Anabasine is an alkaloid frequently quantified by LC–MS/MS to differentiate tobacco use from nicotine replacement therapy. While urine provides reliable measurement, plasma remains a poorly validated and analytically challenging matrix. This study assessed widely used sample preparation strategies for anabasine determination in human plasma. Across all methods, recovery in undiluted plasma was highly variable and largely outside acceptable analytical ranges, with marked ion suppression and poor reproducibility. Matrix dilution increased apparent signal but substantially worsened variability. Experiments in albumin-enriched saline excluded protein binding as the main determinant of analyte loss, indicating broader plasma-related matrix effects. In human plasma, including time-course sampling during and after smoking, anabasine remained consistently below quantifiable levels. None of the tested workflows met the robustness criteria required for quantitative LC–MS/MS analysis, indicating that current preparation approaches do not enable reliable anabasine measurement in plasma, a critical limitation for studies using anabasine as a biomarker of tobacco exposure.
{"title":"Anabasine analysis in human plasma using liquid chromatography coupled to tandem mass spectrometry to Verify tobacco use: empirical challenges and limitations","authors":"Christof Manuel Schönenberger , Aurelie Berthet , Matthias Briel , Alain Amstutz , Matthias Cavassini , Loïc Sartori , Camille Rime , Davide Staedler , Fiorella Lucarini","doi":"10.1016/j.jchromb.2026.124942","DOIUrl":"10.1016/j.jchromb.2026.124942","url":null,"abstract":"<div><div>Anabasine is an alkaloid frequently quantified by LC–MS/MS to differentiate tobacco use from nicotine replacement therapy. While urine provides reliable measurement, plasma remains a poorly validated and analytically challenging matrix. This study assessed widely used sample preparation strategies for anabasine determination in human plasma. Across all methods, recovery in undiluted plasma was highly variable and largely outside acceptable analytical ranges, with marked ion suppression and poor reproducibility. Matrix dilution increased apparent signal but substantially worsened variability. Experiments in albumin-enriched saline excluded protein binding as the main determinant of analyte loss, indicating broader plasma-related matrix effects. In human plasma, including time-course sampling during and after smoking, anabasine remained consistently below quantifiable levels. None of the tested workflows met the robustness criteria required for quantitative LC–MS/MS analysis, indicating that current preparation approaches do not enable reliable anabasine measurement in plasma, a critical limitation for studies using anabasine as a biomarker of tobacco exposure.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1272 ","pages":"Article 124942"},"PeriodicalIF":2.8,"publicationDate":"2026-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146075778","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}
Pub Date : 2026-01-25DOI: 10.1016/j.jchromb.2026.124932
Pengjuan Li , Qiwei Zhang , Hongbo Xu , Yuangui Yang
This study employed an integrated approach combining in vivo metabolism analysis, network pharmacology, experimental validation and quantitative analysis to investigate the anti-inflammatory material basis and mechanism of action of Paris polyphylla var. yunnanensis (PPY). Firstly, rats were orally administered extracts from the roots and leaves of PPY, and plasma, urine, and feces samples were collected at different time points post-administration. Using UHPLC-Q/TOF-HRMS technology, a total of 26 xenobiotics (18 prototypes and 8 metabolites) were identified from the rat samples. Subsequently, network pharmacology analysis predicted 19 core targets and preliminarily screened 10 key bioavailable components with potential anti-inflammatory activity. Further in vitro cell experiments evaluated the bioactivity of these key components, revealing that polyphyllin II exhibited the most significant anti-inflammatory effect, while pennogenin and diosgenin also demonstrated anti-inflammatory activity within certain concentration ranges. Finally, HPLC-DAD analysis was used to compare the content differences of major active components in PPY from different geographical origins. The results showed that samples from Hanzhong (Shaanxi) and Chengdu (Sichuan) contained higher levels of pennogenin-type saponins, whereas those from Honghe (Yunnan) and Yuxi (Yunnan) were richer in diosgenin-type saponins. In summary, this comprehensive strategy significantly contributes to elucidating the anti-inflammatory material basis and mechanism of PPY, while also providing a reference for the systematic discovery of active components in the quality evaluation of traditional Chinese medicines.
