Journal of Chromatography B最新文献

Investigating the mechanism of action of Qianlie Jindan tablets in rats with chronic prostatitis using non-targeted metabolomics

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-04-03 DOI: 10.1016/j.jchromb.2025.124577

Tengfei Chen , Qian Zhang , Zhichao Jia , Zhuozhuo Shi , Junguo Ma , Zhaowang Gao , Chongfu Zhong

Objective

To explore the mechanism of action of Qianlie Jindan tablets by analyzing the metabolomic changes in prostate tissues obtained from rats with chronic prostatitis/chronic pelvic pain (CP/CPPS).

Materials and methods

Male SD rats were randomly divided into three groups: blank control (BC), model control (MC), and treatment (QLJD) groups, with 10 rats in each group. The model was induced using Complete Freund's adjuvant (CFA) and prostate protein purification solution, and the corresponding drug intervention was given. At the end of the experiment, pathological changes in the prostate tissues were observed using hematoxylin and eosin (HE) staining. Differential metabolites were determined by ultra-high-performance liquid chromatography and tandem electrostatic field orbital trap mass spectrometry (UHPLC-Q Exactive HFX), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation was performed. The results were then verified by quantitative real-time polymerase chain reaction (PCR).

Results

QLJD reversed the histopathological damage induced by CP/CPPS. Metabolomics analysis showed that UDP-GlcNAc was the key differential metabolite, and can activate the PI3K/AKT/mTOR pathway. The PCR analysis revealed that the mRNA expression levels of PI3K, AKT and mTOR in the MC group were significantly lower than those in the BC group (P < 0.05). These parameters were increased in the QLJD group compared to the MC group (P < 0.05), validating our metabolomics results.

Conclusion

QLJD exerts a therapeutic effect on CP/CPPS by activating the PI3K/AKT/mTOR pathway through the regulation of UDP-GlcNAc levels.

{"title":"Investigating the mechanism of action of Qianlie Jindan tablets in rats with chronic prostatitis using non-targeted metabolomics","authors":"Tengfei Chen , Qian Zhang , Zhichao Jia , Zhuozhuo Shi , Junguo Ma , Zhaowang Gao , Chongfu Zhong","doi":"10.1016/j.jchromb.2025.124577","DOIUrl":"10.1016/j.jchromb.2025.124577","url":null,"abstract":"<div><h3>Objective</h3><div>To explore the mechanism of action of Qianlie Jindan tablets by analyzing the metabolomic changes in prostate tissues obtained from rats with chronic prostatitis/chronic pelvic pain (CP/CPPS).</div></div><div><h3>Materials and methods</h3><div>Male SD rats were randomly divided into three groups: blank control (BC), model control (MC), and treatment (QLJD) groups, with 10 rats in each group. The model was induced using Complete Freund's adjuvant (CFA) and prostate protein purification solution, and the corresponding drug intervention was given. At the end of the experiment, pathological changes in the prostate tissues were observed using hematoxylin and eosin (HE) staining. Differential metabolites were determined by ultra-high-performance liquid chromatography and tandem electrostatic field orbital trap mass spectrometry (UHPLC-Q Exactive HFX), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation was performed. The results were then verified by quantitative real-time polymerase chain reaction (PCR).</div></div><div><h3>Results</h3><div>QLJD reversed the histopathological damage induced by CP/CPPS. Metabolomics analysis showed that UDP-GlcNAc was the key differential metabolite, and can activate the PI3K/AKT/mTOR pathway. The PCR analysis revealed that the mRNA expression levels of PI3K, AKT and mTOR in the MC group were significantly lower than those in the BC group (<em>P</em> < 0.05). These parameters were increased in the QLJD group compared to the MC group (P < 0.05), validating our metabolomics results.</div></div><div><h3>Conclusion</h3><div>QLJD exerts a therapeutic effect on CP/CPPS by activating the PI3K/AKT/mTOR pathway through the regulation of UDP-GlcNAc levels.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124577"},"PeriodicalIF":2.8,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143791268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development and validation of a stability-indicating reversed phase high-performance liquid chromatography method for quantifying rivaroxaban (XARELTO)

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-04-03 DOI: 10.1016/j.jchromb.2025.124573

Aktham Mestareehi

Rivaroxaban is an anticoagulant medication that targets a key stage in the blood clotting process, preventing the formation and growth of clots. It is commonly used to prevent thrombosis or inhibit the enlargement of existing clots. Rivaroxaban functions as a Factor Xa inhibitor and is indicated for: reducing the risk of stroke and systemic embolism in patients with non-valvular atrial fibrillation, treating deep vein thrombosis (DVT) and pulmonary embolism (PE), as well as reducing the risk of recurrent DVT and PE, and prophylaxis of DVT, which may lead to PE in patients undergoing knee or hip replacement surgery.

