Pub Date : 2025-12-17DOI: 10.1016/j.jchromb.2025.124898
Mengli Chang , Xinghong Li , Fengrong Zhang , Ye Zhao , Xianyu Li , Liying Tang , Hongwei Wu , Hongjun Yang
Gu-Ben-Qing-Hua formula (GBQHF) is a traditional Chinese medicine (TCM) formula that exhibits significant efficacy in the treatment of ulcerative colitis (UC) in a clinical setting. A combination of ultrahigh-performance liquid chromatography with Q Exactive mass spectrometry (UPLC-Q-Exactive-MS) in both positive and negative ionization modes and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in positive ionization mode was used to identify the components of GBQHF and determine its distribution after oral administration. A total of 240 chemical compounds, including 100 flavonoids, 27 saponins, 22 terpenoids, 20 phenolics, 18 alkaloids, 17 volatile compounds, and 12 organic acids, and 24 other compounds, were identified in GBQHF with high mass accuracy (within 5 ppm) and using MS/MS data based on online, local, and self-built databases for GBQHF using UPLC-Q-Exactive-MS. Moreover, 67 prototype compounds were identified in the intestinal tissues. The characteristics of the identified compounds, including the numbers as well as the structure and source in the duodenum, ileum, proximal colon, and distal colon, were elucidated. Among these 67 compounds, the distribution of licoricesaponin A3 and astragaloside IV known for their anti-inflammatory properties was analyzed using MALDI-MSI in 1.5-, 6-, and 24-h postdose sections of the ileum and the proximal and distal colon. Licoricesaponin A3 was significantly distributed in the basolateral region of the proximal colon, especially at 1.5 h. Astragaloside IV showed a significant distribution both in the mucosal side and basolateral region of the distal colon, especially at 6 after administration. This study systematically and comprehensively characterizes the chemical constituents of GBQHF, thereby establishing an effective strategy to characterize compounds in TCM formulae used to treat UC.
{"title":"Chemical composition and spatiotemporal distribution characteristics of Gu-ben-Qing-Hua formula in ulcerative colitis rats based on UPLC-Q-Exactive-MS and MALDI mass spectrometry imaging","authors":"Mengli Chang , Xinghong Li , Fengrong Zhang , Ye Zhao , Xianyu Li , Liying Tang , Hongwei Wu , Hongjun Yang","doi":"10.1016/j.jchromb.2025.124898","DOIUrl":"10.1016/j.jchromb.2025.124898","url":null,"abstract":"<div><div>Gu-Ben-Qing-Hua formula (GBQHF) is a traditional Chinese medicine (TCM) formula that exhibits significant efficacy in the treatment of ulcerative colitis (UC) in a clinical setting. A combination of ultrahigh-performance liquid chromatography with Q Exactive mass spectrometry (UPLC-Q-Exactive-MS) in both positive and negative ionization modes and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in positive ionization mode was used to identify the components of GBQHF and determine its distribution after oral administration. A total of 240 chemical compounds, including 100 flavonoids, 27 saponins, 22 terpenoids, 20 phenolics, 18 alkaloids, 17 volatile compounds, and 12 organic acids, and 24 other compounds, were identified in GBQHF with high mass accuracy (within 5 ppm) and using MS/MS data based on online, local, and self-built databases for GBQHF using UPLC-Q-Exactive-MS. Moreover, 67 prototype compounds were identified in the intestinal tissues. The characteristics of the identified compounds, including the numbers as well as the structure and source in the duodenum, ileum, proximal colon, and distal colon, were elucidated. Among these 67 compounds, the distribution of licoricesaponin A3 and astragaloside IV known for their anti-inflammatory properties was analyzed using MALDI-MSI in 1.5-, 6-, and 24-h postdose sections of the ileum and the proximal and distal colon. Licoricesaponin A3 was significantly distributed in the basolateral region of the proximal colon, especially at 1.5 h. Astragaloside IV showed a significant distribution both in the mucosal side and basolateral region of the distal colon, especially at 6 after administration. This study systematically and comprehensively characterizes the chemical constituents of GBQHF, thereby establishing an effective strategy to characterize compounds in TCM formulae used to treat UC.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124898"},"PeriodicalIF":2.8,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145829591","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 : 2025-12-17DOI: 10.1016/j.jchromb.2025.124901
Claire Andre , Yves Claude Guillaume
In this study, a hydrophilic monolith doped with fumed silica nanoparticles was in-situ prepared in a liquid chromatography column. It was clearly demonstrated the addition of silica nanoparticles in the polymerization mixture improved the formation of mesopores in the low size domain (5.2 nm) and thus the specific surface area and hydrophilicity of the monolith. Stability studies showed this monolith was not affected after slightly swelling or shrinking under different solvent conditions. The potential of this new column was exemplified by its capability in hydrophilic interaction liquid chromatography conditions (1) to separate five standard compounds (nucleotides) and (2) to monitor the main degradation products of topiramate an anticonvulsant molecule used primarily to treat epilepsy. For this second separation, the optimum HPLC gradient conditions were determined with only 18 experiments using a computer assisted design of experiments - simplex algorithm software developed in our laboratory. This column was then used successfully for the quantification of the different doses (in mg) of topiramate in a meglumin pharmaceutical formulation. This novel HPLC analytical shows promise as a stability-indicating assay and may be suitable for the quality control of topiramate in pharmaceutical préparations.
