Pub Date : 2025-02-01DOI: 10.1016/j.jchromb.2025.124460
Yingxia Guo , Meichen Liu , Jiye Tian , Chunpeng Feng , Qingbin Wang , Xuan Zhao , Lei Yin
As an important chemical reagent, methoxy polyethylene glycol amine (mPEG-NH2) is widely used in biomedical field. Unraveling the pharmacokinetic behavior of mPEG-NH2 polymers is essential for revealing the toxicity and efficiency of mPEG-NH2 related drug delivery systems. In this study, a simple analytical assay based on mass spectrometry (MS) was first established and validated for quantification of mPEG5-NH2 in biological matrix. The multiple reaction monitoring (MRM) transitions at m/z 252.2 (precursor ions) → 87.7 (fragment ions) and m/z 371.2 (precursor ions) → 89.2 (fragment ions) were chosen to determine mPEG5-NH2 and OH-PEG8-OH, respectively. The UHPLC-MS/MS assay showed excellent linearity over the range of 0.01–10 μg/mL. Intra-day and inter-day accuracies and precisions of the assay were all within ± 6.44 %. The analytical assay was successfully applied to reveal the in vivo pharmacokinetic behavior of mPEG5-NH2 in rats.
{"title":"Unraveling the in vivo pharmacokinetic behavior of mPEG5-NH2 polymer in rats by UHPLC-MS/MS assay","authors":"Yingxia Guo , Meichen Liu , Jiye Tian , Chunpeng Feng , Qingbin Wang , Xuan Zhao , Lei Yin","doi":"10.1016/j.jchromb.2025.124460","DOIUrl":"10.1016/j.jchromb.2025.124460","url":null,"abstract":"<div><div>As an important chemical reagent, methoxy polyethylene glycol amine (mPEG-NH<sub>2</sub>) is widely used in biomedical field. Unraveling the pharmacokinetic behavior of mPEG-NH<sub>2</sub> polymers is essential for revealing the toxicity and efficiency of mPEG-NH<sub>2</sub> related drug delivery systems. In this study, a simple analytical assay based on mass spectrometry (MS) was first established and validated for quantification of mPEG<sub>5</sub>-NH<sub>2</sub> in biological matrix. The multiple reaction monitoring (MRM) transitions at <em>m</em>/<em>z</em> 252.2 (precursor ions) → 87.7 (fragment ions) and <em>m</em>/<em>z</em> 371.2 (precursor ions) → 89.2 (fragment ions) were chosen to determine mPEG<sub>5</sub>-NH<sub>2</sub> and OH-PEG<sub>8</sub>-OH, respectively. The UHPLC-MS/MS assay showed excellent linearity over the range of 0.01–10 μg/mL. Intra-day and inter-day accuracies and precisions of the assay were all within ± 6.44 %. The analytical assay was successfully applied to reveal the <em>in vivo</em> pharmacokinetic behavior of mPEG<sub>5</sub>-NH<sub>2</sub> in rats.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124460"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142997760","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 : 2025-02-01DOI: 10.1016/j.jchromb.2024.124441
Chulmin Moon, Chi Soo Park, Chang Myeong Jeong, Han Seul Lee, Kyuran Kim, Haeun Byeon, Daeun Eom, Ha Hyung Kim
Chinese hamster ovary (CHO)-K1 cells are widely used in biomedical research relevant to cancer, toxicity screening, and viruses, as well as in the production of recombinant proteins for biopharmaceuticals. In this study, liquid chromatography (LC)-electrospray ionization (ESI)-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS) was used to characterize the surface and lysate N-glycans of CHO-K1 cells and analyze their structures. The relative quantity (%) of each N-glycan and absolute quantity (pmol) of total N-glycans were also obtained. In total, 37 surface and 30 lysate N-glycans were identified. Each of these two fractions contained eight high-mannose type (required for protection against proteolysis and N-glycosylation of recombinant proteins) at 28.8 % (the sum of the relative quantities of each N-glycan) and 66.5 %, respectively. Additionally, the surface and lysate N-glycans differed in their levels of sialyation (affect cell–cell interactions; 48.1 % and 13.5 %), fucosylation (affect cell signaling; 37.9 % and 25.5 %), and terminal-galactosylation (prerequisite for subsequent sialylation; 36.6 % and 20.9 %). These results indicate that the lysate of CHO-K1 cells contained more mannosylated (2.3-fold) N-glycans compared to the surface, which contained relatively more sialylated (3.6-fold), slightly more highly fucosylated (1.5-fold), and more terminal-galactosylated (1.8-fold) N-glycans. The sum of the absolute quantity of each N-glycan was obtained as a ratio of 1 (1,778.7 pmol; surface):2.2 (3,887.3 pmol; lysate) from approximately 5 × 106 CHO-K1 cells. This study is the first to compare the surface and lysate N-glycans of CHO-K1 cells using LC-ESI-HCD-MS/MS. The results can be used to control and optimize biotechnology and biomedical research using CHO-K1 cells.