A robust, precise, and selective reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for analyzing Rivaroxaban in raw materials. Isocratic elution at a flow rate of 1 mL/min was performed using a Thermo ODS Hypersil C18 column (4.6 × 250 mm, 5 μm) at ambient temperature. The mobile phase consisted of monobasic potassium phosphate at pH 2.9 and acetonitrile in a 70:30 (v/v) ratio, with UV detection at 249 nm. Linearity was established in the concentration range of 50–1000 ppm (r² = 0.999), and the retention time for Rivaroxaban was approximately 12 min. The percentage relative standard deviation (RSD) for precision and accuracy was consistently below 2.0 %. Rivaroxaban was subjected to forced degradation under various conditions, including acid and base hydrolysis, hydrogen peroxide oxidation, heat, and UV light exposure. The developed method was validated for specificity, robustness, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ), following International Conference on Harmonisation (ICH) guidelines. The LOD for impurities and degradants was found to be 0.3 ppm, with an LOQ of 1 ppm.

{"title":"Development and validation of a stability-indicating reversed phase high-performance liquid chromatography method for quantifying rivaroxaban (XARELTO)","authors":"Aktham Mestareehi","doi":"10.1016/j.jchromb.2025.124573","DOIUrl":"10.1016/j.jchromb.2025.124573","url":null,"abstract":"<div><div>Rivaroxaban is an anticoagulant medication that targets a key stage in the blood clotting process, preventing the formation and growth of clots. It is commonly used to prevent thrombosis or inhibit the enlargement of existing clots. Rivaroxaban functions as a Factor Xa inhibitor and is indicated for: reducing the risk of stroke and systemic embolism in patients with non-valvular atrial fibrillation, treating deep vein thrombosis (DVT) and pulmonary embolism (PE), as well as reducing the risk of recurrent DVT and PE, and prophylaxis of DVT, which may lead to PE in patients undergoing knee or hip replacement surgery.</div><div>A robust, precise, and selective reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for analyzing Rivaroxaban in raw materials. Isocratic elution at a flow rate of 1 mL/min was performed using a Thermo ODS Hypersil C18 column (4.6 × 250 mm, 5 μm) at ambient temperature. The mobile phase consisted of monobasic potassium phosphate at pH 2.9 and acetonitrile in a 70:30 (<em>v</em>/v) ratio, with UV detection at 249 nm. Linearity was established in the concentration range of 50–1000 ppm (r<sup>2</sup> = 0.999), and the retention time for Rivaroxaban was approximately 12 min. The percentage relative standard deviation (RSD) for precision and accuracy was consistently below 2.0 %. Rivaroxaban was subjected to forced degradation under various conditions, including acid and base hydrolysis, hydrogen peroxide oxidation, heat, and UV light exposure. The developed method was validated for specificity, robustness, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ), following International Conference on Harmonisation (ICH) guidelines. The LOD for impurities and degradants was found to be 0.3 ppm, with an LOQ of 1 ppm.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124573"},"PeriodicalIF":2.8,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143791165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A rapid and sensitive LC–MS/MS method for the simultaneous determination of chlorfenapyr and its major metabolite tralopyril in human plasma: From method development, validation to clinical application

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-04-02 DOI: 10.1016/j.jchromb.2025.124580

Shiyuan Tang , Shunjie Zhang , Hongling Du , Xia Yang , Qunmei Yao , Aihua Peng , Minghai Tang , Yu Cao

Chlorfenapyr (CFN), widely used as an insecticide, is a pro-insecticide that acts after being metabolized to the active metabolite tralopyril (TLP). In clinical practice, CFN poisoning is characterized by atypical symptoms, delayed toxicity and high mortality. The therapeutic importance of assessing TLP levels in the management of CFN poisoning has been demonstrated in recent clinical and toxicokinetic studies. Unfortunately, the simultaneous quantification of CFN and TLP poses technical challenges and is still lacking in clinical practice. In this study, a novel LC-MS/MS method was developed for rapid and accurate simultaneous analysis of CFN and TLP in human plasma within 3 min. Samples were extracted by acetonitrile protein precipitation. Stable CFN anionic adducts [M + HCOO]⁻ were selected to provide satisfactory MS signals. To improve the signal and peak shape, the mobile phase was preferred to consisting of 2 mM ammonium formate containing 0.01 % ammonia and acetonitrile. The optimized multiple reaction monitoring transitions of m/z 451.0 → 346.9, 349.9 → 78.9 and 307.1 → 161.1 were selected in the negative ionization mode to detect CFN, TLP and IS (warfarin), respectively. The method demonstrated satisfactory linearity with low LLOQ (5 ng/mL for CFN and 1 ng/mL for TLP, respectively), and the precision and accuracy were acceptable. Both compounds showed good stability under relevant conditions. Furthermore, the method was successfully applied to 72 clinical plasma samples from 20 patients with CFN poisoning. The results showed that the concentration of TLP in plasma was significantly higher than that of CFN (P < 0.0001), and all five deaths demonstrated high TLP concentrations (>1000 ng/mL). These data demonstrate the potential relationship between toxins concentration and toxicity in CFN poisoning patients.