{"title":"A new organic hydrophilic monolith stationary phase for HILIC - ELSD topiramate quantification and separation with its main polar impurities in infusion solutions","authors":"Claire Andre , Yves Claude Guillaume","doi":"10.1016/j.jchromb.2025.124901","DOIUrl":"10.1016/j.jchromb.2025.124901","url":null,"abstract":"<div><div>In this study, a hydrophilic monolith doped with fumed silica nanoparticles was in-situ prepared in a liquid chromatography column. It was clearly demonstrated the addition of silica nanoparticles in the polymerization mixture improved the formation of mesopores in the low size domain (5.2 nm) and thus the specific surface area and hydrophilicity of the monolith. Stability studies showed this monolith was not affected after slightly swelling or shrinking under different solvent conditions. The potential of this new column was exemplified by its capability in hydrophilic interaction liquid chromatography conditions (1) to separate five standard compounds (nucleotides) and (2) to monitor the main degradation products of topiramate an anticonvulsant molecule used primarily to treat epilepsy. For this second separation, the optimum HPLC gradient conditions were determined with only 18 experiments using a computer assisted design of experiments - simplex algorithm software developed in our laboratory. This column was then used successfully for the quantification of the different doses (in mg) of topiramate in a meglumin pharmaceutical formulation. This novel HPLC analytical shows promise as a stability-indicating assay and may be suitable for the quality control of topiramate in pharmaceutical préparations.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124901"},"PeriodicalIF":2.8,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145788164","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 : 2025-12-16DOI: 10.1016/j.jchromb.2025.124896
Zhen-Yu Zhao , Ya-Ning Che , Qian Huang , Han-Wen Yang , Xian-Hua Wang , Lin-Yi Dong
Candida albicans is the leading cause of invasive fungal disease; candidemia carries 30–40 % mortality. It commonly colonizes human mucosa as a commensal, but immunosuppression or disrupted microbiota enables opportunistic infection. Therefore, rapid, reliable diagnostics are vital to improve public health.
In this study, a magnetic solid-phase microextraction (MSPE) technique based on nystatin-functionalized magnetic nanoparticles (Fe₃O₄@NYS) was developed to achieve the dual objectives of efficiently enriching C. albicans rapidly and extracting its deoxyribonucleic acid (DNA). Optimization of capture conditions enabled rapid enrichment of C. albicans with a maximum recovery rate of approximately 90 %. Subsequent to the capture of C. albicans, DNA extraction was performed on the Fe₃O₄@NYS-C. albicans complexes. Notably, Fe₃O₄@NYS facilitated the extraction of a substantial quantity of DNA, with the extraction yield reaching 1200 ng/mg. Therefore, the integration of DNA extraction with quantitative polymerase chain reaction (qPCR) technology enables sensitive and specific detection of C. albicans in real samples. The strategy demonstrated excellent linearity within the concentration range of 4.20 × 102 to 4.20 × 107 CFU mL−1 for C. albicans. The high sensitivity and accuracy of this method were confirmed in honey samples, showcasing a limit of detection (LOD) 420 CFU mL−1, recoveries between 98.1 % and 99.8 %, and relative standard deviations (RSD) not exceeding 2.51 %. This method presents an innovative strategy for point-of-care clinical diagnosis, disease surveillance, and biosafety management, demonstrating substantial potential for the efficient isolation of DNA from complex biological matrices.
{"title":"Rapid and sensitive detection of Candida albicans using nystatin-functionalized magnetic spheres","authors":"Zhen-Yu Zhao , Ya-Ning Che , Qian Huang , Han-Wen Yang , Xian-Hua Wang , Lin-Yi Dong","doi":"10.1016/j.jchromb.2025.124896","DOIUrl":"10.1016/j.jchromb.2025.124896","url":null,"abstract":"<div><div><em>Candida albicans</em> is the leading cause of invasive fungal disease; candidemia carries 30–40 % mortality. It commonly colonizes human mucosa as a commensal, but immunosuppression or disrupted microbiota enables opportunistic infection. Therefore, rapid, reliable diagnostics are vital to improve public health.</div><div>In this study, a magnetic solid-phase microextraction (MSPE) technique based on nystatin-functionalized magnetic nanoparticles (Fe₃O₄@NYS) was developed to achieve the dual objectives of efficiently enriching <em>C. albicans</em> rapidly and extracting its deoxyribonucleic acid (DNA). Optimization of capture conditions enabled rapid enrichment of <em>C. albicans</em> with a maximum recovery rate of approximately 90 %. Subsequent to the capture of <em>C. albicans,</em> DNA extraction was performed on the Fe₃O₄@NYS-<em>C. albicans</em> complexes. Notably, Fe₃O₄@NYS facilitated the extraction of a substantial quantity of DNA, with the extraction yield reaching 1200 ng/mg. Therefore, the integration of DNA extraction with quantitative polymerase chain reaction (qPCR) technology enables sensitive and specific detection of <em>C. albicans</em> in real samples. The strategy demonstrated excellent linearity within the concentration range of 4.20 × 10<sup>2</sup> to 4.20 × 10<sup>7</sup> CFU mL<sup>−1</sup> for <em>C. albicans</em>. The high sensitivity and accuracy of this method were confirmed in honey samples, showcasing a limit of detection (LOD) 420 CFU mL<sup>−1</sup>, recoveries between 98.1 % and 99.8 %, and relative standard deviations (RSD) not exceeding 2.51 %. This method presents an innovative strategy for point-of-care clinical diagnosis, disease surveillance, and biosafety management, demonstrating substantial potential for the efficient isolation of DNA from complex biological matrices.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124896"},"PeriodicalIF":2.8,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145835848","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 : 2025-12-15DOI: 10.1016/j.jchromb.2025.124897