{"title":"LC-MS/MS analysis of surface and lysate N-glycans of CHO-K1 cells: Structure, relative quantity, and absolute quantity","authors":"Chulmin Moon, Chi Soo Park, Chang Myeong Jeong, Han Seul Lee, Kyuran Kim, Haeun Byeon, Daeun Eom, Ha Hyung Kim","doi":"10.1016/j.jchromb.2024.124441","DOIUrl":"10.1016/j.jchromb.2024.124441","url":null,"abstract":"<div><div>Chinese hamster ovary (CHO)-K1 cells are widely used in biomedical research relevant to cancer, toxicity screening, and viruses, as well as in the production of recombinant proteins for biopharmaceuticals. In this study, liquid chromatography (LC)-electrospray ionization (ESI)-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS) was used to characterize the surface and lysate <em>N</em>-glycans of CHO-K1 cells and analyze their structures. The relative quantity (%) of each <em>N</em>-glycan and absolute quantity (pmol) of total <em>N</em>-glycans were also obtained. In total, 37 surface and 30 lysate <em>N</em>-glycans were identified. Each of these two fractions contained eight high-mannose type (required for protection against proteolysis and <em>N</em>-glycosylation of recombinant proteins) at 28.8 % (the sum of the relative quantities of each <em>N</em>-glycan) and 66.5 %, respectively. Additionally, the surface and lysate <em>N</em>-glycans differed in their levels of sialyation (affect cell–cell interactions; 48.1 % and 13.5 %), fucosylation (affect cell signaling; 37.9 % and 25.5 %), and terminal-galactosylation (prerequisite for subsequent sialylation; 36.6 % and 20.9 %). These results indicate that the lysate of CHO-K1 cells contained more mannosylated (2.3-fold) <em>N</em>-glycans compared to the surface, which contained relatively more sialylated (3.6-fold), slightly more highly fucosylated (1.5-fold), and more terminal-galactosylated (1.8-fold) <em>N</em>-glycans. The sum of the absolute quantity of each <em>N</em>-glycan was obtained as a ratio of 1 (1,778.7 pmol; surface):2.2 (3,887.3 pmol; lysate) from approximately 5 × 10<sup>6</sup> CHO-K1 cells. This study is the first to compare the surface and lysate <em>N</em>-glycans of CHO-K1 cells using LC-ESI-HCD-MS/MS. The results can be used to control and optimize biotechnology and biomedical research using CHO-K1 cells.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124441"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142930265","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 : 2025-02-01DOI: 10.1016/j.jchromb.2025.124470
Gerard Aime Kenfack Teponnou , Anton Joubert , Saskia Spaltman , Marthinus van der Merwe , Edda Zangenberg , Sharon Sawe , Paolo Denti , Sandra Castel , Francesca Conradie , Richard Court , Gary Maartens , Lubbe Wiesner
Dried blood spot (DBS) assays to quantify novel and repurposed drugs for the treatment of rifampicin-resistant tuberculosis (RR-TB) would facilitate pharmacokinetic studies and therapeutic drug monitoring in low-middle income settings, considering their ease of application and simple sample storage requirements. We describe a DBS method for the simultaneous quantification of bedaquiline and metabolite N-desmethyl bedaquiline, linezolid, levofloxacin, and clofazimine. The analytes were extracted from the matrix and isolated by solid-phase extraction. Two LC-MS/MS systems were used, optimized for the separate analysis of the more polar compounds (linezolid and levofloxacin), and less polar compounds (bedaquiline, N-desmethyl bedaquiline, and clofazimine), employing gradient elution. Electrospray ionization and multiple reaction monitoring were used to quantify the analytes on a Sciex API3200 and an API5500 triple quadrupole mass spectrometer, for the more polar and less polar analytes, respectively. Isotopically labelled internal standards were used to compensate for variability in the quantification of each analyte. The method was validated according to international guidelines and applied to samples from a clinical trial. We performed correlation and agreement analysis of the DBS assay and in-house plasma methods using Deming regressions and Bland–Altman plots. Coefficients of correlation between measured plasma and DBS concentrations ranged from 0.866 (95% CI: 0.817–0.902) to 0.989 (95% CI: 0.985–0.992). More than 67% of the samples showed a difference between the observed and estimated plasma concentrations within 20% of their means, meeting EMA requirements for method reproducibility and demonstrating the interchangeability of our DBS and plasma LC-MS/MS methods.