{"title":"A rapid and sensitive LC–MS/MS method for the simultaneous determination of chlorfenapyr and its major metabolite tralopyril in human plasma: From method development, validation to clinical application","authors":"Shiyuan Tang , Shunjie Zhang , Hongling Du , Xia Yang , Qunmei Yao , Aihua Peng , Minghai Tang , Yu Cao","doi":"10.1016/j.jchromb.2025.124580","DOIUrl":"10.1016/j.jchromb.2025.124580","url":null,"abstract":"<div><div>Chlorfenapyr (CFN), widely used as an insecticide, is a pro-insecticide that acts after being metabolized to the active metabolite tralopyril (TLP). In clinical practice, CFN poisoning is characterized by atypical symptoms, delayed toxicity and high mortality. The therapeutic importance of assessing TLP levels in the management of CFN poisoning has been demonstrated in recent clinical and toxicokinetic studies. Unfortunately, the simultaneous quantification of CFN and TLP poses technical challenges and is still lacking in clinical practice. In this study, a novel LC-MS/MS method was developed for rapid and accurate simultaneous analysis of CFN and TLP in human plasma within 3 min. Samples were extracted by acetonitrile protein precipitation. Stable CFN anionic adducts [M + HCOO]<sup>−</sup> were selected to provide satisfactory MS signals. To improve the signal and peak shape, the mobile phase was preferred to consisting of 2 mM ammonium formate containing 0.01 % ammonia and acetonitrile. The optimized multiple reaction monitoring transitions of <em>m</em>/<em>z</em> 451.0 → 346.9, 349.9 → 78.9 and 307.1 → 161.1 were selected in the negative ionization mode to detect CFN, TLP and IS (warfarin), respectively. The method demonstrated satisfactory linearity with low LLOQ (5 ng/mL for CFN and 1 ng/mL for TLP, respectively), and the precision and accuracy were acceptable. Both compounds showed good stability under relevant conditions. Furthermore, the method was successfully applied to 72 clinical plasma samples from 20 patients with CFN poisoning. The results showed that the concentration of TLP in plasma was significantly higher than that of CFN (<em>P</em> < 0.0001), and all five deaths demonstrated high TLP concentrations (>1000 ng/mL). These data demonstrate the potential relationship between toxins concentration and toxicity in CFN poisoning patients.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124580"},"PeriodicalIF":2.8,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143777477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Size exclusion chromatography: An efficient tool for hsEV isolation from insect Haemolymph 尺寸排阻色谱法：从昆虫血淋巴中分离 hsEV 的有效工具

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-03-29 DOI: 10.1016/j.jchromb.2025.124570

Jaspreet Kaur , Janvi Aggarwal , Mukesh Behera , Tannu Rachna Dahiya , Dhiraj Kumar , Narender K. Dhania

Extracellular vesicles (EV) serve an important role in biological system as they can change the phenotype and function of the cell. The discovery of EVs has led to the development of novel vaccines, associated with immunosuppressive and immunostimulatory effects during disease progression. To address the opportunities in EVs, here we used insect as model system. The insect also has medicinal properties as it produces antimicrobial peptides which are found circulating fluid called as hemolymph. Hemolymph consists of defensins, gloverins, proline-rich peptides, attacins, cecropins, moricins, anionic antimicrobial peptides, and several other components that create robust defense mechanisms against a broad spectrum of pathogens. Here, the insect hemolymph was used for EV isolation which encapsulates such kinds of proteins and acts as cargo transporters. As the biogenesis was unknown, we have defined it as hemolymph-derived small extracellular vesicles (hsEV). The present paper deals with the appropriate methodology for the isolation of hsEV. The hemolymph was processed for isolation via size exclusion chromatography. Molecular and physical characterization was performed using western blotting utilizing anti-CD63, anti-Flotillin-2, TEM, FESEM, confocal microscopy, and DLS. The isolated hsEV was used to assess the antibacterial property by measuring bacterial growth.