Jianzhu Han , Na Guo , Sijia Zhou, Yijia Chen, Wenqiang Yang, Zhaoyan Wang
The incidence of obesity has increased in recent years, and studies have shown that obese individuals often exhibit elevated branched chain amino acids (BCAAs) in blood. However, the relationship between the salivary BCAA content and obesity remains to be clarified. In this work, BCAAs in saliva were directly determined by capillary electrophoresis with ultraviolet detection (CE-UV) within 15 min, and a sequential stacking enhance combining sweeping and acid barrage stacking (ABS) was employed to enhance sensitivity. Under the optimized conditions, enrichment factors of 49.3, 58.2 and 56.4 were achieved for valine (Val), leucine (Leu), and isoleucine (Ile), respectively. The limits of detection (LODs) for Val, Leu and Ile were 0.28, 0.25 and 0.26 μg/mL, and the limits of quantification (LOQs) were 0.94, 0.83 and 0.88 μg/mL, respectively. Based on salivary BCAA measurements from 16 volunteers, a preliminary positive correlation between salivary BCAA levels and body mass index (BMI) was observed.
{"title":"Online sequential stacking enrichment to verify the relationship between branched chain amino acids in saliva and body mass index by capillary electrophoresis","authors":"Jianzhu Han , Na Guo , Sijia Zhou, Yijia Chen, Wenqiang Yang, Zhaoyan Wang","doi":"10.1016/j.jchromb.2025.124897","DOIUrl":"10.1016/j.jchromb.2025.124897","url":null,"abstract":"<div><div>The incidence of obesity has increased in recent years, and studies have shown that obese individuals often exhibit elevated branched chain amino acids (BCAAs) in blood. However, the relationship between the salivary BCAA content and obesity remains to be clarified. In this work, BCAAs in saliva were directly determined by capillary electrophoresis with ultraviolet detection (CE-UV) within 15 min, and a sequential stacking enhance combining sweeping and acid barrage stacking (ABS) was employed to enhance sensitivity. Under the optimized conditions, enrichment factors of 49.3, 58.2 and 56.4 were achieved for valine (Val), leucine (Leu), and isoleucine (Ile), respectively. The limits of detection (LODs) for Val, Leu and Ile were 0.28, 0.25 and 0.26 μg/mL, and the limits of quantification (LOQs) were 0.94, 0.83 and 0.88 μg/mL, respectively. Based on salivary BCAA measurements from 16 volunteers, a preliminary positive correlation between salivary BCAA levels and body mass index (BMI) was observed.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124897"},"PeriodicalIF":2.8,"publicationDate":"2025-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145788165","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 : 2025-12-13DOI: 10.1016/j.jchromb.2025.124894
Avni Yıldızbaş , Parham Taslimi , Burak Tüzün , Nastaran Sadeghian , Rıfat Kurt , Abdullah İstek
<div><div>Since ancient times, <em>Sideritis taurica</em> and other <em>Sideritis</em> species have been used in traditional medicine in Türkiye and beyond for treating a variety of ailments, including coughs, sore throats, gastrointestinal, respiratory, and urogenital disorders, as well as wounds, colds, and flu, and are believed to possess numerous therapeutic properties such as antispasmodic, analgesic, antibacterial, anti-inflammatory, and antioxidant effects. This study aimed to evaluate the enzyme inhibition and antioxidant activities of various extracts from <em>S. taurica</em> leaves collected from Kurucaşile, Bartın, Türkiye. The extracts were prepared using ethanol, methanol, and hot/cold water extraction methods from leaves that were dried at room temperature and stored in a freezer. Enzyme inhibition activities were assessed against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-glucosidase, and α-amylase, with IC<sub>50</sub> values calculated. Antioxidant activities were measured using DPPH, ABTS, and CUPRAC assays. Furthermore, ADME/T (Absorption, Distribution, Metabolism, Excretion, and Toxicity) and molecular docking calculations were performed on the phytochemical components of <em>S. taurica</em> to investigate their effects and interactions with human metabolism. These calculations were performed on a number of proteins, including alpha-amylase protein (PDB ID: <span><span>1HNY</span><svg><path></path></svg></span>), AChE protein (PDB ID: <span><span>4M0E</span><svg><path></path></svg></span>), and BChE protein (PDB ID: <span><span>5NN0</span><svg><path></path></svg></span>). The purpose of these calculations was to investigate the interaction between these substances and human metabolism. The results indicated that the ethanol and methanol extracts exhibited the highest inhibition on AChE and BChE (IC<sub>50</sub> values of 73.99 μg/mL and 5.04 μg/mL, respectively). The methanol extracts also demonstrated potent inhibition against α-glucosidase (IC<sub>50</sub> value of 25.81 μg/mL) and α-amylase (IC<sub>50</sub> value of 70.42 μg/mL). Regarding antioxidant activity, the methanol extracts showed the highest radical scavenging activity in the DPPH (87.88 %) and ABTS (99.97 %) tests. Additionally, the methanol extracts stored in the freezer exhibited the best-reducing power in the CUPRAC assay (2.436 ± 0.1669). These findings underscore the potential of <em>S. taurica</em> as a source of natural antioxidants and enzyme inhibitors, suggesting its applicability in the treatment of neurodegenerative diseases such as Alzheimer's disease. In conclusion, extracts obtained from <em>S. taurica</em> leaves, particularly those derived from room temperature-dried leaves, demonstrate significant enzyme inhibition and antioxidant properties. Also, the findings support the consideration of <em>S. taurica</em> as a natural therapeutic source for neurodegenerative diseases and emphasize for further investigation into its active c