{"title":"Integrated metabolomics, network pharmacology, experimental validation and quantitative analysis to reveal and evaluate the pharmacological substances of Paris polyphylla var. yunnanensis on anti-inflammatory","authors":"Pengjuan Li , Qiwei Zhang , Hongbo Xu , Yuangui Yang","doi":"10.1016/j.jchromb.2026.124932","DOIUrl":"10.1016/j.jchromb.2026.124932","url":null,"abstract":"<div><div>This study employed an integrated approach combining <em>in vivo</em> metabolism analysis, network pharmacology, experimental validation and quantitative analysis to investigate the anti-inflammatory material basis and mechanism of action of <em>Paris polyphylla</em> var. <em>yunnanensis</em> (PPY). Firstly, rats were orally administered extracts from the roots and leaves of PPY, and plasma, urine, and feces samples were collected at different time points post-administration. Using UHPLC-Q/TOF-HRMS technology, a total of 26 xenobiotics (18 prototypes and 8 metabolites) were identified from the rat samples. Subsequently, network pharmacology analysis predicted 19 core targets and preliminarily screened 10 key bioavailable components with potential anti-inflammatory activity. Further <em>in vitro</em> cell experiments evaluated the bioactivity of these key components, revealing that polyphyllin II exhibited the most significant anti-inflammatory effect, while pennogenin and diosgenin also demonstrated anti-inflammatory activity within certain concentration ranges. Finally, HPLC-DAD analysis was used to compare the content differences of major active components in PPY from different geographical origins. The results showed that samples from Hanzhong (Shaanxi) and Chengdu (Sichuan) contained higher levels of pennogenin-type saponins, whereas those from Honghe (Yunnan) and Yuxi (Yunnan) were richer in diosgenin-type saponins. In summary, this comprehensive strategy significantly contributes to elucidating the anti-inflammatory material basis and mechanism of PPY, while also providing a reference for the systematic discovery of active components in the quality evaluation of traditional Chinese medicines.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1272 ","pages":"Article 124932"},"PeriodicalIF":2.8,"publicationDate":"2026-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146075777","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}
Pub Date : 2026-01-24DOI: 10.1016/j.jchromb.2026.124941
Mengyuan Wang, Yunuo Fan, Ye Zhang, Xingyu Li, Bin Li, Ping Li
The liver plays a central role in drug metabolism, and understanding its metabolic processes is crucial for effective drug development. Gegen Qinlian Decoction (GQD) is widely used in clinical therapy, but its systematic liver metabolic characteristics are still unclear. By integrating UPLC-QTOF-MS with mass defect filtering (MDF), in-house database screening, and feature-based molecular network (FBMN), this study established a multi-dimensional data mining strategy to systematically characterize the in vivo and in vitro liver metabolism characteristics of GQD and clarify its biotransformation pathways. A total of 220 GQD-related components were annotated, of which 179 and 140 were identified in rat liver tissue and human liver microsomes, respectively, and 99 components overlapped between these two biological matrices. Comprehensive analysis shows that alkaloids mainly undergo demethylation and hydroxylation, flavonoids are mainly metabolized by glucuronidation and sulfation, and triterpenoids mainly undergo glucuronidation, hydroxylation, and hydrolysis. The interspecies comparison further demonstrates that alkaloids show more diverse metabolic reactions in rat liver microsomes (RLMs), and the metabolite characteristics largely include those in HLMs and show comparable metabolic rates. In contrast, flavonoids are metabolized faster and extensively in HLMs, resulting in greater product diversity. In vitro and in vivo identification data further support these observations. These findings show that the rat model provides a reasonable representation for the study of alkaloid metabolism, while flavonoid metabolism needs to be verified using HLMs or humanized liver models to improve the clinical translatability of predictions.