{"title":"Size exclusion chromatography: An efficient tool for hsEV isolation from insect Haemolymph","authors":"Jaspreet Kaur , Janvi Aggarwal , Mukesh Behera , Tannu Rachna Dahiya , Dhiraj Kumar , Narender K. Dhania","doi":"10.1016/j.jchromb.2025.124570","DOIUrl":"10.1016/j.jchromb.2025.124570","url":null,"abstract":"<div><div>Extracellular vesicles (EV) serve an important role in biological system as they can change the phenotype and function of the cell. The discovery of EVs has led to the development of novel vaccines, associated with immunosuppressive and immunostimulatory effects during disease progression. To address the opportunities in EVs, here we used insect as model system. The insect also has medicinal properties as it produces antimicrobial peptides which are found circulating fluid called as hemolymph. Hemolymph consists of defensins, gloverins, proline-rich peptides, attacins, cecropins, moricins, anionic antimicrobial peptides, and several other components that create robust defense mechanisms against a broad spectrum of pathogens. Here, the insect hemolymph was used for EV isolation which encapsulates such kinds of proteins and acts as cargo transporters. As the biogenesis was unknown, we have defined it as hemolymph-derived small extracellular vesicles (hsEV). The present paper deals with the appropriate methodology for the isolation of hsEV. The hemolymph was processed for isolation via size exclusion chromatography. Molecular and physical characterization was performed using western blotting utilizing anti-CD63, anti-Flotillin-2, TEM, FESEM, confocal microscopy, and DLS. The isolated hsEV was used to assess the antibacterial property by measuring bacterial growth.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124570"},"PeriodicalIF":2.8,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143783397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Pentafluorobenzyl bromide – A versatile derivatization agent in chromatography and mass spectrometry: II. Analysis of organic acids and bases, and comparison with other perfluorinated reagents

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-03-28 DOI: 10.1016/j.jchromb.2025.124578

Dimitrios Tsikas

Analytical derivatization is an important for the vast majority of substances an indispensable sample preparation step for their quantitative GC–MS and GC–MS/MS analysis in biological samples. Pentafluorobenzyl bromide (PFB-Br), pentafluorobenzoyl chloride (PFB-COCl), pentafluorobenzyl hydroxylamine (PFB-NHNH₂), pentafluorophenyl hydrazine (PFPh-ONH₂), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric anhydride (HFBA) are versatile derivatization reagents in analytical chemistry. In the present work, the utility of the above mentioned derivatization reagents for the GC–MS analysis of carboxylic, aldehydic, hydroxylic and amine groups containing analytes including amino acids is reviewed and discussed. Derivatization requires different conditions for solvents, reaction temperature and time, and possibly for catalysts. The perfluorinated derivatives are electrically neutral and best soluble in water-immiscible organic solvents such as toluene. Under negative-ion chemical ionization (NICI) conditions, the perfluorinated derivatives readily and abundantly ionize that allows for sensitive analysis. In addition, the perfluorinated analyte derivatives emerge earlier from GC columns than protiated, thus enabling shorter analysis times. Externally added ²H-, ¹³C-, ¹⁵N and ¹⁸O-isotopologs for use as internal standards undergo similar changes during derivatization, extraction by organic solvents, ionization in the ion-source of GC–MS apparatus and have almost identical retention times with the analytes. Due to selective analytical derivatization, almost all classes of endogenous and exogenous low-molecular-mass analytes, including drugs and inorganic anions such as nitrite, nitrate, carbonate, and (pseudo)halogenides, become accessible to quantitative GC–MS and GC–MS/MS analysis. Thanks the high sensitivity of quantitative analytical methods based on GC–MS and GC–MS/MS, very low amounts of perfluorinated derivatization reagents are consumed. In consideration of the enormously high global warming potential (GWP) of F-containing derivatization reagents, this article discussed a potential abandonment of the use of perfluorinated reagents and their replacement by F-free reagents in GC–MS and GC–MS/MS.

{"title":"Pentafluorobenzyl bromide – A versatile derivatization agent in chromatography and mass spectrometry: II. Analysis of organic acids and bases, and comparison with other perfluorinated reagents","authors":"Dimitrios Tsikas","doi":"10.1016/j.jchromb.2025.124578","DOIUrl":"10.1016/j.jchromb.2025.124578","url":null,"abstract":"<div><div>Analytical derivatization is an important for the vast majority of substances an indispensable sample preparation step for their quantitative GC–MS and GC–MS/MS analysis in biological samples. Pentafluorobenzyl bromide (PFB-Br), pentafluorobenzoyl chloride (PFB-COCl), pentafluorobenzyl hydroxylamine (PFB-NHNH<sub>2</sub>), pentafluorophenyl hydrazine (PFPh-ONH<sub>2</sub>), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric anhydride (HFBA) are versatile derivatization reagents in analytical chemistry. In the present work, the utility of the above mentioned derivatization reagents for the GC–MS analysis of carboxylic, aldehydic, hydroxylic and amine groups containing analytes including amino acids is reviewed and discussed. Derivatization requires different conditions for solvents, reaction temperature and time, and possibly for catalysts. The perfluorinated derivatives are electrically neutral and best soluble in water-immiscible organic solvents such as toluene. Under negative-ion chemical ionization (NICI) conditions, the perfluorinated derivatives readily and abundantly ionize that allows for sensitive analysis. In addition, the perfluorinated analyte derivatives emerge earlier from GC columns than protiated, thus enabling shorter analysis times. Externally added <sup>2</sup>H-, <sup>13</sup>C-, <sup>15</sup>N and <sup>18</sup>O-isotopologs for use as internal standards undergo similar changes during derivatization, extraction by organic solvents, ionization in the ion-source of GC–MS apparatus and have almost identical retention times with the analytes. Due to selective analytical derivatization, almost all classes of endogenous and exogenous low-molecular-mass analytes, including drugs and inorganic anions such as nitrite, nitrate, carbonate, and (pseudo)halogenides, become accessible to quantitative GC–MS and GC–MS/MS analysis. Thanks the high sensitivity of quantitative analytical methods based on GC–MS and GC–MS/MS, very low amounts of perfluorinated derivatization reagents are consumed. In consideration of the enormously high global warming potential (GWP) of F-containing derivatization reagents, this article discussed a potential abandonment of the use of perfluorinated reagents and their replacement by F-free reagents in GC–MS and GC–MS/MS.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124578"},"PeriodicalIF":2.8,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734726","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}

