Pub Date : 2025-03-27DOI: 10.1016/j.jpba.2025.116842
Junxin Chen , Xue Yang , Shuaile Jia, Shuo Zhang, Yuchuan Wang
Gallium-based metallic drugs and agents have been widely applied for the diagnosis and treatment of diseases such as non-Hodgkin's lymphoma (NHL), but there are few reports on the potential Ga(III)-binding proteins and the related cytotoxic mechanisms for Ga(III). Herein, by using human urinary bladder cancer T24 cells as a model, we identify and report that tubulin is a Ga(III)-binding protein target in T24 cells. Our analyses, including the employment of a series of methods based on immobilized metal affinity chromatography (IMAC), cellular thermal shift assay (CETSA), and immunofluorescence experiments, collectively explained this finding. Our results suggest that the binding of Ga(III) to tubulin led to significant changes in the morphology and distribution of microtubules in cells. The blocked microtubule formation or microtubule depolymerization as a result of the binding of Ga(III) to tubulin may be an important molecular mechanism by which Ga(III) exerts its cytotoxic effects in T24 cells to inhibit tumor cell growth.
{"title":"Identification and characterization of tubulin as Ga(III)-binding protein in T24 cells","authors":"Junxin Chen , Xue Yang , Shuaile Jia, Shuo Zhang, Yuchuan Wang","doi":"10.1016/j.jpba.2025.116842","DOIUrl":"10.1016/j.jpba.2025.116842","url":null,"abstract":"<div><div>Gallium-based metallic drugs and agents have been widely applied for the diagnosis and treatment of diseases such as non-Hodgkin's lymphoma (NHL), but there are few reports on the potential Ga(III)-binding proteins and the related cytotoxic mechanisms for Ga(III). Herein, by using human urinary bladder cancer T24 cells as a model, we identify and report that tubulin is a Ga(III)-binding protein target in T24 cells. Our analyses, including the employment of a series of methods based on immobilized metal affinity chromatography (IMAC), cellular thermal shift assay (CETSA), and immunofluorescence experiments, collectively explained this finding. Our results suggest that the binding of Ga(III) to tubulin led to significant changes in the morphology and distribution of microtubules in cells. The blocked microtubule formation or microtubule depolymerization as a result of the binding of Ga(III) to tubulin may be an important molecular mechanism by which Ga(III) exerts its cytotoxic effects in T24 cells to inhibit tumor cell growth.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116842"},"PeriodicalIF":3.1,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734794","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}
This work presents a generic SFC-MS method for the simultaneous analysis of 22 anticancer drugs (fluorouracil, busulfan, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, gemcitabine, idarubicin, ifosfamide, irinotecan, methotrexate, paclitaxel, pemetrexed, raltitrexed, topotecan, treosulfan, vinblastine, vincristine). The separation conditions were optimized by screening nine stationary phases (2-picolylamine, bare hybrid silica, 2-ethylpyridine, fluoro-phenyl, octadecyl, diethylamine, diol, 1-aminoanthracene, zwitterionic modification), evaluating additives effects (2–5 % water, 20–50 mM ammonium formate, 0–1 mM ammonium fluoride), and adjusting the organic modifier composition (methanol, ethanol, isopropanol, acetonitrile). The optimized SFC-MS method successfully analyzed 22 anticancer drugs, along with 5 additional challenging compounds (azacitidine, mitomycin, cisplatin, oxaliplatin, carboplatin), in 12 min, using a diol column (100 × 3 mm, 1.7 µm) and a gradient of 2–100 % methanol containing 2 % water and 50 mM ammonium formate. To overcome overpressure generated by high organic solvent content, a backpressure gradient (110–150 bar) and a flow rate gradient (0.6–1.5 mL/min) were applied. The diol column was selected as the most promising based on five predefined chromatographic criteria. Additives with 5 % water or ammonium fluoride were excluded due to overpressure and signal loss, respectively. Increasing ammonium formate concentration improved peak symmetry by 29 %. For the organic modifier, pure methanol was chosen since ternary mixtures led to system overpressure without improving separation. Comparison with the LC-MS method using real samples confirmed the potential applicability of the SFC method, as the same trace compounds were detected with comparable concentrations. Sensitivity optimization and method validation will be discussed separately in a later paper.