肝脏在药物代谢中起着核心作用，了解其代谢过程对有效的药物开发至关重要。葛根芩连汤（GQD）在临床治疗中应用广泛，但其系统肝脏代谢特性尚不清楚。本研究将UPLC-QTOF-MS与质量缺陷过滤（mass defect filtering， MDF）、内部数据库筛选和基于特征的分子网络（feature based molecular network， FBMN）相结合，建立了多维数据挖掘策略，系统表征GQD的体内和体外肝脏代谢特征，阐明其生物转化途径。共注释了220个gqd相关成分，其中分别在大鼠肝组织和人肝微粒体中鉴定到179个和140个，其中99个成分在这两种生物基质之间存在重叠。综合分析表明，生物碱主要进行去甲基化和羟基化，黄酮类主要进行葡萄糖醛酸化和硫酸化代谢，三萜主要进行葡萄糖醛酸化、羟基化和水解代谢。物种间比较进一步表明，生物碱在大鼠肝微粒体（RLMs）中的代谢反应更加多样化，代谢产物特征在很大程度上包括肝微粒体中的代谢产物特征，并且具有相似的代谢率。相比之下，黄酮类化合物在HLMs中代谢更快、更广泛，导致更大的产品多样性。体外和体内鉴定数据进一步支持这些观察结果。这些发现表明，大鼠模型为生物碱代谢的研究提供了合理的表征，而黄酮类代谢需要使用HLMs或人源化肝脏模型进行验证，以提高预测结果的临床可翻译性。
{"title":"Unravelling hepatic metabolism patterns of Gegen Qinlian decoction through a comparative in vivo and in vitro study","authors":"Mengyuan Wang, Yunuo Fan, Ye Zhang, Xingyu Li, Bin Li, Ping Li","doi":"10.1016/j.jchromb.2026.124941","DOIUrl":"10.1016/j.jchromb.2026.124941","url":null,"abstract":"<div><div>The liver plays a central role in drug metabolism, and understanding its metabolic processes is crucial for effective drug development. Gegen Qinlian Decoction (GQD) is widely used in clinical therapy, but its systematic liver metabolic characteristics are still unclear. By integrating UPLC-QTOF-MS with mass defect filtering (MDF), in-house database screening, and feature-based molecular network (FBMN), this study established a multi-dimensional data mining strategy to systematically characterize the <em>in vivo</em> and <em>in vitro</em> liver metabolism characteristics of GQD and clarify its biotransformation pathways. A total of 220 GQD-related components were annotated, of which 179 and 140 were identified in rat liver tissue and human liver microsomes, respectively, and 99 components overlapped between these two biological matrices. Comprehensive analysis shows that alkaloids mainly undergo demethylation and hydroxylation, flavonoids are mainly metabolized by glucuronidation and sulfation, and triterpenoids mainly undergo glucuronidation, hydroxylation, and hydrolysis. The interspecies comparison further demonstrates that alkaloids show more diverse metabolic reactions in rat liver microsomes (RLMs), and the metabolite characteristics largely include those in HLMs and show comparable metabolic rates. In contrast, flavonoids are metabolized faster and extensively in HLMs, resulting in greater product diversity. <em>In vitro</em> and <em>in vivo</em> identification data further support these observations. These findings show that the rat model provides a reasonable representation for the study of alkaloid metabolism, while flavonoid metabolism needs to be verified using HLMs or humanized liver models to improve the clinical translatability of predictions.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1272 ","pages":"Article 124941"},"PeriodicalIF":2.8,"publicationDate":"2026-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146075780","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}
Pub Date : 2026-01-19DOI: 10.1016/j.jchromb.2026.124931
Yang Liu , Shujing Chen , Xue Meng , Rui Wang , Kunze Du , Yanxu Chang , Jin Li
The dried stamen of Nelumbo nucifera Gaertn., holds significant value and is widely used in health teas, functional foods and herbal medicine. Nelumbinis Stamen possesses anti-inflammatory, antioxidant and anti-melanin properties. However, the blood-absorbed components and their mechanisms of action remain unclear. This study aimed to identify the blood-absorbed components and elucidate the anti-inflammatory mechanisms of Nelumbinis Stamen by integrating pharmacokinetics with network pharmacology. A validated UHPLC-MS/MS method was developed to quantify eleven flavonoids in rat plasma following the administration of the aqueous and alcohol extracts for pharmacokinetic comparisons between the two extracts. Five flavonoids were detected in plasma. Kaempferol-3-O-β-D-glucuronide and kaempferol-3-O-β-galactoside present in both extracts while hyperoside, isorhamnetin and isorhamnetin-3-O-β-D-glucoside were exclusive to the alcohol extract. Significant differences in the pharmacokinetic characteristics of these components were observed between the two extracts. Network pharmacology analysis identified PI3K-AKT, TNF and IL-17 signaling pathways as key pathways mediating the anti-inflammatory effects. It was further validated that the alcohol extracts can regulate the PI3K-AKT pathway through in vitro experiments. These findings highlight the effectiveness of integrating pharmacokinetics with network pharmacology in clarifying the bioactive components and mechanisms of Nelumbinis Stamen, providing a scientific basis for its therapeutic potential in inflammatory diseases.