引用次数: 0

A novel ultra-sensitive LC-MS/MS method for determination of elobixibat in human plasma; Application to a bioequivalence study on healthy volunteers

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-03-27 DOI: 10.1016/j.jchromb.2025.124576

Mamdouh R. Rezk , Michael Soliman , Huda Shawky , Kamal A. Badr , Mina Wadie

Towards a new era for alleviating chronic idiopathic constipation, elobixibat has been recently approved in Japan and completed phase III in clinical trials as a highly potent inhibitor for ileal bile acid transporter. This work provides the field of biomedical analysis and clinical studies with the first bioanalytical method for elobixibat quantitation in real human plasma. A new ultra-sensitive liquid chromatography-tandem mass spectrometric method was developed using elobixibat-d5 as an internal standard. First of all, several preliminary efforts were exerted and directed towards optimizing sample preparation procedures to extract the desired drug at a very low concentration level and to avoid any matrix interference or masking. The trials settled on adopting liquid-liquid extraction using methyl tertiary butyl ether after adding 200 μL of 10 % formic acid to 500 μL plasma sample. Chromatographic separation was then conducted using Kinetex® EVO C₁₈ column with mobile phase composed of acetonitrile and 20 mM ammonium format acidified with 0.1 % formic acid in ratio of 80:20 (v/v) and pumped at 0.6 mL/min. Positive electrospray ionization was adopted for mass acquisition, operated in multiple reactions monitoring (MRM) mode at m/z 696 → 593.1 for elobixibat and m/z 701 → 598.1 for the IS. Full bioanalytical validation as per FDA guidance was done over a range of 20.0–1500.0 pg/mL. The proposed method was successfully exploited for determination of elobixibat in human plasma samples and extended to estimate the pharmacokinetic parameters after administration of a single oral dose of 5 mg elobixibat tablet to thirty healthy volunteers.

{"title":"A novel ultra-sensitive LC-MS/MS method for determination of elobixibat in human plasma; Application to a bioequivalence study on healthy volunteers","authors":"Mamdouh R. Rezk , Michael Soliman , Huda Shawky , Kamal A. Badr , Mina Wadie","doi":"10.1016/j.jchromb.2025.124576","DOIUrl":"10.1016/j.jchromb.2025.124576","url":null,"abstract":"<div><div>Towards a new era for alleviating chronic idiopathic constipation, elobixibat has been recently approved in Japan and completed phase III in clinical trials as a highly potent inhibitor for ileal bile acid transporter. This work provides the field of biomedical analysis and clinical studies with the first bioanalytical method for elobixibat quantitation in real human plasma. A new ultra-sensitive liquid chromatography-tandem mass spectrometric method was developed using elobixibat-d5 as an internal standard. First of all, several preliminary efforts were exerted and directed towards optimizing sample preparation procedures to extract the desired drug at a very low concentration level and to avoid any matrix interference or masking. The trials settled on adopting liquid-liquid extraction using methyl tertiary butyl ether after adding 200 μL of 10 % formic acid to 500 μL plasma sample. Chromatographic separation was then conducted using Kinetex® EVO C<sub>18</sub> column with mobile phase composed of acetonitrile and 20 mM ammonium format acidified with 0.1 % formic acid in ratio of 80:20 (<em>v</em>/v) and pumped at 0.6 mL/min. Positive electrospray ionization was adopted for mass acquisition, operated in multiple reactions monitoring (MRM) mode at <em>m</em>/<em>z</em> 696 → 593.1 for elobixibat and m/z 701 → 598.1 for the IS. Full bioanalytical validation as per FDA guidance was done over a range of 20.0–1500.0 pg/mL. The proposed method was successfully exploited for determination of elobixibat in human plasma samples and extended to estimate the pharmacokinetic parameters after administration of a single oral dose of 5 mg elobixibat tablet to thirty healthy volunteers.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124576"},"PeriodicalIF":2.8,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734736","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Comparative pharmacokinetics of seven propargyl-linked antifolate antibiotics in the mouse