{"title":"Simultaneous analysis of various anticancer drugs by supercritical fluid chromatography-mass spectrometry. Part I: Optimization of chromatographic separation","authors":"Nathalie Nguyen , Davy Guillarme , Serge Rudaz , Pascal Bonnabry , Sandrine Fleury-Souverain","doi":"10.1016/j.jpba.2025.116838","DOIUrl":"10.1016/j.jpba.2025.116838","url":null,"abstract":"<div><div>This work presents a generic SFC-MS method for the simultaneous analysis of 22 anticancer drugs (fluorouracil, busulfan, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, gemcitabine, idarubicin, ifosfamide, irinotecan, methotrexate, paclitaxel, pemetrexed, raltitrexed, topotecan, treosulfan, vinblastine, vincristine). The separation conditions were optimized by screening nine stationary phases (2-picolylamine, bare hybrid silica, 2-ethylpyridine, fluoro-phenyl, octadecyl, diethylamine, diol, 1-aminoanthracene, zwitterionic modification), evaluating additives effects (2–5 % water, 20–50 mM ammonium formate, 0–1 mM ammonium fluoride), and adjusting the organic modifier composition (methanol, ethanol, isopropanol, acetonitrile). The optimized SFC-MS method successfully analyzed 22 anticancer drugs, along with 5 additional challenging compounds (azacitidine, mitomycin, cisplatin, oxaliplatin, carboplatin), in 12 min, using a diol column (100 × 3 mm, 1.7 µm) and a gradient of 2–100 % methanol containing 2 % water and 50 mM ammonium formate. To overcome overpressure generated by high organic solvent content, a backpressure gradient (110–150 bar) and a flow rate gradient (0.6–1.5 mL/min) were applied. The diol column was selected as the most promising based on five predefined chromatographic criteria. Additives with 5 % water or ammonium fluoride were excluded due to overpressure and signal loss, respectively. Increasing ammonium formate concentration improved peak symmetry by 29 %. For the organic modifier, pure methanol was chosen since ternary mixtures led to system overpressure without improving separation. Comparison with the LC-MS method using real samples confirmed the potential applicability of the SFC method, as the same trace compounds were detected with comparable concentrations. Sensitivity optimization and method validation will be discussed separately in a later paper.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116838"},"PeriodicalIF":3.1,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143739982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-27DOI: 10.1016/j.jpba.2025.116841
Qiuyi Jing , Hong Pan , Xiaoli Li , Yaya Fan , Jingshan Shi , Chao Fang , Fuguo Shi
Catalpol, an iridoid glycoside, exhibits potent and versatile pharmacological effects. It is a promising drug candidate for treating ischemic stroke. However, its drug metabolism and disposition remain poorly understood, which hinders a better understanding of its mode of action. Here, we elucidated the intriguing metabolic characteristics of catalpol in rats. Catalpol underwent sequential metabolism mainly in the intestine, resulting in 31 stable metabolites and 8 unstable dialdehyde metabolites. Twelve glucosyl-containing metabolites were generated through the direct metabolism of catalpol. Eleven deglycosylated metabolites were primarily derived from catalpol aglycone metabolism. Seven N-heterocyclic metabolites originated from aglycone metabolites. Eight unstable and reactive dialdehyde metabolites were formed by the ring-opening of the hemiacetal reaction in aglycone metabolites. Eleven metabolic pathways were involved in catalpol metabolism. All eight dialdehyde metabolites were produced in the intestine through the sequential metabolism of catalpol. Notably, five dialdehyde metabolites could covalently bind to the proteins in the intestine. The dialdehyde metabolites were primarily derived from didehydroxylated and acetylated aglycone. Catalpol could improve the levels of gut bacterial metabolites, short-chain fatty acids. In conclusion, catalpol underwent extensive and sequential metabolism, generating 31 stable metabolites and 8 reactive dialdehyde metabolites. Five dialdehyde metabolites enable covalent protein modification in the intestine, which may be vital to the potent and versatile pharmacological effects of catalpol.
