meiling Qi ,&nbsp;Chenyue Zhu ,&nbsp;Yi Chen ,&nbsp;Zhifeng Du ,&nbsp;Zhongzhe Cheng ,&nbsp;Hongliang Jiang","doi":"10.1016/j.jchromb.2025.124505","DOIUrl":"10.1016/j.jchromb.2025.124505","url":null,"abstract":"<div><div>Oligonucleotide (ON) is one of the rapidly developing fields in biotherapeutics. Ultra high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) has been widely used for the identification of metabolites due to its high sensitivity, high resolution, and ability to provide structural information. The identification of ON metabolites in matrix has been reported by UHPLC-HRMS, however, manual data processing is time-consuming. In this study, an analytical strategy based on UHPLC-QTOF-MS/MS and molecule profiler software was established and employed for the automatic identification of metabolites of ONs. Fomivirsen (FMVS), a 21-mer antisense oligonucleotide with 20 phosphorothioate linkages, was selected as proof of concept. Firstly, the sample preparation and UHPLC-QTOF-MS/MS condition were optimized. Secondly, the feasibility of the automatic identification of ON metabolites by this strategy was verified using enzymatic digests of FMVS. Finally, in <em>vitro</em> and in <em>vivo</em> metabolites of FMVS were identified. As a result, the enzymatic digests of FMVS were successfully identified by the established strategy, and a total of 17 metabolites were identified from serum, plasma and tissues. FMVS was mainly metabolized by 3′-exonuclease in plasma, liver and kidney. This is the first study on metabolites identification of FMVS, and the proposed strategy would simplify the identification of ON metabolites, thus could be used for other ON metabolites.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1254 ","pages":"Article 124505"},"PeriodicalIF":2.8,"publicationDate":"2025-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143372030","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}

{"title":"Enrichment and purification of trace substances from yew extracum by twin-column recycling chromatography with a step solvent gradient","authors":"Wei Xie ,&nbsp;Yuxue Wu ,&nbsp;Guangxia Jin ,&nbsp;Jiarong Sang ,&nbsp;Feng Wei","doi":"10.1016/j.jchromb.2025.124467","DOIUrl":"10.1016/j.jchromb.2025.124467","url":null,"abstract":"<div><div>The concentration and purification of trace substances from natural products represent a bottleneck in current chromatographic separations. In this study, a twin-column recycling chromatography with a step solvent gradient method was proposed and successfully applied in the purification of unknown substances with tiny content in yew extracum. The method consists of two steps. Initially, the target substances were enriched from the yew extracum stock solution, followed by further separation and purification of the enriched substances in the second step. Ultimately, the target substances A and B were obtained with a purity of 99.7 % and 96.6 %, respectively. The proposed method has the potential to be applied in the preparation of trace substances for toxicological evaluation in natural product studies.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124467"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142997700","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}