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-03-26 DOI: 10.1016/j.jchromb.2025.124575

John Hoody , Jeremy B. Alverson , Santosh Keshipeddy , Patrick A. Barney , Larissa Walker , Nathan D. Gibson , Grant J. Sormunen , Stephen C. Bergmeier , Amy C. Anderson , Dennis L. Wright , Nigel D. Priestley

Antimicrobial resistance (AMR) to existing antibiotics poses a critical global health challenge, with significant morbidity and mortality from bacterial infections. Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains (VISA, VRSA) are among the most pressing threats, particularly for vulnerable populations. To combat this crisis, the development of novel therapeutic strategies is imperative.

We report the pharmacokinetic evaluation of a promising class of propargyl-linked diaminopyrimidine dihydrofolate reductase (DHFR) inhibitors with potent activity against drug-resistant bacteria, including MRSA and VISA strains. Previous studies have demonstrated minimum inhibitory concentration (MIC) values below 1 μg.mL⁻¹ for several compounds in this series. Here, we detail the development and validation of an LC-QQQ bioanalytical method for seven propargyl-linked diaminopyrimidine analogues. Pharmacokinetic studies in a murine model across intravenous (IV), intraperitoneal (IP), and oral (PO) routes revealed substantial variability in parameters such as half-life (t₁_/₂), area under the curve (AUC), and peak plasma concentration (Cmax).

Compound 38C1 demonstrated favorable solubility, a higher maximum tolerated dose, and oral bioavailability of 20 %, making it a lead candidate. Pharmacokinetic-to-MIC ratio analyses showed that 38C1 maintained plasma concentrations significantly above MIC values for multiple S. aureus strains, including MRSA and VISA.

These findings highlight 38C1 as a promising antifolate candidate for further development. Ongoing studies will assess its efficacy in infection models and refine delivery strategies to maximize therapeutic potential while mitigating resistance development.

{"title":"Comparative pharmacokinetics of seven propargyl-linked antifolate antibiotics in the mouse","authors":"John Hoody , Jeremy B. Alverson , Santosh Keshipeddy , Patrick A. Barney , Larissa Walker , Nathan D. Gibson , Grant J. Sormunen , Stephen C. Bergmeier , Amy C. Anderson , Dennis L. Wright , Nigel D. Priestley","doi":"10.1016/j.jchromb.2025.124575","DOIUrl":"10.1016/j.jchromb.2025.124575","url":null,"abstract":"<div><div>Antimicrobial resistance (AMR) to existing antibiotics poses a critical global health challenge, with significant morbidity and mortality from bacterial infections. Methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) and vancomycin-resistant strains (VISA, VRSA) are among the most pressing threats, particularly for vulnerable populations. To combat this crisis, the development of novel therapeutic strategies is imperative.</div><div>We report the pharmacokinetic evaluation of a promising class of propargyl-linked diaminopyrimidine dihydrofolate reductase (DHFR) inhibitors with potent activity against drug-resistant bacteria, including MRSA and VISA strains. Previous studies have demonstrated minimum inhibitory concentration (MIC) values below 1 μg.mL<sup>−1</sup> for several compounds in this series. Here, we detail the development and validation of an LC-QQQ bioanalytical method for seven propargyl-linked diaminopyrimidine analogues. Pharmacokinetic studies in a murine model across intravenous (IV), intraperitoneal (IP), and oral (PO) routes revealed substantial variability in parameters such as half-life (t₁<sub>/</sub>₂), area under the curve (AUC), and peak plasma concentration (Cmax).</div><div>Compound <strong>38C1</strong> demonstrated favorable solubility, a higher maximum tolerated dose, and oral bioavailability of 20 %, making it a lead candidate. Pharmacokinetic-to-MIC ratio analyses showed that <strong>38C1</strong> maintained plasma concentrations significantly above MIC values for multiple <em>S. aureus</em> strains, including MRSA and VISA.</div><div>These findings highlight <strong>38C1</strong> as a promising antifolate candidate for further development. Ongoing studies will assess its efficacy in infection models and refine delivery strategies to maximize therapeutic potential while mitigating resistance development.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124575"},"PeriodicalIF":2.8,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143799928","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A novel quality-by-design assisted HPLC-DAD method for the simultaneous quantification of tryptophan, tryptophol, and voriconazole for early diagnosis and prognosis of fungal infections decoding quorum sensing phenomenon

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-03-25 DOI: 10.1016/j.jchromb.2025.124571

Ahmed M. Saleh , Ola A. Saleh , Rabeay Y.A. Hassan , Amr M. Badawey , Hoda M. Marzouk