{"title":"Development and validation of an LC-MS/MS multiplex assay for the quantification of bedaquiline, n-desmethyl bedaquiline, linezolid, levofloxacin, and clofazimine in dried blood spots","authors":"Gerard Aime Kenfack Teponnou , Anton Joubert , Saskia Spaltman , Marthinus van der Merwe , Edda Zangenberg , Sharon Sawe , Paolo Denti , Sandra Castel , Francesca Conradie , Richard Court , Gary Maartens , Lubbe Wiesner","doi":"10.1016/j.jchromb.2025.124470","DOIUrl":"10.1016/j.jchromb.2025.124470","url":null,"abstract":"<div><div>Dried blood spot (DBS) assays to quantify novel and repurposed drugs for the treatment of rifampicin-resistant tuberculosis (RR-TB) would facilitate pharmacokinetic studies and therapeutic drug monitoring in low-middle income settings, considering their ease of application and simple sample storage requirements. We describe a DBS method for the simultaneous quantification of bedaquiline and metabolite <em>N</em>-desmethyl bedaquiline, linezolid, levofloxacin, and clofazimine. The analytes were extracted from the matrix and isolated by solid-phase extraction. Two LC-MS/MS systems were used, optimized for the separate analysis of the more polar compounds (linezolid and levofloxacin), and less polar compounds (bedaquiline, <em>N</em>-desmethyl bedaquiline, and clofazimine), employing gradient elution. Electrospray ionization and multiple reaction monitoring were used to quantify the analytes on a Sciex API3200 and an API5500 triple quadrupole mass spectrometer, for the more polar and less polar analytes, respectively. Isotopically labelled internal standards were used to compensate for variability in the quantification of each analyte. The method was validated according to international guidelines and applied to samples from a clinical trial. We performed correlation and agreement analysis of the DBS assay and in-house plasma methods using Deming regressions and Bland–Altman plots. Coefficients of correlation between measured plasma and DBS concentrations ranged from 0.866 (95% CI: 0.817–0.902) to 0.989 (95% CI: 0.985–0.992). More than 67% of the samples showed a difference between the observed and estimated plasma concentrations within 20% of their means, meeting EMA requirements for method reproducibility and demonstrating the interchangeability of our DBS and plasma LC-MS/MS methods.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124470"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142997732","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 : 2025-02-01DOI: 10.1016/j.jchromb.2025.124461
Zhuo Mi, Wenkang Zhang, Han Wang, Xingyi Qi, Shuo Wang, Jiayi Song, Ping Su, Yi Yang
Glycopeptides are an important biomarker, which play a crucial role in various biological processes. Due to their low abundance and the presence of interfering macromolecular proteins, enrichment of glycopeptides is necessary before testing. However, most materials for enriching glycopeptides have high site resistance, relatively low surface area, and limited recognition sites. Herein, a highly hydrophilic two-dimensional (2-D) covalent organic framework (NUS-10) loaded with chitosan (CS) (denoted as NUS-10@CS) had been synthesized. After enrichment with NUS-10@CS, a total of 34 glycopeptides from horseradish peroxidase (HRP) tryptic digests were detected, demonstrating a high enrichment efficiency for glycopeptides from model glycoprotein digestion. Meanwhile, the material exhibited ultra-high adsorption capacity (1 fmol/μL HRP), excellent selectivity (HRP tryptic digest/bovine serum albumin (BSA) tryptic digest = 1:2000), macromolecular protein anti-interference ability (HRP tryptic digest/BSA = 1:2000) and good binding capacity (200 mg/g). Additionally, 712 glycopeptides corresponding to 200 glycoproteins were identified from 3 µL human serum. NUS-10@CS was promising for glycopeptide analysis, helping to identify potential disease biomarkers more efficiently, and leading to easier and more accurate diagnosis of diseases, which was essential for early intervention and treatment.