{"title":"Chemical composition and bioactivity of Sideritis taurica Stephan ex Wild. (Lamiaceae) leaves: GC/MS analysis, antioxidant and enzyme inhibition activities, and in silico studies","authors":"Avni Yıldızbaş , Parham Taslimi , Burak Tüzün , Nastaran Sadeghian , Rıfat Kurt , Abdullah İstek","doi":"10.1016/j.jchromb.2025.124894","DOIUrl":"10.1016/j.jchromb.2025.124894","url":null,"abstract":"<div><div>Since ancient times, <em>Sideritis taurica</em> and other <em>Sideritis</em> species have been used in traditional medicine in Türkiye and beyond for treating a variety of ailments, including coughs, sore throats, gastrointestinal, respiratory, and urogenital disorders, as well as wounds, colds, and flu, and are believed to possess numerous therapeutic properties such as antispasmodic, analgesic, antibacterial, anti-inflammatory, and antioxidant effects. This study aimed to evaluate the enzyme inhibition and antioxidant activities of various extracts from <em>S. taurica</em> leaves collected from Kurucaşile, Bartın, Türkiye. The extracts were prepared using ethanol, methanol, and hot/cold water extraction methods from leaves that were dried at room temperature and stored in a freezer. Enzyme inhibition activities were assessed against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-glucosidase, and α-amylase, with IC<sub>50</sub> values calculated. Antioxidant activities were measured using DPPH, ABTS, and CUPRAC assays. Furthermore, ADME/T (Absorption, Distribution, Metabolism, Excretion, and Toxicity) and molecular docking calculations were performed on the phytochemical components of <em>S. taurica</em> to investigate their effects and interactions with human metabolism. These calculations were performed on a number of proteins, including alpha-amylase protein (PDB ID: <span><span>1HNY</span><svg><path></path></svg></span>), AChE protein (PDB ID: <span><span>4M0E</span><svg><path></path></svg></span>), and BChE protein (PDB ID: <span><span>5NN0</span><svg><path></path></svg></span>). The purpose of these calculations was to investigate the interaction between these substances and human metabolism. The results indicated that the ethanol and methanol extracts exhibited the highest inhibition on AChE and BChE (IC<sub>50</sub> values of 73.99 μg/mL and 5.04 μg/mL, respectively). The methanol extracts also demonstrated potent inhibition against α-glucosidase (IC<sub>50</sub> value of 25.81 μg/mL) and α-amylase (IC<sub>50</sub> value of 70.42 μg/mL). Regarding antioxidant activity, the methanol extracts showed the highest radical scavenging activity in the DPPH (87.88 %) and ABTS (99.97 %) tests. Additionally, the methanol extracts stored in the freezer exhibited the best-reducing power in the CUPRAC assay (2.436 ± 0.1669). These findings underscore the potential of <em>S. taurica</em> as a source of natural antioxidants and enzyme inhibitors, suggesting its applicability in the treatment of neurodegenerative diseases such as Alzheimer's disease. In conclusion, extracts obtained from <em>S. taurica</em> leaves, particularly those derived from room temperature-dried leaves, demonstrate significant enzyme inhibition and antioxidant properties. Also, the findings support the consideration of <em>S. taurica</em> as a natural therapeutic source for neurodegenerative diseases and emphasize for further investigation into its active c","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124894"},"PeriodicalIF":2.8,"publicationDate":"2025-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145777090","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 : 2025-12-10DOI: 10.1016/j.jchromb.2025.124893
Kayla Moehnke, Pragya Sharma, Jennifer Kemp, Anthony Maus
Accurate quantification of steroid hormones such as estrogens (estradiol, estrone) and glucocorticoids (cortisol, cortisone) is essential for diagnosing and monitoring endocrine disorders. However, their structural similarity and low physiological concentrations pose analytical challenges in clinical laboratories. This study evaluates the utility of differential mobility spectrometry (DMS) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for improving the specificity and sensitivity of steroid measurements in serum (estrogens) and scalp hair (glucocorticoids) using a SelexION-equipped Sciex 6500+ mass spectrometer coupled to a high throughput Thermo Scientific TLX-2 LC system. DMS significantly reduced chromatographic interferences and enhanced signal-to-noise (S/N) ratios—up to 420 % for estradiol and 210 % for estrone. For cortisol and cortisone in hair, DMS also reduced interferences and increased S/N, as well as improving fragment ion agreement as evidenced by the reduction in fragment ion calculated concentration discrepancies exceeding ±20 % from 18 to 8 samples for cortisol and from 23 to 2 samples for cortisone. These findings support DMS as a complementary technique to LC-MS/MS, offering orthogonal separation and improved analytical performance for steroid hormone quantification in complex biological matrices.