莲属植物的干雄蕊。，具有重要的价值，广泛用于保健茶、功能食品和草药中。莲蓬雄蕊具有抗炎、抗氧化和抗黑色素的特性。然而，血液吸收成分及其作用机制尚不清楚。本研究旨在通过结合药代动力学和网络药理学的方法，鉴定莲蓬的血吸收成分，阐明其抗炎机制。采用高效液相色谱-质谱联用（UHPLC-MS/MS）方法对水提液和醇提液给药后大鼠血浆中的11种黄酮类化合物进行了定量分析，并对两种提取物进行了药动学比较。血浆中检测到5种黄酮类化合物。两种提取物中均含有山奈酚-3- 0 -β- d -葡萄糖苷和山奈酚-3- 0 -β-半乳糖苷，而醇提取物中只含有金丝桃苷、异鼠李素和异鼠李素-3- 0 -β- d -葡萄糖苷。这些成分的药代动力学特征在两种提取物之间有显著差异。网络药理学分析发现PI3K-AKT、TNF和IL-17信号通路是介导抗炎作用的关键通路。通过体外实验进一步验证了醇提物对PI3K-AKT通路的调节作用。这些发现强调了将药代动力学与网络药理学相结合在阐明莲心的生物活性成分和作用机制方面的有效性，为其治疗炎症性疾病的潜力提供了科学依据。
{"title":"The pharmacokinetics and network pharmacology of blood absorbed components for clarifying the potential anti-inflammatory action mechanism of Nelumbinis stamen","authors":"Yang Liu , Shujing Chen , Xue Meng , Rui Wang , Kunze Du , Yanxu Chang , Jin Li","doi":"10.1016/j.jchromb.2026.124931","DOIUrl":"10.1016/j.jchromb.2026.124931","url":null,"abstract":"<div><div>The dried stamen of <em>Nelumbo nucifera</em> Gaertn., holds significant value and is widely used in health teas, functional foods and herbal medicine. <em>Nelumbinis</em> Stamen possesses anti-inflammatory, antioxidant and anti-melanin properties. However, the blood-absorbed components and their mechanisms of action remain unclear. This study aimed to identify the blood-absorbed components and elucidate the anti-inflammatory mechanisms of <em>Nelumbinis</em> Stamen by integrating pharmacokinetics with network pharmacology. A validated UHPLC-MS/MS method was developed to quantify eleven flavonoids in rat plasma following the administration of the aqueous and alcohol extracts for pharmacokinetic comparisons between the two extracts. Five flavonoids were detected in plasma. Kaempferol-3-<em>O</em>-<em>β</em>-D-glucuronide and kaempferol-3-<em>O</em>-<em>β</em>-galactoside present in both extracts while hyperoside, isorhamnetin and isorhamnetin-3-<em>O</em>-<em>β</em>-D-glucoside were exclusive to the alcohol extract. Significant differences in the pharmacokinetic characteristics of these components were observed between the two extracts. Network pharmacology analysis identified PI3K-AKT, TNF and IL-17 signaling pathways as key pathways mediating the anti-inflammatory effects. It was further validated that the alcohol extracts can regulate the PI3K-AKT pathway through <em>in vitro</em> experiments. These findings highlight the effectiveness of integrating pharmacokinetics with network pharmacology in clarifying the bioactive components and mechanisms of <em>Nelumbinis</em> Stamen, providing a scientific basis for its therapeutic potential in inflammatory diseases.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1272 ","pages":"Article 124931"},"PeriodicalIF":2.8,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146006743","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}
Pub Date : 2026-01-19DOI: 10.1016/j.jchromb.2026.124927
Hazim M. Ali , Lateefa A. Al-Khateeb , Mohammed M. Ghoneim , Maha M. Abdelrahman , Ismail M. Ahmed , Mohammed Gamal
Sensitive and accurate analytical strategies for the simultaneous quantification of Baricitinib (BAR) and Remdesivir (REM) in biological fluids are essential for supporting their combined use in COVID-19 therapy. Currently, no analytical methods exist for the simultaneous quantification of both drugs in human biological fluids. To bridge this gap, we developed, optimized, and validated novel RP-HPLC-fluorescence and LC-MS methods for the concurrent assay of BAR and REM in serum and urine samples. A high-performance liquid chromatography method with fluorescence detection (HPLC-fluorescence) was optimized using an isocratic mobile phase of phosphoric acid (pH = 3) and ethanol (30:70, v/v) at a flow rate of 0.6 mL/min, with detection at excitation/emission wavelengths of 245/400 nm. Furthermore, an LC-MS approach was developed using a mobile phase of 0.1% formic acid with 0.05 M ammonium formate in water and methanol (80:20, v/v) at 0.5 mL/min in an isocratic approach. The two LC approaches were successfully validated and applied for the simultaneous analysis of spiked BAR and REM mixtures in human urine and serum samples within 5 and 6 min, respectively. In biological human samples, accuracy varied from 96.41% to 98.10% for the HPLC-fluorescence strategy and from 95.17% to 98.47% for the LC-MS approach. Additionally, the precision ranged from 0.83% to 1.23% for the HPLC-fluorescence approach and from 0.66% to 1.55% for the LC-MS strategy. The HPLC-FLD technique demonstrated higher sensitivity for quantifying both BAR and REM at trace levels, with a lower limit of quantification of 0.1 ng/mL and 0.5 ng/mL, respectively, whereas the LC-MS method offered a wider linear range, extending up to 2000 ng/mL for BAR and 1000 ng/mL for REM.