{"title":"Chitosan functionalized two-dimensional covalent organic framework nanosheets with high hydrophilicity for efficient glycopeptide enrichment","authors":"Zhuo Mi, Wenkang Zhang, Han Wang, Xingyi Qi, Shuo Wang, Jiayi Song, Ping Su, Yi Yang","doi":"10.1016/j.jchromb.2025.124461","DOIUrl":"10.1016/j.jchromb.2025.124461","url":null,"abstract":"<div><div>Glycopeptides are an important biomarker, which play a crucial role in various biological processes. Due to their low abundance and the presence of interfering macromolecular proteins, enrichment of glycopeptides is necessary before testing. However, most materials for enriching glycopeptides have high site resistance, relatively low surface area, and limited recognition sites. Herein, a highly hydrophilic two-dimensional (2-D) covalent organic framework (NUS-10) loaded with chitosan (CS) (denoted as NUS-10@CS) had been synthesized. After enrichment with NUS-10@CS, a total of 34 glycopeptides from horseradish peroxidase (HRP) tryptic digests were detected, demonstrating a high enrichment efficiency for glycopeptides from model glycoprotein digestion. Meanwhile, the material exhibited ultra-high adsorption capacity (1 fmol/μL HRP), excellent selectivity (HRP tryptic digest/bovine serum albumin (BSA) tryptic digest = 1:2000), macromolecular protein anti-interference ability (HRP tryptic digest/BSA = 1:2000) and good binding capacity (200 mg/g). Additionally, 712 glycopeptides corresponding to 200 glycoproteins were identified from 3 µL human serum. NUS-10@CS was promising for glycopeptide analysis, helping to identify potential disease biomarkers more efficiently, and leading to easier and more accurate diagnosis of diseases, which was essential for early intervention and treatment.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124461"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142997685","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}
Xanthohumol(Xn) is isolated from female inflorescences of Humulus lupulus. It has been discovered that Xn and its formulation are useful in the treatment of cancer. As this bioactive compound has medicinal importance, hence, a novel, precise, and sensitive HPLC method should be developed. In the present study, an RP-HPLC method has been developed and validated as per ICH Q2(R1) guidelines using column C18 having particle size 5 µm and dimension 250 × 4.6 mm. The detection wavelength (λ) used was 370 nm. The mobile phase consisted of a combination of HPLC grade Methanol and HPLC grade water buffer at a ratio of 95:5 v/v, with a flow rate of 1.0 mL/min. The total run time was 10 min with Xn retention time (Rt) at 4.5 min. The calibration plot was found linear in the range 2–10 μg/mL. The recovery of 97.65 % indicated good accuracy of method. The method’s precision is within 2 % of acceptable limits. LOD and LOQ values of 0.85 and 2.6 μg/mL indicated good sensitivity of method. The uniqueness of the research work is relying on achieving peak of Xn at lesser retention time as compared to existing methods. Further the results of specificity studies revealed absence of any interference of Xn peak with the excipients used in the nanostructured lipid carriers (NLCs). Overall, the study provided an accurate, precise, sensitive and specific method to quantify Xn in bulk and NLCs.