{"title":"Assessment of the benefits of adding differential mobility spectrometry to LC-MS/MS workflows for challenging steroid measurements","authors":"Kayla Moehnke, Pragya Sharma, Jennifer Kemp, Anthony Maus","doi":"10.1016/j.jchromb.2025.124893","DOIUrl":"10.1016/j.jchromb.2025.124893","url":null,"abstract":"<div><div>Accurate quantification of steroid hormones such as estrogens (estradiol, estrone) and glucocorticoids (cortisol, cortisone) is essential for diagnosing and monitoring endocrine disorders. However, their structural similarity and low physiological concentrations pose analytical challenges in clinical laboratories. This study evaluates the utility of differential mobility spectrometry (DMS) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for improving the specificity and sensitivity of steroid measurements in serum (estrogens) and scalp hair (glucocorticoids) using a SelexION-equipped Sciex 6500+ mass spectrometer coupled to a high throughput Thermo Scientific TLX-2 LC system. DMS significantly reduced chromatographic interferences and enhanced signal-to-noise (S/N) ratios—up to 420 % for estradiol and 210 % for estrone. For cortisol and cortisone in hair, DMS also reduced interferences and increased S/N, as well as improving fragment ion agreement as evidenced by the reduction in fragment ion calculated concentration discrepancies exceeding ±20 % from 18 to 8 samples for cortisol and from 23 to 2 samples for cortisone. These findings support DMS as a complementary technique to LC-MS/MS, offering orthogonal separation and improved analytical performance for steroid hormone quantification in complex biological matrices.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124893"},"PeriodicalIF":2.8,"publicationDate":"2025-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145777067","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 : 2025-12-08DOI: 10.1016/j.jchromb.2025.124892
Doudou Lou , Jingke Fan , Lin Fan , Yang Song , Jinlin Zhang
N-glycosylation of monoclonal antibodies (mAbs) critically affects their efficacy, stability, and immunogenicity, necessitating efficient and sensitive glycan analysis. Hydrophilic Interaction Liquid Chromatography with Fluorescence Detection (HILIC-FLD) is widely used for antibody N-glycan profiling and glycosylation heterogeneity control due to its high sensitivity and strong applicability, primarily by cleaving N-glycans from antibodies with PNGase F followed by liquid chromatography or coupled mass spectrometry (MS) analysis. Conventional PNGase F digestion protocols often require protein denaturation before digestion and overnight incubation, which may cause the preparation process cumbersome, time-consuming and toxic. To address these limitations, we developed a rapid, non-denaturing enzymatic cleavage method by selecting an appropriate buffer system and regulating the pH of the reaction system for mAbs. The method requires only a single-step digestion and enhances enzyme access to N-glycans by modulating antibody conformation and surface charge through pH adjustment, enabling efficient cleavage.
This method achieves N-glycan release within 20 min—48 times faster than conventional overnight digestion—while eliminating toxic denaturants, delivering superior performance in time efficiency, operational simplicity, safety, and environmental friendliness. Validation with trastuzumab (IgG1) and sintilimab (IgG4) showed nearly identical N-glycan profiles to conventional methods, retaining chromatographic properties and yielding comparable FA2 peak areas and glycoform abundances. MS revealed consistent glycoform profiles between methods (27 vs. 25 glycoforms in optimized vs. conventional), with strong abundance correlation. These results demonstrate that the method is not only applicable to multi-sample scenarios but also fully MS compatible, offering a robust tool for biopharmaceutical development, batch consistency assessments, and biomarker discovery in glycobiology research.