The assessment of green features using the Analytical GREEnness Metric Approach (AGREE) revealed that the HPLC-fluorescence approach, particularly due to its use of ethanol, is more sustainable (AGREE score: 0.68) than the LC-MS approach (AGREE score: 0.56). The practical functionality of both novel approaches was confirmed with high Blue Applicability Grade Index (BAGI) scores (77.5 and 75, respectively). Without bio-matrices interference, the optimized LC approaches showed very high levels of sensitivity and accuracy. Therefore, these methods are highly suitable for therapeutic drug monitoring and pharmacokinetic studies of the BAR-REM combination.
{"title":"High-sensitivity dual analysis of Baricitinib and remdesivir in serum and urine using HPLC-fluorescence and LC-MS approaches in COVID-19 therapy","authors":"Hazim M. Ali , Lateefa A. Al-Khateeb , Mohammed M. Ghoneim , Maha M. Abdelrahman , Ismail M. Ahmed , Mohammed Gamal","doi":"10.1016/j.jchromb.2026.124927","DOIUrl":"10.1016/j.jchromb.2026.124927","url":null,"abstract":"<div><div>Sensitive and accurate analytical strategies for the simultaneous quantification of Baricitinib (BAR) and Remdesivir (REM) in biological fluids are essential for supporting their combined use in COVID-19 therapy. Currently, no analytical methods exist for the simultaneous quantification of both drugs in human biological fluids. To bridge this gap, we developed, optimized, and validated novel RP-HPLC-fluorescence and LC-MS methods for the concurrent assay of BAR and REM in serum and urine samples. A high-performance liquid chromatography method with fluorescence detection (HPLC-fluorescence) was optimized using an isocratic mobile phase of phosphoric acid (pH = 3) and ethanol (30:70, <em>v</em>/v) at a flow rate of 0.6 mL/min, with detection at excitation/emission wavelengths of 245/400 nm. Furthermore, an LC-MS approach was developed using a mobile phase of 0.1% formic acid with 0.05 M ammonium formate in water and methanol (80:20, v/v) at 0.5 mL/min in an isocratic approach. The two LC approaches were successfully validated and applied for the simultaneous analysis of spiked BAR and REM mixtures in human urine and serum samples within 5 and 6 min, respectively. In biological human samples, accuracy varied from 96.41% to 98.10% for the HPLC-fluorescence strategy and from 95.17% to 98.47% for the LC-MS approach. Additionally, the precision ranged from 0.83% to 1.23% for the HPLC-fluorescence approach and from 0.66% to 1.55% for the LC-MS strategy. The HPLC-FLD technique demonstrated higher sensitivity for quantifying both BAR and REM at trace levels, with a lower limit of quantification of 0.1 ng/mL and 0.5 ng/mL, respectively, whereas the LC-MS method offered a wider linear range, extending up to 2000 ng/mL for BAR and 1000 ng/mL for REM.</div><div>The assessment of green features using the Analytical GREEnness Metric Approach (AGREE) revealed that the HPLC-fluorescence approach, particularly due to its use of ethanol, is more sustainable (AGREE score: 0.68) than the LC-MS approach (AGREE score: 0.56). The practical functionality of both novel approaches was confirmed with high Blue Applicability Grade Index (BAGI) scores (77.5 and 75, respectively). Without bio-matrices interference, the optimized LC approaches showed very high levels of sensitivity and accuracy. Therefore, these methods are highly suitable for therapeutic drug monitoring and pharmacokinetic studies of the BAR-REM combination.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1272 ","pages":"Article 124927"},"PeriodicalIF":2.8,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146037396","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}
Dihydropyrimidine dehydrogenase (DPD) is a key phase I drug-metabolizing enzyme responsible for catabolizing fluoropyrimidine chemotherapeutics such as 5-fluorouracil, capecitabine and tegafur. Its activity shows marked interindividual variability due to both genetic polymorphisms and environmental factors. Reduced DPD activity results in excessive fluoropyrimidine exposure, potentially causing severe and life-threatening toxicities. Although DPD testing is recommended before initiating fluoropyrimidine therapy, no single analytical strategy has been universally adopted, leading to substantial variability in clinical practice. While genotyping offers high specificity, phenotyping provides greater sensitivity by capturing both genetic and non-genetic influences on enzyme activity. In this study, we developed and validated an HPLC-MS/MS assay for the simultaneous quantification of endogenous uracil and dihydrouracil in human plasma, widely used endogenous biomarkers for DPD phenotyping. Sample preparation involved extraction with lipid removal, followed by chromatographic separation on porous graphitic carbon (100 × 2.1 mm, 5 μm; Hypercarb™, Thermo Scientific). Validation was performed according to international guidelines, demonstrating appropriate selectivity, sensitivity, linearity, accuracy, precision, carry-over, and stability. The method was applied to 28 human plasma samples, with uracil concentrations compared against an external laboratory using an independent LC–MS/MS platform. Deming regression showed no significant constant bias at the 95% confidence level, although underestimation occurred at higher concentrations (y = 0.63× + 4.86). Bland–Altman analysis indicated a small mean difference of −0.02 ng/mL but wide limits of agreement (−9.61 to +9.57 ng/mL), highlighting the impact of analytical variability near clinical decision thresholds. Further studies are warranted to evaluate the clinical utility of this approach in larger cohorts and to assess its correlation and complementarity with DPD genotyping and clinical outcomes. This method represents a valuable analytical tool to support personalized fluoropyrimidine-based chemotherapy in patients with solid tumors.