{"title":"Method development and validation on RP-HPLC method for estimation of xanthohumol in nanostructured lipid carriers drug delivery systems","authors":"Shubham Singh , Himani Sharma , Vijay Kumar , Gaurav Gupta , Samir Patel , Archita Patel , Kamal Dua , Sachin Kumar Singh","doi":"10.1016/j.jchromb.2024.124437","DOIUrl":"10.1016/j.jchromb.2024.124437","url":null,"abstract":"<div><div>Xanthohumol(Xn) is isolated from female inflorescences of <em>Humulus lupulus</em>. It has been discovered that Xn and its formulation are useful in the treatment of cancer. As this bioactive compound has medicinal importance, hence, a novel, precise, and sensitive HPLC method should be developed. In the present study, an RP-HPLC method has been developed and validated as per ICH Q2(R1) guidelines using column C18 having particle size 5 µm and dimension 250 × 4.6 mm. The detection wavelength (λ) used was 370 nm. The mobile phase consisted of a combination of HPLC grade Methanol and HPLC grade water buffer at a ratio of 95:5 v/v, with a flow rate of 1.0 mL/min. The total run time was 10 min with Xn retention time (Rt) at 4.5 min. The calibration plot was found linear in the range 2–10 μg/mL. The recovery of 97.65 % indicated good accuracy of method. The method’s precision is within 2 % of acceptable limits. LOD and LOQ values of 0.85 and 2.6 μg/mL indicated good sensitivity of method. The uniqueness of the research work is relying on achieving peak of Xn at lesser retention time as compared to existing methods. Further the results of specificity studies revealed absence of any interference of Xn peak with the excipients used in the nanostructured lipid carriers (NLCs). Overall, the study provided an accurate, precise, sensitive and specific method to quantify Xn in bulk and NLCs.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124437"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142925895","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 : 2025-02-01DOI: 10.1016/j.jchromb.2025.124449
Meryem Ermis , Fatma Kir , Selma Sahin
A new reversed phase high-performance liquid chromatography (RP-HPLC) method, with a short analysis time and easy to apply, was developed for the simultaneous detection of cimetidine (CIM), metoprolol tartrate (MT) and phenol red (PR) for use in intestinal perfusion studies. The analysis was performed with phosphate buffer (pH 5.0, 12.5 mM)-acetonitrile mixture as mobile phase and C18 column (Inertsil ODS-3; 5 µm, 4.6 × 250 mm) as stationary phase. Gradient analysis conditions were used and the acetonitrile ratio in the mobile phase varied from 10 to 50% in 10 min. Total run time for analysis was 10 min and the injection volume was 20 µL. Detection of compounds was performed at 207 nm. Under optimum HPLC conditions, retention times were 4.03 min for CIM, 6.99 min for MT and 8.49 min for PR. The method was validated according to ICH Q2 (R1) guideline for specificity, linearity, sensitivity, precision, accuracy, stability and robustness. Developed method was linear and determination coefficients of the calibration curves were 0.9993, 0.9991 and 1.0 for CIM, MT and PR, respectively. The limits of quantification were 6.20, 2.78 and 0.45 μg/mL for CIM, MT and PR, respectively. The precision and accuracy values of the developed analytical method met the ICH Q2 (R1) limits. The applicability of the method was demonstrated by preliminary in-situ intestinal perfusion studies. In conclusion, samples obtained from in-situ intestinal perfusion studies performed to examine the absorption/permeability of CIM, MT, and PR can be analyzed with the developed HPLC method.