For the first time, a comprehensive analytical approach is introduced that simultaneously quantifies a metabolic precursor (tryptophan), a quorum-sensing biomarker for fungal infections (tryptophol), and an antifungal drug (voriconazole) within a single platform using reversed-phase high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). The method utilizes a Pursuit PFP column featuring a unique pentafluorinated structure, with a mobile phase of methanol: water (60:40, v/v), a flow rate of 1.0 mL/min, and a detection wavelength set at 254.0 nm. An Analytical Quality-by-Design (AQbD) methodology was employed, incorporating a full factorial design for optimal method development. Validation was performed in accordance with ICH guidelines, demonstrating exceptional linearity (2.0–60.0 μg/mL) for all target analytes, along with high precision, accuracy, and system suitability. Furthermore, the method proved robust and versatile when applied to complex matrices, including spiked human serum and pharmaceutical tablet formulations. Noteworthy is the integration of green and white chemistry principles for evaluating the method's sustainability, representing a significant advancement in analytical technique development. Assessment of greenness, blueness, and whiteness with AGREE, ComplexGAPI index, BAGI and RGB 12 Tools, respectively. This innovative analytical platform provides a powerful tool for the early detection and real-time therapeutic monitoring of fungal infections. By enabling the simultaneous analysis of a metabolic marker, a quorum-sensing specific biomarker, and an antifungal agent, the method advances personalized medicine. It offers a novel, efficient, and sustainable solution for the personalized management of fungal infections, enhancing both diagnostic and therapeutic strategies.

{"title":"A novel quality-by-design assisted HPLC-DAD method for the simultaneous quantification of tryptophan, tryptophol, and voriconazole for early diagnosis and prognosis of fungal infections decoding quorum sensing phenomenon","authors":"Ahmed M. Saleh , Ola A. Saleh , Rabeay Y.A. Hassan , Amr M. Badawey , Hoda M. Marzouk","doi":"10.1016/j.jchromb.2025.124571","DOIUrl":"10.1016/j.jchromb.2025.124571","url":null,"abstract":"<div><div>For the first time, a comprehensive analytical approach is introduced that simultaneously quantifies a metabolic precursor (tryptophan), a quorum-sensing biomarker for fungal infections (tryptophol), and an antifungal drug (voriconazole) within a single platform using reversed-phase high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). The method utilizes a Pursuit PFP column featuring a unique pentafluorinated structure, with a mobile phase of methanol: water (60:40, v/v), a flow rate of 1.0 mL/min, and a detection wavelength set at 254.0 nm. An Analytical Quality-by-Design (AQbD) methodology was employed, incorporating a full factorial design for optimal method development. Validation was performed in accordance with ICH guidelines, demonstrating exceptional linearity (2.0–60.0 μg/mL) for all target analytes, along with high precision, accuracy, and system suitability. Furthermore, the method proved robust and versatile when applied to complex matrices, including spiked human serum and pharmaceutical tablet formulations. Noteworthy is the integration of green and white chemistry principles for evaluating the method's sustainability, representing a significant advancement in analytical technique development. Assessment of greenness, blueness, and whiteness with AGREE, ComplexGAPI index, BAGI and RGB 12 Tools, respectively. This innovative analytical platform provides a powerful tool for the early detection and real-time therapeutic monitoring of fungal infections. By enabling the simultaneous analysis of a metabolic marker, a quorum-sensing specific biomarker, and an antifungal agent, the method advances personalized medicine. It offers a novel, efficient, and sustainable solution for the personalized management of fungal infections, enhancing both diagnostic and therapeutic strategies.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124571"},"PeriodicalIF":2.8,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143768008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Liquid chromatography in determination of pharmacokinetic properties of compounds in drug discovery process

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-03-24 DOI: 10.1016/j.jchromb.2025.124574

Mihajlo V. Jakanovski , Marko D. Jović , Mirjana D. Mosić , Dušanka M. Milojković-Opsenica , Sandra B. Šegan

Liquid chromatography plays a pivotal role in the determination of pharmacokinetic properties during drug discovery, particularly through the evaluation of lipophilicity. This parameter is essential in drug development, significantly influencing pharmacokinetic and pharmacodynamic behavior of potential drugs. It affects membrane permeability, solubility, distribution, and interaction with biological targets, making it a central focus in the early stages of drug design. Poor lipophilicity-related characteristics are often associated with drug failures, inefficacy, toxicity, and increased development costs. Experimental and computational methods, such as chromatographic techniques and theoretical calculations, are vital for accurately determining lipophilicity. These approaches enable the simulation of biological processes, providing insights into how lipophilicity impacts ADME (absorption, distribution, metabolism, and excretion) properties and supporting the optimization of drug candidates. In silico tools further enhance the efficiency of ADME evaluations, reducing the risk of pharmacokinetic-related failures and streamlining the drug discovery process.