{"title":"An optimized HPLC-MS/MS assay for uracil and dihydrouracil in plasma: Improving dihydropyrimidine dehydrogenase phenotyping for individualized fluoropyrimidine treatment","authors":"Yahia Bennani , Carla Chehade , Caroline Samer , Thibaud Kössler , Aurélien Thomas , Youssef Daali","doi":"10.1016/j.jchromb.2026.124926","DOIUrl":"10.1016/j.jchromb.2026.124926","url":null,"abstract":"<div><div>Dihydropyrimidine dehydrogenase (DPD) is a key phase I drug-metabolizing enzyme responsible for catabolizing fluoropyrimidine chemotherapeutics such as 5-fluorouracil, capecitabine and tegafur. Its activity shows marked interindividual variability due to both genetic polymorphisms and environmental factors. Reduced DPD activity results in excessive fluoropyrimidine exposure, potentially causing severe and life-threatening toxicities. Although DPD testing is recommended before initiating fluoropyrimidine therapy, no single analytical strategy has been universally adopted, leading to substantial variability in clinical practice. While genotyping offers high specificity, phenotyping provides greater sensitivity by capturing both genetic and non-genetic influences on enzyme activity. In this study, we developed and validated an HPLC-MS/MS assay for the simultaneous quantification of endogenous uracil and dihydrouracil in human plasma, widely used endogenous biomarkers for DPD phenotyping. Sample preparation involved extraction with lipid removal, followed by chromatographic separation on porous graphitic carbon (100 × 2.1 mm, 5 μm; Hypercarb™, Thermo Scientific). Validation was performed according to international guidelines, demonstrating appropriate selectivity, sensitivity, linearity, accuracy, precision, carry-over, and stability. The method was applied to 28 human plasma samples, with uracil concentrations compared against an external laboratory using an independent LC–MS/MS platform. Deming regression showed no significant constant bias at the 95% confidence level, although underestimation occurred at higher concentrations (y = 0.63× + 4.86). Bland–Altman analysis indicated a small mean difference of −0.02 ng/mL but wide limits of agreement (−9.61 to +9.57 ng/mL), highlighting the impact of analytical variability near clinical decision thresholds. Further studies are warranted to evaluate the clinical utility of this approach in larger cohorts and to assess its correlation and complementarity with DPD genotyping and clinical outcomes. This method represents a valuable analytical tool to support personalized fluoropyrimidine-based chemotherapy in patients with solid tumors.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1272 ","pages":"Article 124926"},"PeriodicalIF":2.8,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146047065","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}
Pub Date : 2026-01-16DOI: 10.1016/j.jchromb.2026.124930
Jian Zhong , Xiaoli Ma , Danchen Wang , Wei Luo , Yicong Yin , Yutong Zou , Ying Zhu , Ming Li , Shaowei Xie , Songlin Yu , Ling Qiu
Steroid hormones are essential regulators of physiological homeostasis, which can help in diagnosing primary aldosteronism (PA). However, current methodologies for urinary steroid analysis face critical limitations, including narrow analyte coverage and tedious sample preparation workflows. Since the liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the preferred technique for steroid measurement, there is a great need to establish an LC-MS/MS method combined with simplified and efficient sample pretreatment for multi-steroid profiling. We establish a novel LC-MS/MS method for quantifying 35 steroid hormones within a 25-min runtime. 40 μL of urine samples can be efficiently hydrolyzed at room temperature in just 0.5 h using transgenic β-glucuronidase. Detection is performed on the Waters Acquity I-Class UPLC-tandem with a Waters TQ-S triple quadrupole MS/MS system, and the method performance is systematically evaluated. Linearity is excellent, and the recovery rates range from 80.0 to 120.6%. Acceptable intra-assay and inter-assay precisions are achieved, with the coefficients of variation ranging from 1.86 to 15.10% and 3.91 to 19.75%, respectively. For clinical application, patients with PA (n = 37) exhibit significantly higher levels of aldosterone, tetrahydroaldosterone, 18-hydroxycorticosterone, 18-oxocortisol, and 18-hydroxycortisol compared to non-PA patients (n = 104). Combined steroid profiling demonstrates strong diagnostic performance for PA, yielding an area under the curve of 0.914, with a sensitivity of 0.87 and a specificity of 0.88. In conclusion, this study establishes a technically advanced LC-MS/MS method that integrates efficient enzymatic hydrolysis to enable the accurate quantification of urinary steroids for the diagnosis of endocrine disorders.