{"title":"Development and validation of a RP-HPLC method for simultaneous determination of cimetidine, metoprolol tartrate and phenol red for intestinal perfusion studies","authors":"Meryem Ermis , Fatma Kir , Selma Sahin","doi":"10.1016/j.jchromb.2025.124449","DOIUrl":"10.1016/j.jchromb.2025.124449","url":null,"abstract":"<div><div>A new reversed phase high-performance liquid chromatography (RP-HPLC) method, with a short analysis time and easy to apply, was developed for the simultaneous detection of cimetidine (CIM), metoprolol tartrate (MT) and phenol red (PR) for use in intestinal perfusion studies. The analysis was performed with phosphate buffer (pH 5.0, 12.5 mM)-acetonitrile mixture as mobile phase and C<sub>18</sub> column (Inertsil ODS-3; 5 µm, 4.6 × 250 mm) as stationary phase. Gradient analysis conditions were used and the acetonitrile ratio in the mobile phase varied from 10 to 50% in 10 min. Total run time for analysis was 10 min and the injection volume was 20 µL. Detection of compounds was performed at 207 nm. Under optimum HPLC conditions, retention times were 4.03 min for CIM, 6.99 min for MT and 8.49 min for PR. The method was validated according to ICH Q2 (R1) guideline for specificity, linearity, sensitivity, precision, accuracy, stability and robustness. Developed method was linear and determination coefficients of the calibration curves were 0.9993, 0.9991 and 1.0 for CIM, MT and PR, respectively. The limits of quantification were 6.20, 2.78 and 0.45 μg/mL for CIM, MT and PR, respectively. The precision and accuracy values of the developed analytical method met the ICH Q2 (R1) limits. The applicability of the method was demonstrated by preliminary <em>in-situ</em> intestinal perfusion studies. In conclusion, samples obtained from <em>in-situ</em> intestinal perfusion studies performed to examine the absorption/permeability of CIM, MT, and PR can be analyzed with the developed HPLC method.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124449"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142942171","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 : 2025-02-01DOI: 10.1016/j.jchromb.2024.124443
Paweł K. Kunicki , Maciej T. Grymm , Tomasz Pawiński , Daniel Szulczyk , Marcin Waligóra , Grzegorz Kopeć
A considerable percentage of ineffective treatment in pulmonary arterial hypertension (PAH) may be related to subtherapeutic dosage or non-adherence. The aim of the study was to develop a simple analytical method suitable for plasma determination of selected drugs: riociguat (RIO), bosentan (BOS) and macitentan (MAC) administered to PAH patients. An isocratic HPLC-UV system (Spectra Physics – Shimadzu) with a manual injector (50 μL loop) was applied. Chromatographic analysis was performed using a Suplecosil LC-CN column (150 × 4.6 mm, 5 μm) protected with a Supelguard precolumn at room temperature. The separation was carried out using the mobile phase: CH3CN:H2O:0.5 M KH2PO4:85 % H3PO4 (172:324.2:3.7:0.1, v/v) at a flow rate of 1.8 mL/min. Ethyl acetate (4 mL) was used for 10-min liquid–liquid extraction from 0.4 mL alkalized plasma sample. Detection was performed at λ = 245 nm chosen as a compromise between signal intensity and matrix interference. The analytes were eluted at retention times of 4.4 min (RIO), 5.4 min (BOS), 8.9 min (MAC) and 7.8 min for gallopamil (internal standard, GAL). The method was found linear and calibrated in the ranges: 5–1000 ng/mL for RIO, 10–2000 ng/mL for BOS and 20–2000 ng/mL for MAC, with r2 of 0.9991 for RIO, 0.9983 for BOS, and 0.9949 for MAC, respectively. Within the given ranges, the method ensured reliable results with the required precision and accuracy: ≤15 % (≤20 % for LLOQ). There was no significant carryover effect. The method has been successfully used in pilot study on adherence in patients treated for PAH, enabling monitoring of RIO, BOS and MAC. Drug concentrations were assessed in samples taken before (C0) and 3 h after drug administration (C3). For RIO, BOS and MAC, the developed method was suitable for both C0 and C3 samples, allowing steady-state drug determination if used. The presented method can be recommended to laboratories equipped with basic HPLC apparatus as an attractive analytical tool for both TDM and adherence studies.