{"title":"Covalent protein modification by multiple reactive dialdehyde metabolites of catalpol via intestinal bioactivation","authors":"Qiuyi Jing , Hong Pan , Xiaoli Li , Yaya Fan , Jingshan Shi , Chao Fang , Fuguo Shi","doi":"10.1016/j.jpba.2025.116841","DOIUrl":"10.1016/j.jpba.2025.116841","url":null,"abstract":"<div><div>Catalpol, an iridoid glycoside, exhibits potent and versatile pharmacological effects. It is a promising drug candidate for treating ischemic stroke. However, its drug metabolism and disposition remain poorly understood, which hinders a better understanding of its mode of action. Here, we elucidated the intriguing metabolic characteristics of catalpol in rats. Catalpol underwent sequential metabolism mainly in the intestine, resulting in 31 stable metabolites and 8 unstable dialdehyde metabolites. Twelve glucosyl-containing metabolites were generated through the direct metabolism of catalpol. Eleven deglycosylated metabolites were primarily derived from catalpol aglycone metabolism. Seven <em>N</em>-heterocyclic metabolites originated from aglycone metabolites. Eight unstable and reactive dialdehyde metabolites were formed by the ring-opening of the hemiacetal reaction in aglycone metabolites. Eleven metabolic pathways were involved in catalpol metabolism. All eight dialdehyde metabolites were produced in the intestine through the sequential metabolism of catalpol. Notably, five dialdehyde metabolites could covalently bind to the proteins in the intestine. The dialdehyde metabolites were primarily derived from didehydroxylated and acetylated aglycone. Catalpol could improve the levels of gut bacterial metabolites, short-chain fatty acids. In conclusion, catalpol underwent extensive and sequential metabolism, generating 31 stable metabolites and 8 reactive dialdehyde metabolites. Five dialdehyde metabolites enable covalent protein modification in the intestine, which may be vital to the potent and versatile pharmacological effects of catalpol.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116841"},"PeriodicalIF":3.1,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143725989","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 : 2025-03-27DOI: 10.1016/j.jpba.2025.116837
Karoline D. do Amaral, Pamela S.M. Rocha, André S. Araújo, Ricardo J. Cassella
The determination of metals in urine using spectrometric techniques presents challenges due to the complex matrix of substances in the samples. This work introduces a novel method for determining Ag(I), Cu(II), and Ni(II) in urine using a semipermeable membrane device (SPMD) filled with chloroform (CHCl₃). The analytes were separated after complexation with ammonium pyrrolidinedithiocarbamate (APDC), forming a substance capable of migrating through the membrane and being absorbed by the internal phase. After extraction, the internal phase (solvent) was transferred to a volumetric flask, diluted with ethanol, and analyzed by graphite furnace atomic absorption spectrometry (GF AAS). Optimization of the extraction process included the acceptor phase, sample pH, APDC concentration, extraction time, and GF AAS temperature program. The limits of detection were 0.19, 0.23, and 0.15 µg L⁻¹ for Ag(I), Cu(II), and Ni(II), respectively, while the limits of quantification were 0.64, 0.77, and 0.51 µg L⁻¹ . Analysis of five urine samples and recovery tests, involving spiking with 5 and 10 µg L⁻¹ of each analyte, resulted in recovery percentages between 85 % and 106 %, confirming the suitability of the method for quantifying metals in complex samples like urine.
{"title":"Development of a novel strategy for the determination of Ag(I), Cu(II) and Ni(II) in urine after the extraction mediated by a semipermeable membrane","authors":"Karoline D. do Amaral, Pamela S.M. Rocha, André S. Araújo, Ricardo J. Cassella","doi":"10.1016/j.jpba.2025.116837","DOIUrl":"10.1016/j.jpba.2025.116837","url":null,"abstract":"<div><div>The determination of metals in urine using spectrometric techniques presents challenges due to the complex matrix of substances in the samples. This work introduces a novel method for determining Ag(I), Cu(II), and Ni(II) in urine using a semipermeable membrane device (SPMD) filled with chloroform (CHCl₃). The analytes were separated after complexation with ammonium pyrrolidinedithiocarbamate (APDC), forming a substance capable of migrating through the membrane and being absorbed by the internal phase. After extraction, the internal phase (solvent) was transferred to a volumetric flask, diluted with ethanol, and analyzed by graphite furnace atomic absorption spectrometry (GF AAS). Optimization of the extraction process included the acceptor phase, sample pH, APDC concentration, extraction time, and GF AAS temperature program. The limits of detection were 0.19, 0.23, and 0.15 µg L⁻¹ for Ag(I), Cu(II), and Ni(II), respectively, while the limits of quantification were 0.64, 0.77, and 0.51 µg L⁻¹ . Analysis of five urine samples and recovery tests, involving spiking with 5 and 10 µg L⁻¹ of each analyte, resulted in recovery percentages between 85 % and 106 %, confirming the suitability of the method for quantifying metals in complex samples like urine.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116837"},"PeriodicalIF":3.1,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734854","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 : 2025-03-26DOI: 10.1016/j.jpba.2025.116840
Sil Thanh Nguyen , Huyen Thu Thi Nguyen , Duyen Cam Thi Nguyen , Phuc Thien Lu , Thu Huynh Dang , Trinh To Huynh Ma , Thi Anh Huynh Huynh , Tho Vinh Minh Chau Do