{"title":"Liquid chromatography-tandem mass spectrometry-based urinary steroid profiling applied to primary aldosteronism diagnosis","authors":"Jian Zhong , Xiaoli Ma , Danchen Wang , Wei Luo , Yicong Yin , Yutong Zou , Ying Zhu , Ming Li , Shaowei Xie , Songlin Yu , Ling Qiu","doi":"10.1016/j.jchromb.2026.124930","DOIUrl":"10.1016/j.jchromb.2026.124930","url":null,"abstract":"<div><div>Steroid hormones are essential regulators of physiological homeostasis, which can help in diagnosing primary aldosteronism (PA). However, current methodologies for urinary steroid analysis face critical limitations, including narrow analyte coverage and tedious sample preparation workflows. Since the liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the preferred technique for steroid measurement, there is a great need to establish an LC-MS/MS method combined with simplified and efficient sample pretreatment for multi-steroid profiling. We establish a novel LC-MS/MS method for quantifying 35 steroid hormones within a 25-min runtime. 40 μL of urine samples can be efficiently hydrolyzed at room temperature in just 0.5 h using transgenic β-glucuronidase. Detection is performed on the Waters Acquity I-Class UPLC-tandem with a Waters TQ-S triple quadrupole MS/MS system, and the method performance is systematically evaluated. Linearity is excellent, and the recovery rates range from 80.0 to 120.6%. Acceptable intra-assay and inter-assay precisions are achieved, with the coefficients of variation ranging from 1.86 to 15.10% and 3.91 to 19.75%, respectively. For clinical application, patients with PA (<em>n</em> = 37) exhibit significantly higher levels of aldosterone, tetrahydroaldosterone, 18-hydroxycorticosterone, 18-oxocortisol, and 18-hydroxycortisol compared to non-PA patients (<em>n</em> = 104). Combined steroid profiling demonstrates strong diagnostic performance for PA, yielding an area under the curve of 0.914, with a sensitivity of 0.87 and a specificity of 0.88. In conclusion, this study establishes a technically advanced LC-MS/MS method that integrates efficient enzymatic hydrolysis to enable the accurate quantification of urinary steroids for the diagnosis of endocrine disorders.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1273 ","pages":"Article 124930"},"PeriodicalIF":2.8,"publicationDate":"2026-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146116632","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}
Pub Date : 2026-01-16DOI: 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.
{"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 , Marta Pelcová , Pavel Šmak , Ondřej Strýček , Jana Gregorová , Ondřej Peš , Zdeněk Glatz , Pavel Šištík , 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> < 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":"2026-01-16","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}
Pub Date : 2026-01-16DOI: 10.1016/j.jchromb.2026.124928
Aslıhan Gürbüzer , Halil İbrahim Ulusoy , İbrahim Narin , Abuzar Kabir , Marcello Locatelli
This study presents a sustainable analytical approach centered on the characterization and environmental evaluation of a sol-gel Carbowax 20M-coated fabric-phase sorptive extraction (FPSE) membrane designed for detecting pharmaceutical adulterants—specifically fluoxetine and sibutramine—in herbal slimming products. The membrane was synthesized via in situ sol-gel immobilization and characterized using FTIR and SEM, confirming the formation of a uniform and porous sorbent layer that maintained the structural integrity and permeability of the cellulose fabric substrate. Under optimized conditions, the developed FPSE-HPLC-DAD method achieved limits of detection (LOD) of 4.28 ng mL−1 for fluoxetine and 5.71 ng mL−1 for sibutramine, with recoveries ranging between 94.2% and 112.5% and relative standard deviations (RSD) below 5.2%. The linear ranges extended from 15 to 900 ng mL−1 for fluoxetine and 20–1200 ng mL−1 for sibutramine, demonstrating strong linearity (R2 > 0.99). Beyond analytical validation, the environmental performance of the method was systematically assessed using six recognized greenness and applicability metrics: AGREE (score = 0.63), MoGAPI, AGSA, BAGI (> 60), CACI (62%), and AGREEprep, all confirming high sustainability and practical applicability. These findings highlight that the sol-gel Carbowax 20M coated FPSE membrane provides a low-cost, solvent-efficient, and environmentally responsible platform for the routine screening of undeclared pharmaceutical compounds in complex herbal matrices.