{"title":"Glycan profiling of therapeutic monoclonal antibodies: Rapid, non-denaturing single-step digestion via pH-tuned enzymatic cleavage","authors":"Doudou Lou , Jingke Fan , Lin Fan , Yang Song , Jinlin Zhang","doi":"10.1016/j.jchromb.2025.124892","DOIUrl":"10.1016/j.jchromb.2025.124892","url":null,"abstract":"<div><div>N-glycosylation of monoclonal antibodies (mAbs) critically affects their efficacy, stability, and immunogenicity, necessitating efficient and sensitive glycan analysis. Hydrophilic Interaction Liquid Chromatography with Fluorescence Detection (HILIC-FLD) is widely used for antibody N-glycan profiling and glycosylation heterogeneity control due to its high sensitivity and strong applicability, primarily by cleaving N-glycans from antibodies with PNGase F followed by liquid chromatography or coupled mass spectrometry (MS) analysis. Conventional PNGase F digestion protocols often require protein denaturation before digestion and overnight incubation, which may cause the preparation process cumbersome, time-consuming and toxic. To address these limitations, we developed a rapid, non-denaturing enzymatic cleavage method by selecting an appropriate buffer system and regulating the pH of the reaction system for mAbs. The method requires only a single-step digestion and enhances enzyme access to N-glycans by modulating antibody conformation and surface charge through pH adjustment, enabling efficient cleavage.</div><div>This method achieves N-glycan release within 20 min—48 times faster than conventional overnight digestion—while eliminating toxic denaturants, delivering superior performance in time efficiency, operational simplicity, safety, and environmental friendliness. Validation with trastuzumab (IgG1) and sintilimab (IgG4) showed nearly identical N-glycan profiles to conventional methods, retaining chromatographic properties and yielding comparable FA2 peak areas and glycoform abundances. MS revealed consistent glycoform profiles between methods (27 vs. 25 glycoforms in optimized vs. conventional), with strong abundance correlation. These results demonstrate that the method is not only applicable to multi-sample scenarios but also fully MS compatible, offering a robust tool for biopharmaceutical development, batch consistency assessments, and biomarker discovery in glycobiology research.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124892"},"PeriodicalIF":2.8,"publicationDate":"2025-12-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145735275","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 : 2025-12-05DOI: 10.1016/j.jchromb.2025.124891
Ahmed M. Saleh , Ola A. Saleh , Rabeay Y.A. Hassan , Ahmed A. Abd-Rabou , Amr M. Badawey , Hoda M. Marzouk
For the first time, an integrated dual analytical–therapeutic platform is presented for concurrent investigation of hematological (chronic myeloid leukemia, CML) and solid (hepatocellular carcinoma, HCC) malignancies. A six-dimensional analytical framework combining RP-HPLC–DAD, UV–Vis spectrophotometry, ELISA, MTT assay, single-cell fluorescence imaging, and flow cytometry enabled comprehensive biochemical profiling. A novel QbD-driven RP-HPLC–DAD method was developed for the simultaneous quantification of Nilotinib (NIL, tyrosine kinase inhibitor, TKI), Upadacitinib (UPA, Janus kinase inhibitor, JAKI) and tryptophan (TRP) as a critical metabolic biomarker, successfully applied in serum matrices. Because CML and immune-mediated inflammatory diseases (IMIDs) share aberrant JAK–STAT signaling, and both NIL and UPA are metabolized via CYP3A4, continuous quantification is essential for safe co-therapy. Chromatographic separation was achieved on a carefully selected fluorinated Pursuit PFP column (150 × 4.6 mm, 3 μm), a key factor enabling efficient separation of the fluorinated drugs, using methanol–water (75,25, v/v) at 1.0 m/min and 230 nm detection, within a QbD 23 full factorial framework, exhibiting excellent linearity (1.0–50.0 μg/mL). Fluorescence-based determination using flow cytometry with ELISA revealed a pioneer significant pro-apoptotic effect of Upadacitinib in HCC cells via modulation of the Bax/Bcl-2 axis with combination therapy showing superior anticancer effect compared to standard chemotherapy of Doxorubicin (DOX). Sustainability evaluation using AGREE (greenness) and BAGI (blueness) with RGB12 algorithm (whiteness) and spider-diagram visualization, in addition to the recently launched Multi-Color Assessment (MA) tool to confirm the method's multidimensional eco-efficiency in strong alignment with the United Nations Sustainable Development Goals (SDGs). This study further harnesses the Need–Quality–Sustainability (NQS) index and Koel's pyramid principles for holistic evaluation and benchmarking against reported approaches toward sustainable analytical oncology with personalized medicine.
{"title":"Simultaneous chromatographic quantification of upadacitinib, nilotinib, and tryptophan decoding myeloid leukemia and liver cancer integrated with ELISA and Flow cytometry","authors":"Ahmed M. Saleh , Ola A. Saleh , Rabeay Y.A. Hassan , Ahmed A. Abd-Rabou , Amr M. Badawey , Hoda M. Marzouk","doi":"10.1016/j.jchromb.2025.124891","DOIUrl":"10.1016/j.jchromb.2025.124891","url":null,"abstract":"<div><div>For the first time, an integrated dual analytical–therapeutic platform is presented for concurrent investigation of hematological (chronic myeloid leukemia, CML) and solid (hepatocellular carcinoma, HCC) malignancies. A six-dimensional analytical framework combining RP-HPLC–DAD, UV–Vis spectrophotometry, ELISA, MTT assay, single-cell fluorescence imaging, and flow cytometry enabled comprehensive biochemical profiling. A novel QbD-driven RP-HPLC–DAD method was developed for the simultaneous quantification of Nilotinib (NIL, tyrosine kinase inhibitor, TKI), Upadacitinib (UPA, Janus kinase inhibitor, JAKI) and tryptophan (TRP) as a critical metabolic biomarker, successfully applied