Lotus (Nelumbo nucifera Gaertn.) is widely utilized in traditional medicine and cuisine throughout Asia, with its leaves containing valuable bioactive alkaloids and flavonoids. However, Lotus leaves are often considered as by-products, urging the need for methods to analyze these components and enhance biological and economic value. In this study, a novel, rapid, and reliable UPLC-MS/MS method was developed to evaluate the quality of Folium Nelumbinis by establishing the first chromatographic fingerprint of simultaneously determination of bioactive alkaloids (nuciferine, O-nornuciferine, N-nornuciferine) and flavonoids (kaempferol, quercetin, isoquercitrin, quercetin-3-O-glucuronide). The method was validated according to the Association of Official Analytical Chemists and European Commission Decision 2021/808/EC guidelines, demonstrating high sensitivity, specificity, and reproducibility, with detection and quantification limits below 0.48 ng/mL and 1.50 ng/mL, respectively. When the procedure was applied to 49 Lotus leaf samples collected across various regions and growth stages in Vietnam, revealing significant regional and developmental variations in alkaloid and flavonoid content. Nuciferine and quercetin-3-O-glucuronide were the most abundant compounds, with the highest alkaloid concentration found in Hanoi and the highest flavonoid content in Lam Dong. Besides, this study contributes not only to the quality control of Lotus leaves but also to the understanding of geographical and developmental impacts on their bioactive composition. The chromatographic fingerprinting initiative described here highlights the application of the approach as a potential standard for the quality and regularity of traditional herbal medicines and hence the sustainable and optimal utilization of Lotus leaves in pharmaceutical and nutraceutical applications.
{"title":"Advancing UPLC-MS/MS for mapping the chemical fingerprint of bioactive compounds in lotus leaves (Folium Nelumbinis)","authors":"Sil Thanh Nguyen , Huyen Thu Thi Nguyen , Duyen Cam Thi Nguyen , Phuc Thien Lu , Thu Huynh Dang , Trinh To Huynh Ma , Thi Anh Huynh Huynh , Tho Vinh Minh Chau Do","doi":"10.1016/j.jpba.2025.116840","DOIUrl":"10.1016/j.jpba.2025.116840","url":null,"abstract":"<div><div>Lotus (<em>Nelumbo nucifera</em> Gaertn.) is widely utilized in traditional medicine and cuisine throughout Asia, with its leaves containing valuable bioactive alkaloids and flavonoids. However, Lotus leaves are often considered as by-products, urging the need for methods to analyze these components and enhance biological and economic value. In this study, a novel, rapid, and reliable UPLC-MS/MS method was developed to evaluate the quality of <em>Folium Nelumbinis</em> by establishing the first chromatographic fingerprint of simultaneously determination of bioactive alkaloids (nuciferine, <em>O</em>-nornuciferine, <em>N</em>-nornuciferine) and flavonoids (kaempferol, quercetin, isoquercitrin, quercetin-3-<em>O</em>-glucuronide). The method was validated according to the Association of Official Analytical Chemists and European Commission Decision 2021/808/EC guidelines, demonstrating high sensitivity, specificity, and reproducibility, with detection and quantification limits below 0.48 ng/mL and 1.50 ng/mL, respectively. When the procedure was applied to 49 Lotus leaf samples collected across various regions and growth stages in Vietnam, revealing significant regional and developmental variations in alkaloid and flavonoid content. Nuciferine and quercetin-3-<em>O</em>-glucuronide were the most abundant compounds, with the highest alkaloid concentration found in Hanoi and the highest flavonoid content in Lam Dong. Besides, this study contributes not only to the quality control of Lotus leaves but also to the understanding of geographical and developmental impacts on their bioactive composition. The chromatographic fingerprinting initiative described here highlights the application of the approach as a potential standard for the quality and regularity of traditional herbal medicines and hence the sustainable and optimal utilization of Lotus leaves in pharmaceutical and nutraceutical applications.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116840"},"PeriodicalIF":3.1,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143746447","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 : 2025-03-22DOI: 10.1016/j.jpba.2025.116834
Ziyan Ma , Minghai Tang , Linyu Yang , Lijuan Chen
Purinostat Mesylate (PM) is a novel and highly efficient selective histone deacetylase (HDAC) I/IIb inhibitor for hematologic tumor treatment that was granted Investigational New Drug (IND) approval for clinical investigation by the National Medical Products Administration and is currently in phase IIb clinical trials for relapsed/refractory diffuse large B-cell lymphoma. In this paper, the excretion, distribution, and metabolism properties of this IND were researched by High-Performance Liquid Chromatography coupled with LTQ Orbitrap Mass Spectrometry/Radioactivity Monitoring (HPLC-LTQ-Orbitrap-MS/RAM) and liquid scintillation counting. Following a single intravenous dose of [14C] PM to rats, a total of 98.49 % of the dose was recovered from intact rats within 0–168 h post-dose, with 14.16 % in urine and 83.15 % in feces, most of which was recovered within the first 24 h post-dose. For bile duct cannulated rats, a total of 95.54 % of the dose was recovered, with 62.37 % in bile, 23.37 % in urine and 8.58 % in feces within 0–72 h post-dose, suggesting that [14C] PM was excreted mainly into feces via biliary excretion. [14C] PM was distributed widely and eliminated rapidly throughout the body, with the lung, liver, kidney and intestine as the main organs. Interestingly, slow elimination was observed in the spleen, which could benefit the functional restoration of the spleen in hematological tumors. In terms of metabolism, [14C] PM underwent an extensive metabolic transformation in rats. Fourteen metabolites were tentatively identified, with major phase I metabolic pathways encompassing reduction, N-dealkylation, and oxidative deamination. Concomitantly, the primary phase II metabolic routes involved acetylation and glucuronic acid conjugation. This study was the first comprehensive PM pharmacokinetic study utilizing [14C] isotope labeling technology.