本研究提出了一种以溶胶-凝胶Carbowax 20m涂层织物相吸附萃取（FPSE）膜的表征和环境评价为中心的可持续分析方法，该膜设计用于检测草药减肥产品中的药物掺杂-特别是氟西汀和西布曲明。通过原位溶胶-凝胶固定化法合成了该膜，并用FTIR和SEM对其进行了表征，证实了该膜形成了均匀多孔的吸附层，保持了纤维素织物基底的结构完整性和渗透性。在优化条件下，该方法的检出限（LOD）为氟西汀4.28 ng mL−1，西布曲明5.71 ng mL−1，加样回收率为94.2% ~ 112.5%，相对标准偏差（RSD）小于5.2%。氟西汀的线性范围为15 ~ 900 ng mL - 1，西布曲明的线性范围为20 ~ 1200 ng mL - 1，显示出很强的线性关系（R2 > 0.99）。除了分析验证之外，还使用六个公认的绿色和适用性指标对该方法的环境绩效进行了系统评估：AGREE（得分= 0.63），MoGAPI， AGSA, BAGI (> 60)， CACI（62%）和AGREEprep，均证实了该方法的高可持续性和实用性。这些发现表明，溶胶-凝胶Carbowax 20M包覆的FPSE膜为复杂草药基质中未申报的药物化合物的常规筛选提供了一种低成本、溶剂效率高、环保的平台。
{"title":"Sustainable fabric-phase sorptive extraction membrane characterization and comprehensive greenness assessment for detecting pharmaceutical adulterants in herbal slimming products","authors":"Aslıhan Gürbüzer , Halil İbrahim Ulusoy , İbrahim Narin , Abuzar Kabir , Marcello Locatelli","doi":"10.1016/j.jchromb.2026.124928","DOIUrl":"10.1016/j.jchromb.2026.124928","url":null,"abstract":"<div><div>This study presents a sustainable analytical approach centered on the characterization and environmental evaluation of a sol-gel Carbowax 20M-coated fabric-phase sorptive extraction (FPSE) membrane designed for detecting pharmaceutical adulterants—specifically fluoxetine and sibutramine—in herbal slimming products. The membrane was synthesized via in situ sol-gel immobilization and characterized using FTIR and SEM, confirming the formation of a uniform and porous sorbent layer that maintained the structural integrity and permeability of the cellulose fabric substrate. Under optimized conditions, the developed FPSE-HPLC-DAD method achieved limits of detection (LOD) of 4.28 ng mL<sup>−1</sup> for fluoxetine and 5.71 ng mL<sup>−1</sup> for sibutramine, with recoveries ranging between 94.2% and 112.5% and relative standard deviations (RSD) below 5.2%. The linear ranges extended from 15 to 900 ng mL<sup>−1</sup> for fluoxetine and 20–1200 ng mL<sup>−1</sup> for sibutramine, demonstrating strong linearity (R<sup>2</sup> > 0.99). Beyond analytical validation, the environmental performance of the method was systematically assessed using six recognized greenness and applicability metrics: AGREE (score = 0.63), MoGAPI, AGSA, BAGI (> 60), CACI (62%), and AGREEprep, all confirming high sustainability and practical applicability. These findings highlight that the sol-gel Carbowax 20M coated FPSE membrane provides a low-cost, solvent-efficient, and environmentally responsible platform for the routine screening of undeclared pharmaceutical compounds in complex herbal matrices.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1272 ","pages":"Article 124928"},"PeriodicalIF":2.8,"publicationDate":"2026-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146006748","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}
Pub Date : 2026-01-12DOI: 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 , Xiaofang He , Yueling Gong , Mingxiao Li , Lili Sheng , Ningning Zheng , Jianbo Wan , Houkai Li , 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":"2026-01-12","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}
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