in serum matrices. Because CML and immune-mediated inflammatory diseases (IMIDs) share aberrant JAK–STAT signaling, and both NIL and UPA are metabolized via CYP3A4, continuous quantification is essential for safe co-therapy. Chromatographic separation was achieved on a carefully selected fluorinated Pursuit PFP column (150 × 4.6 mm, 3 μm), a key factor enabling efficient separation of the fluorinated drugs, using methanol–water (75,25, <em>v</em>/v) at 1.0 m/min and 230 nm detection, within a QbD 2<sup>3</sup> full factorial framework, exhibiting excellent linearity (1.0–50.0 μg/mL). Fluorescence-based determination using flow cytometry with ELISA revealed a pioneer significant pro-apoptotic effect of Upadacitinib in HCC cells via modulation of the Bax/Bcl-2 axis with combination therapy showing superior anticancer effect compared to standard chemotherapy of Doxorubicin (DOX). Sustainability evaluation using AGREE (greenness) and BAGI (blueness) with RGB12 algorithm (whiteness) and spider-diagram visualization, in addition to the recently launched Multi-Color Assessment (MA) tool to confirm the method's multidimensional eco-efficiency in strong alignment with the United Nations Sustainable Development Goals (SDGs). This study further harnesses the Need–Quality–Sustainability (NQS) index and Koel's pyramid principles for holistic evaluation and benchmarking against reported approaches toward sustainable analytical oncology with personalized medicine.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124891"},"PeriodicalIF":2.8,"publicationDate":"2025-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145735274","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 : 2025-12-04DOI: 10.1016/j.jchromb.2025.124890
Keran Chen , Orwa Siddig , Jucui Yang , Mohammed Dhawelbate , Alrazi Eisa Shogar , Mohammed Abdelrahman , Xinrui Wu , Tai-jun Hang
A validated LC-MS/MS analytical method was established for quantifying evocalcet in human plasma, using evocalcet-d4 as the internal standard. Chromatographic separation was performed on a Thermo ODS C8 column (150 × 4.6 mm, 5 μm) with gradient elution of water and methanol, both containing 0. formic acid, as the mobile phases. Detection was carried out in positive ion mode using an electrospray ionization source. The plasma sample was spiked with an internal standard solution, followed by protein precipitation, and the resulting supernatant was injected into the LC-MS/MS system. The method, validated under ICH guidelines for quantitative biological analysis, demonstrated linearity from 1 to 1000 ng/mL (R2 > 0.99) with an LLOQ of 1 ng/mL. Accuracy and precision were confirmed by recovery studies, and all other validation parameters were within acceptable limits. This validated method was successfully applied to characterize the pharmacokinetic profile of evocalcet in fasting and postprandial healthy Chinese volunteers after oral administration. Results showed significantly higher Cmax and AUC values in the fasting group compared to the postprandial group, as well as delayed Tmax (p < 0.01). The pharmacokinetic parameters, including Cmax, Tmax, t1/2, and AUC0-∞, were consistent with those reported in earlier studies. Moreover, hyperthyroid patients exhibited longer Tmax and t1/2, along with increased AUC0-t and AUC0-∞ values, compared to healthy individuals. These findings offer valuable methodological insights and pharmacokinetic data to advance clinical research on evocalcet, thereby contributing to a deeper understanding of its safety and efficacy profile.
{"title":"Quantitative LC-MS/MS Assay for Evocalcet in Human Plasma: Comparative Pharmacokinetic Evaluation in Fasted and Fed Healthy Chinese Volunteers","authors":"Keran Chen , Orwa Siddig , Jucui Yang , Mohammed Dhawelbate , Alrazi Eisa Shogar , Mohammed Abdelrahman , Xinrui Wu , Tai-jun Hang","doi":"10.1016/j.jchromb.2025.124890","DOIUrl":"10.1016/j.jchromb.2025.124890","url":null,"abstract":"<div><div>A validated LC-MS/MS analytical method was established for quantifying evocalcet in human plasma, using evocalcet-d4 as the internal standard. Chromatographic separation was performed on a Thermo ODS C8 column (150 × 4.6 mm, 5 μm) with gradient elution of water and methanol, both containing 0. formic acid, as the mobile phases. Detection was carried out in positive ion mode using an electrospray ionization source. The plasma sample was spiked with an internal standard solution, followed by protein precipitation, and the resulting supernatant was injected into the LC-MS/MS system. The method, validated under ICH guidelines for quantitative biological analysis, demonstrated linearity from 1 to 1000 ng/mL (R<sup>2</sup> > 0.99) with an LLOQ of 1 ng/mL. Accuracy and precision were confirmed by recovery studies, and all other validation parameters were within acceptable limits. This validated method was successfully applied to characterize the pharmacokinetic profile of evocalcet in fasting and postprandial healthy Chinese volunteers after oral administration. Results showed significantly higher C<sub>max</sub> and AUC values in the fasting group compared to the postprandial group, as well as delayed T<sub>max</sub> (<em>p</em> < 0.01). The pharmacokinetic parameters, including C<sub>max</sub>, T<sub>max</sub>, t<sub>1/2</sub>, and AUC<sub>0-∞</sub>, were consistent with those reported in earlier studies. Moreover, hyperthyroid patients exhibited longer T<sub>max</sub> and t<sub>1/2</sub>, along with increased AUC<sub>0-t</sub> and AUC<sub>0-∞</sub> values, compared to healthy individuals. These findings offer valuable methodological insights and pharmacokinetic data to advance clinical research on evocalcet, thereby contributing to a deeper understanding of its safety and efficacy profile.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124890"},"PeriodicalIF":2.8,"publicationDate":"2025-12-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145716836","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 : 2025-12-03DOI: 10.1016/j.jchromb.2025.124888