{"title":"Distribution, metabolism, and excretion of [14C] purinostat mesylate, a novel selective HDAC I/IIb inhibitor, in rats analyzed by high-performance liquid chromatography coupled with LTQ orbitrap mass spectrometry/radioactivity monitoring","authors":"Ziyan Ma , Minghai Tang , Linyu Yang , Lijuan Chen","doi":"10.1016/j.jpba.2025.116834","DOIUrl":"10.1016/j.jpba.2025.116834","url":null,"abstract":"<div><div>Purinostat Mesylate (PM) is a novel and highly efficient selective histone deacetylase (HDAC) I/IIb inhibitor for hematologic tumor treatment that was granted Investigational New Drug (IND) approval for clinical investigation by the National Medical Products Administration and is currently in phase IIb clinical trials for relapsed/refractory diffuse large B-cell lymphoma. In this paper, the excretion, distribution, and metabolism properties of this IND were researched by High-Performance Liquid Chromatography coupled with LTQ Orbitrap Mass Spectrometry/Radioactivity Monitoring (HPLC-LTQ-Orbitrap-MS/RAM) and liquid scintillation counting. Following a single intravenous dose of [<sup>14</sup>C] PM to rats, a total of 98.49 % of the dose was recovered from intact rats within 0–168 h post-dose, with 14.16 % in urine and 83.15 % in feces, most of which was recovered within the first 24 h post-dose. For bile duct cannulated rats, a total of 95.54 % of the dose was recovered, with 62.37 % in bile, 23.37 % in urine and 8.58 % in feces within 0–72 h post-dose, suggesting that [<sup>14</sup>C] PM was excreted mainly into feces via biliary excretion. [<sup>14</sup>C] PM was distributed widely and eliminated rapidly throughout the body, with the lung, liver, kidney and intestine as the main organs. Interestingly, slow elimination was observed in the spleen, which could benefit the functional restoration of the spleen in hematological tumors. In terms of metabolism, [<sup>14</sup>C] PM underwent an extensive metabolic transformation in rats. Fourteen metabolites were tentatively identified, with major phase I metabolic pathways encompassing reduction, <em>N-</em>dealkylation, and oxidative deamination. Concomitantly, the primary phase II metabolic routes involved acetylation and glucuronic acid conjugation. This study was the first comprehensive PM pharmacokinetic study utilizing [<sup>14</sup>C] isotope labeling technology.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116834"},"PeriodicalIF":3.1,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734793","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 : 2025-03-22DOI: 10.1016/j.jpba.2025.116836
Elena Ferri , Andrea Cerrato , Cristian Caprari , Fabiana Russo , Aldo Laganà , Anna Laura Capriotti , Giorgio Faggiana , Arcangelo Moro , Cinzia Citti , Giuseppe Cannazza
The present study investigates the phytocannabinoid profiles of Cannabis sativa L. by analyzing individual glandular trichomes from both a medicinal cannabis variety (FM2) and a high-CBD (cannabidiol) hemp variety. The analysis was performed using an untargeted metabolomics approach (phytocannabinomics) that enabled the identification of nearly 70 phytocannabinoids in single trichomes, a significant improvement over previous studies, which typically analyzed pooled or enriched trichome fractions. For comparison, the phytocannabinoid profiles of whole inflorescences were also examined. Results showed that the profiles of single trichomes and whole inflorescences were similar in terms of the number and distribution of cannabinoids, with CBD-type compounds being the most abundant. The study revealed a diverse array of carboxylated and decarboxylated cannabinoids, including several homologs of CBD, tetrahydrocannabinol (THC), and cannabigerol (CBG). Although the absolute quantities of cannabinoids varied, the CBD to THC ratio was found to remain consistent across both single trichomes and whole inflorescences, suggesting that the genetic chemotype largely dictates the cannabinoid composition. Statistical analysis indicated minor significant differences between single trichomes and inflorescences, likely due to developmental stage or other biological factors influencing cannabinoid accumulation. This work represents the first successful analysis of phytocannabinoids at the single-trichome level and provides new insights into the chemical diversity of Cannabis sativa L. at a microscopic scale. Additionally, the three characteristic cannabis flavonoids, cannflavin A, B, and C, with important biological activities, including anti-inflammatory, anti-viral, anti-cancer, and anti-parasitic effects, were surprisingly found in the isolated trichomes. These findings offer new insights into the metabolic diversity of cannabis trichomes and the distribution of biologically active metabolites.