{"title":"Liquid chromatography in determination of pharmacokinetic properties of compounds in drug discovery process","authors":"Mihajlo V. Jakanovski , Marko D. Jović , Mirjana D. Mosić , Dušanka M. Milojković-Opsenica , Sandra B. Šegan","doi":"10.1016/j.jchromb.2025.124574","DOIUrl":"10.1016/j.jchromb.2025.124574","url":null,"abstract":"<div><div>Liquid chromatography plays a pivotal role in the determination of pharmacokinetic properties during drug discovery, particularly through the evaluation of lipophilicity. This parameter is essential in drug development, significantly influencing pharmacokinetic and pharmacodynamic behavior of potential drugs. It affects membrane permeability, solubility, distribution, and interaction with biological targets, making it a central focus in the early stages of drug design. Poor lipophilicity-related characteristics are often associated with drug failures, inefficacy, toxicity, and increased development costs. Experimental and computational methods, such as chromatographic techniques and theoretical calculations, are vital for accurately determining lipophilicity. These approaches enable the simulation of biological processes, providing insights into how lipophilicity impacts ADME (absorption, distribution, metabolism, and excretion) properties and supporting the optimization of drug candidates. <em>In silico</em> tools further enhance the efficiency of ADME evaluations, reducing the risk of pharmacokinetic-related failures and streamlining the drug discovery process.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1257 ","pages":"Article 124574"},"PeriodicalIF":2.8,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143734787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Determination of disulfiram and diethyldithiocarbamate in plasma by high performance liquid chromatography with an electrochemical detection using a diamond electrode

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-03-22 DOI: 10.1016/j.jchromb.2025.124568

Masahiro Komeno , Arisa Ohta , Naoaki Tanabe , Hiroko Abe , Yuya Terashima , Akiyoshi Saitoh , Kyohei Higashi

Disulfiram (DSF), a drug approved for the treatment of alcoholism, has been increasing attention as an anti-inflammatory agent via inhibit cytoplasmic FROUNT, a common regulator of chemokine receptor CCR2 and CCR5 signaling. Although the determination of DSF in biological fluids, particularly plasma, is important for evaluating drug efficacy, rapid degradation of DSF by albumin often interferes with its accuracy and reproducibility. In this study, cost-effective, selective, and reproducible high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) equipped with a diamond electrode was used to quantify DSF and its metabolite, diethyldithiocarbamate (DDTC). The limit of quantification (LOQ) for DSF in an albumin-free solution using the HPLC-ECD method was 0.04 μg/mL; however, DSF was not detected in the serum spiked with DSF. Two forms of DSF metabolites, the free and albumin bound forms of DDTC, were detected as methyl-DDTC (MeDDTC) by methyl iodide treatment. The LOQ of MeDDTC in acetonitrile and serum determined using the HPLC-ECD method were 0.41 μg/mL and 1.22 μg/mL, respectively. Plasma MeDDTC was detected in rats that were orally administered DSF (1000 mg/kg) up to 48 h after treatment. This result suggests that HPLC-ECD with a diamond electrode is useful for determining MeDDTC for DSF monitoring in plasma.

{"title":"Determination of disulfiram and diethyldithiocarbamate in plasma by high performance liquid chromatography with an electrochemical detection using a diamond electrode","authors":"Masahiro Komeno , Arisa Ohta , Naoaki Tanabe , Hiroko Abe , Yuya Terashima , Akiyoshi Saitoh , Kyohei Higashi","doi":"10.1016/j.jchromb.2025.124568","DOIUrl":"10.1016/j.jchromb.2025.124568","url":null,"abstract":"<div><div>Disulfiram (DSF), a drug approved for the treatment of alcoholism, has been increasing attention as an anti-inflammatory agent via inhibit cytoplasmic FROUNT, a common regulator of chemokine receptor CCR2 and CCR5 signaling. Although the determination of DSF in biological fluids, particularly plasma, is important for evaluating drug efficacy, rapid degradation of DSF by albumin often interferes with its accuracy and reproducibility. In this study, cost-effective, selective, and reproducible high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) equipped with a diamond electrode was used to quantify DSF and its metabolite, diethyldithiocarbamate (DDTC). The limit of quantification (LOQ) for DSF in an albumin-free solution using the HPLC-ECD method was 0.04 μg/mL; however, DSF was not detected in the serum spiked with DSF. Two forms of DSF metabolites, the free and albumin bound forms of DDTC, were detected as methyl-DDTC (MeDDTC) by methyl iodide treatment. The LOQ of MeDDTC in acetonitrile and serum determined using the HPLC-ECD method were 0.41 μg/mL and 1.22 μg/mL, respectively. Plasma MeDDTC was detected in rats that were orally administered DSF (1000 mg/kg) up to 48 h after treatment. This result suggests that HPLC-ECD with a diamond electrode is useful for determining MeDDTC for DSF monitoring in plasma.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1256 ","pages":"Article 124568"},"PeriodicalIF":2.8,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143698138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0