Maryam Soliman , Noha Mostafa , Sally Tarek Mahmoud , Dalia S. El-Gamil , Mohammad Abdel-Halim , Marwa Fouad
Compound BHBC-01, a promising multi-target inhibitor of Dyrk1A, Dyrk1B, and Clk1, which is based on 5-hydroxybenzo[b]thiophene-2-carboxylic acid benzylamide scaffold, has demonstrated its potential as a novel anticancer agent. To support its further development, this study investigated its pharmacokinetic and stability profiles. An ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the analysis of compound BHBC-01. Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column with gradient elution, employing a mobile phase consisting of 0.1 % formic acid in water and acetonitrile at a flow rate of 0.4 mL/min. Diphenhydramine was used as the internal standard. Electrospray ionization in positive ion mode allowed multiple reaction monitoring detection using parent ions ([M + H]+) at m/z 314.096 and m/z 256.14, and daughter ions at m/z 120.94 and m/z 167.04 for compound BHBC-01 and diphenhydramine, respectively. The bioanalytical method was validated according to US-FDA guidelines, demonstrating good linearity (50–1000 ng/mL), accuracy, precision, specificity, and stability, with a lower limit of quantification of 50 ng/mL. This method was applied to evaluate the in vitro metabolic stability of compound BHBC-01 in rat liver S9 fraction, revealing a half-life time of 1.95 h and an intrinsic clearance of 11.94 mL/min/kg, indicating stability against biotransformation. Compound BHBC-01 also exhibited significant stability in human plasma, with a half-life time of 57.7 h. Furthermore, gastrointestinal stability studies in simulated gastric and intestinal fluids demonstrated half-lives of 13.75 and 14.11 h, respectively, supporting its suitability for oral administration. In vivo pharmacokinetic studies were conducted in Sprague-Dawley rats following intravenous (5 mg/kg) and oral (30 mg/kg) administration. According to the findings, the compound demonstrates adequate pharmacokinetic characteristics through both intravenous and oral routes. The intravenous route demonstrated a Cmax of 545.40 ng/mL, an AUC0-t of 434.01 ng*h/mL, and a half-life time of 0.66 h. Oral administration showed higher Cmax (657 ng/mL), and a half-life time of 7.18 h. The oral bioavailability (F) was calculated to be 19.62 %. Collectively, these findings highlight compound BHBC-01's favorable pharmacokinetic and stability profiles, supporting its potential as a drug candidate for further clinical development as a multi-target anticancer agent.
{"title":"Pharmacokinetic assessment and metabolic stability of a novel multi-targeted Clk/Dyrk inhibitor using a validated LC-MS/MS method: In vitro and in vivo insights","authors":"Maryam Soliman , Noha Mostafa , Sally Tarek Mahmoud , Dalia S. El-Gamil , Mohammad Abdel-Halim , Marwa Fouad","doi":"10.1016/j.jchromb.2025.124888","DOIUrl":"10.1016/j.jchromb.2025.124888","url":null,"abstract":"<div><div>Compound <strong>BHBC-01</strong>, a promising multi-target inhibitor of Dyrk1A, Dyrk1B, and Clk1, which is based on 5-hydroxybenzo[<em>b</em>]thiophene-2-carboxylic acid benzylamide scaffold, has demonstrated its potential as a novel anticancer agent. To support its further development, this study investigated its pharmacokinetic and stability profiles. An ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the analysis of compound <strong>BHBC-01</strong>. Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column with gradient elution, employing a mobile phase consisting of 0.1 % formic acid in water and acetonitrile at a flow rate of 0.4 mL/min. Diphenhydramine was used as the internal standard. Electrospray ionization in positive ion mode allowed multiple reaction monitoring detection using parent ions ([M + H]<sup>+</sup>) at <em>m</em>/<em>z</em> 314.096 and m/z 256.14, and daughter ions at m/z 120.94 and m/z 167.04 for compound <strong>BHBC-01</strong> and diphenhydramine, respectively. The bioanalytical method was validated according to US-FDA guidelines, demonstrating good linearity (50–1000 ng/mL), accuracy, precision, specificity, and stability, with a lower limit of quantification of 50 ng/mL. This method was applied to evaluate the <em>in vitro</em> metabolic stability of compound <strong>BHBC-01</strong> in rat liver S9 fraction, revealing a half-life time of 1.95 h and an intrinsic clearance of 11.94 mL/min/kg, indicating stability against biotransformation. Compound <strong>BHBC-01</strong> also exhibited significant stability in human plasma, with a half-life time of 57.7 h. Furthermore, gastrointestinal stability studies in simulated gastric and intestinal fluids demonstrated half-lives of 13.75 and 14.11 h, respectively, supporting its suitability for oral administration. <em>In vivo</em> pharmacokinetic studies were conducted in Sprague-Dawley rats following intravenous (5 mg/kg) and oral (30 mg/kg) administration. According to the findings, the compound demonstrates adequate pharmacokinetic characteristics through both intravenous and oral routes. The intravenous route demonstrated a C<sub>max</sub> of 545.40 ng/mL, an AUC<sub>0-t</sub> of 434.01 ng*h/mL, and a half-life time of 0.66 h. Oral administration showed higher C<sub>max</sub> (657 ng/mL), and a half-life time of 7.18 h. The oral bioavailability (F) was calculated to be 19.62 %. Collectively, these findings highlight compound <strong>BHBC-01</strong>'s favorable pharmacokinetic and stability profiles, supporting its potential as a drug candidate for further clinical development as a multi-target anticancer agent.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124888"},"PeriodicalIF":2.8,"publicationDate":"2025-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145703708","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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