{"title":"Single trichome phytocannabinomics of two different cannabis varieties","authors":"Elena Ferri , Andrea Cerrato , Cristian Caprari , Fabiana Russo , Aldo Laganà , Anna Laura Capriotti , Giorgio Faggiana , Arcangelo Moro , Cinzia Citti , Giuseppe Cannazza","doi":"10.1016/j.jpba.2025.116836","DOIUrl":"10.1016/j.jpba.2025.116836","url":null,"abstract":"<div><div>The present study investigates the phytocannabinoid profiles of <em>Cannabis sativa</em> L. by analyzing individual glandular trichomes from both a medicinal cannabis variety (FM2) and a high-CBD (cannabidiol) hemp variety. The analysis was performed using an untargeted metabolomics approach (<em>phytocannabinomics</em>) that enabled the identification of nearly 70 phytocannabinoids in single trichomes, a significant improvement over previous studies, which typically analyzed pooled or enriched trichome fractions. For comparison, the phytocannabinoid profiles of whole inflorescences were also examined. Results showed that the profiles of single trichomes and whole inflorescences were similar in terms of the number and distribution of cannabinoids, with CBD-type compounds being the most abundant. The study revealed a diverse array of carboxylated and decarboxylated cannabinoids, including several homologs of CBD, tetrahydrocannabinol (THC), and cannabigerol (CBG). Although the absolute quantities of cannabinoids varied, the CBD to THC ratio was found to remain consistent across both single trichomes and whole inflorescences, suggesting that the genetic chemotype largely dictates the cannabinoid composition. Statistical analysis indicated minor significant differences between single trichomes and inflorescences, likely due to developmental stage or other biological factors influencing cannabinoid accumulation. This work represents the first successful analysis of phytocannabinoids at the single-trichome level and provides new insights into the chemical diversity of <em>Cannabis sativa</em> L. at a microscopic scale. Additionally, the three characteristic cannabis flavonoids, cannflavin A, B, and C, with important biological activities, including anti-inflammatory, anti-viral, anti-cancer, and anti-parasitic effects, were surprisingly found in the isolated trichomes. These findings offer new insights into the metabolic diversity of cannabis trichomes and the distribution of biologically active metabolites.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116836"},"PeriodicalIF":3.1,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143696920","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 : 2025-03-22DOI: 10.1016/j.jpba.2025.116833
Marcel Grapp , Christoph Kaufmann , Andreas Peschel , Meike Potzscher , Marek Dziadosz , Lisa Marquenie , Jörg Teske
Hexahydrocannabinol (HHC) is a phytocannabinoid that has been known since 1940, but has only recently appeared on the recreational drug market. For the analytical detection of HHC consumption, a GC-MS method for the quantification of cannabinoids was extended and validated by adding (9S)-HHC, (9 R)-HHC, (9S)-carboxy-HHC (HHC-COOH) and (9 R)-HHC-COOH. Both HHC and HHC-COOH epimers were chromatographically separated and the validation data were convincing for forensic toxicological routine analysis. This method was used to analyze 599 serum samples from forensic cases where cannabis use was suspected. Results were classified into three consumption groups: Δ9-THC only (n = 574), Δ9-THC and HHC (n = 19), and HHC only (n = 6). The concentration in serum was between 0.15 ng/mL to 14.4 ng/mL for (9 R)-HHC and 0.14 ng/mL to 5.76 ng/mL for (9S)-HHC. In all HHC positive samples, (9 R)-HHC-COOH could be detected in concentrations between 1.0 and 314 ng/mL. Of note, in the cases that tested positive for Δ9-THC only, (9 R)-HHC-COOH was also unambiguously detected in serum samples as a metabolite not only of HHC but also of Δ9-THC. In six urine samples of THC users that were examined by GC-MS and LC-MS/MS both epimers of HHC-COOH and their glucuronides could be detected. (9S)-HHC-COOH was the predominant epimer in urine which was not detected in serum. Results suggest that detection of HHC-COOH epimers alone cannot prove prior HHC consumption. With the data presented, we tentatively recommend a cut-off of 30 % of the (9 R)-HHC-COOH/THC-COOH ratio in serum to distinguish the intake of both substances from the intake of Δ9-THC alone.
{"title":"Detection of 11-nor-9-carboxy-hexahydrocannabinol (HHC-COOH) as metabolite of both hexahydrocannabinol (HHC) and Δ9-tetrahydrocannabinol (Δ9-THC) in routine forensic samples","authors":"Marcel Grapp , Christoph Kaufmann , Andreas Peschel , Meike Potzscher , Marek Dziadosz , Lisa Marquenie , Jörg Teske","doi":"10.1016/j.jpba.2025.116833","DOIUrl":"10.1016/j.jpba.2025.116833","url":null,"abstract":"<div><div>Hexahydrocannabinol (HHC) is a phytocannabinoid that has been known since 1940, but has only recently appeared on the recreational drug market. For the analytical detection of HHC consumption, a GC-MS method for the quantification of cannabinoids was extended and validated by adding (<em>9S</em>)-HHC, (<em>9 R</em>)-HHC, (<em>9S</em>)-carboxy-HHC (HHC-COOH) and (<em>9 R</em>)-HHC-COOH. Both HHC and HHC-COOH epimers were chromatographically separated and the validation data were convincing for forensic toxicological routine analysis. This method was used to analyze 599 serum samples from forensic cases where cannabis use was suspected. Results were classified into three consumption groups: Δ<sup>9</sup>-THC only (n = 574), Δ<sup>9</sup>-THC and HHC (n = 19), and HHC only (n = 6). The concentration in serum was between 0.15 ng/mL to 14.4 ng/mL for (9 <em>R</em>)-HHC and 0.14 ng/mL to 5.76 ng/mL for (9<em>S</em>)-HHC. In all HHC positive samples, (9 <em>R</em>)-HHC-COOH could be detected in concentrations between 1.0 and 314 ng/mL. Of note, in the cases that tested positive for Δ<sup>9</sup>-THC only, (9 <em>R</em>)-HHC-COOH was also unambiguously detected in serum samples as a metabolite not only of HHC but also of Δ<sup>9</sup>-THC. In six urine samples of THC users that were examined by GC-MS and LC-MS/MS both epimers of HHC-COOH and their glucuronides could be detected. (<em>9S</em>)-HHC-COOH was the predominant epimer in urine which was not detected in serum. Results suggest that detection of HHC-COOH epimers alone cannot prove prior HHC consumption. With the data presented, we tentatively recommend a cut-off of 30 % of the (<em>9 R</em>)-HHC-COOH/THC-COOH ratio in serum to distinguish the intake of both substances from the intake of Δ<sup>9</sup>-THC alone.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116833"},"PeriodicalIF":3.1,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143696922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-21DOI: 10.1016/j.jpba.2025.116820
Xiaona Zhao , Tong gong Liu , Hongfang Chen , Xi Chen , Liwen Zhu , Jie Wen , Dayong Gu
C-Reactive protein (CRP) is a key biomarker for evaluating inflammation levels and estimating cardiovascular risk. However, current CRP detection methods rely on monoclonal antibodies (mAb), which possess shortcomings such as a lengthy preparation cycle, high cost, and poor repeatability. To address these challenges, we explored the potential of peptide aptamers as an alternative to mAb for CRP detection. Using some bioinformatics approaches, we designed and optimized peptide aptamers, selecting the dominant peptide aptamer C9m (KWRWRFRLSR) through experimental validation for its specific recognition of CRP. We then established a sandwich ELISA detection system combining C9m with CRP mAb. This system demonstrated a detection limit of 22.275 ng/mL CRP and exhibited excellent specificity, with no cross-reactivity observed with human serum albumin or γ-globulin. The method also showed high reproducibility, with intra- and inter-assay coefficients of variation (CV) less than 15 %, meeting laboratory testing standards. Furthermore, comparison with clinically used immunoturbidimetry revealed high consistency (r = 0.9891).
{"title":"From bioinformatics to clinical application: A new strategy in CRP detection with peptide aptamer","authors":"Xiaona Zhao , Tong gong Liu , Hongfang Chen , Xi Chen , Liwen Zhu , Jie Wen , Dayong Gu","doi":"10.1016/j.jpba.2025.116820","DOIUrl":"10.1016/j.jpba.2025.116820","url":null,"abstract":"<div><div>C-Reactive protein (CRP) is a key biomarker for evaluating inflammation levels and estimating cardiovascular risk. However, current CRP detection methods rely on monoclonal antibodies (mAb), which possess shortcomings such as a lengthy preparation cycle, high cost, and poor repeatability. To address these challenges, we explored the potential of peptide aptamers as an alternative to mAb for CRP detection. Using some bioinformatics approaches, we designed and optimized peptide aptamers, selecting the dominant peptide aptamer C9m (KWRWRFRLSR) through experimental validation for its specific recognition of CRP. We then established a sandwich ELISA detection system combining C9m with CRP mAb. This system demonstrated a detection limit of 22.275 ng/mL CRP and exhibited excellent specificity, with no cross-reactivity observed with human serum albumin or γ-globulin. The method also showed high reproducibility, with intra- and inter-assay coefficients of variation (CV) less than 15 %, meeting laboratory testing standards. Furthermore, comparison with clinically used immunoturbidimetry revealed high consistency (r = 0.9891).</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116820"},"PeriodicalIF":3.1,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143697999","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 : 2025-03-20DOI: 10.1016/j.jpba.2025.116826
Xiujuan Jia , James Z. Deng , Michael A. Winters , Mellie June Paulines , Weidong Tong , Erin Cannon , Mirlinda Biba , Ping Zhuang
Pneumococcal conjugate vaccines (PCVs) are developed by conjugating pneumococcal polysaccharides to a carrier protein, such as CRM197. Size and molecular weight are important attributes of each monovalent conjugate in a PCV, making accurate monitoring of molecular weights crucial during the conjugation process. While size-exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS), refractive index (RI), and ultraviolet (UV) detectors (SEC-MALS) is the gold standard used for absolute molecular weight characterization, this study presents the development of a multi-detection (MALS, UV and RI) hydrodynamic chromatography (HDC-MALS) method and its utility for comprehensive PCV characterization. The optimized HDC-MALS method is employed for in-depth understanding of vaccine conjugation process and effective characterization of heterogeneous, large conjugates through granular molar mass distribution analysis. Compared to other mild separation techniques such as field flow fractionation (FFF), HDC allows for high mobile phase flow rates without compromising separation efficiency, enabling faster run times that meet the demands of in-process control with rapid turn-around times.
{"title":"Characterization of pneumococcal conjugates in vaccine process development by multi-detection hydrodynamic chromatography","authors":"Xiujuan Jia , James Z. Deng , Michael A. Winters , Mellie June Paulines , Weidong Tong , Erin Cannon , Mirlinda Biba , Ping Zhuang","doi":"10.1016/j.jpba.2025.116826","DOIUrl":"10.1016/j.jpba.2025.116826","url":null,"abstract":"<div><div>Pneumococcal conjugate vaccines (PCVs) are developed by conjugating pneumococcal polysaccharides to a carrier protein, such as CRM<sub>197</sub>. Size and molecular weight are important attributes of each monovalent conjugate in a PCV, making accurate monitoring of molecular weights crucial during the conjugation process. While size-exclusion chromatography (SEC) coupled with multi-angle light scattering (MALS), refractive index (RI), and ultraviolet (UV) detectors (SEC-MALS) is the gold standard used for absolute molecular weight characterization, this study presents the development of a multi-detection (MALS, UV and RI) hydrodynamic chromatography (HDC-MALS) method and its utility for comprehensive PCV characterization. The optimized HDC-MALS method is employed for in-depth understanding of vaccine conjugation process and effective characterization of heterogeneous, large conjugates through granular molar mass distribution analysis. Compared to other mild separation techniques such as field flow fractionation (FFF), HDC allows for high mobile phase flow rates without compromising separation efficiency, enabling faster run times that meet the demands of in-process control with rapid turn-around times.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"261 ","pages":"Article 116826"},"PeriodicalIF":3.1,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143682401","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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