相当大比例的肺动脉高压（PAH）无效治疗可能与亚治疗剂量或不依从性有关。本研究的目的是建立一种简单的分析方法，适用于PAH患者血浆中所选药物的测定：瑞西奎特（里约热内卢）、波生坦（BOS）和马西坦（MAC）。采用手动进样（50 μL回路）等密度HPLC-UV体系（Spectra Physics - Shimadzu）。色谱分析采用Supelguard预柱保护的Suplecosil LC-CN柱（150 × 4.6 mm, 5 μm），室温下进行。采用流动相CH3CN:H2O:0.5 M kh2po4: 85% H3PO4 （172:324.2:3.7:0.1, v/v）进行分离，流速为1.8 mL/min。0.4 mL碱化血浆样品，用乙酸乙酯（4ml）液液萃取10min。在λ = 245 nm处进行检测，选择信号强度和矩阵干扰之间的折衷。洗脱时间为4.4 min（里约热内卢）、5.4 min （BOS）、8.9 min （MAC）和7.8 min （GAL）。该方法在里约热内卢的5-1000 ng/mL、BOS的10-2000 ng/mL和MAC的20-2000 ng/mL范围内呈线性关系，里约热内卢、BOS和MAC的r2分别为0.9991、0.9983和0.9949。在给定的范围内，该方法保证了可靠的结果，所需的精密度和准确度：≤15% （LLOQ≤20%）。没有明显的结转效应。该方法已成功用于PAH治疗患者依从性的试点研究，可以监测里约热内卢，BOS和MAC。在给药前（C0）和给药后3小时（C3）的样品中评估药物浓度。对于里约热内卢、BOS和MAC，该方法适用于C0和C3样品，使用该方法可以进行稳态药物测定。该方法可推荐给配备基本HPLC设备的实验室，作为TDM和粘附性研究的有吸引力的分析工具。
{"title":"A simple HPLC-UV method for monitoring therapeutic adherence in pulmonary arterial hypertension","authors":"Paweł K. Kunicki , Maciej T. Grymm , Tomasz Pawiński , Daniel Szulczyk , Marcin Waligóra , Grzegorz Kopeć","doi":"10.1016/j.jchromb.2024.124443","DOIUrl":"10.1016/j.jchromb.2024.124443","url":null,"abstract":"<div><div>A considerable percentage of ineffective treatment in pulmonary arterial hypertension (PAH) may be related to subtherapeutic dosage or non-adherence. The aim of the study was to develop a simple analytical method suitable for plasma determination of selected drugs: riociguat (RIO), bosentan (BOS) and macitentan (MAC) administered to PAH patients. An isocratic HPLC-UV system (Spectra Physics – Shimadzu) with a manual injector (50 μL loop) was applied. Chromatographic analysis was performed using a Suplecosil LC-CN column (150 × 4.6 mm, 5 μm) protected with a Supelguard precolumn at room temperature. The separation was carried out using the mobile phase: CH<sub>3</sub>CN:H<sub>2</sub>O:0.5 M KH<sub>2</sub>PO<sub>4</sub>:85 % H<sub>3</sub>PO<sub>4</sub> (172:324.2:3.7:0.1, v/v) at a flow rate of 1.8 mL/min. Ethyl acetate (4 mL) was used for 10-min liquid–liquid extraction from 0.4 mL alkalized plasma sample. Detection was performed at λ = 245 nm chosen as a compromise between signal intensity and matrix interference. The analytes were eluted at retention times of 4.4 min (RIO), 5.4 min (BOS), 8.9 min (MAC) and 7.8 min for gallopamil (internal standard, GAL). The method was found linear and calibrated in the ranges: 5–1000 ng/mL for RIO, 10–2000 ng/mL for BOS and 20–2000 ng/mL for MAC, with r<sup>2</sup> of 0.9991 for RIO, 0.9983 for BOS, and 0.9949 for MAC, respectively. Within the given ranges, the method ensured reliable results with the required precision and accuracy: ≤15 % (≤20 % for LLOQ). There was no significant carryover effect. The method has been successfully used in pilot study on adherence in patients treated for PAH, enabling monitoring of RIO, BOS and MAC. Drug concentrations were assessed in samples taken before (C0) and 3 h after drug administration (C3). For RIO, BOS and MAC, the developed method was suitable for both C0 and C3 samples, allowing steady-state drug determination if used. The presented method can be recommended to laboratories equipped with basic HPLC apparatus as an attractive analytical tool for both TDM and adherence studies.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124443"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142942014","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 : 2025-02-01DOI: 10.1016/j.jchromb.2025.124468
Wanyu Wang , Zhichao Yin , Pengfei Du , Jianbang Wu , Minhui Wang , Yaqin Wang , Ping Wu , Xiaocui Xia , Lin Zhang , Yamin Liu , Zhenyue Gao , Jie Shen , Yuanwei Jia
Eldecalcitol is a novel analog of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] for the treatment of patients with osteoporosis. A highly sensitive and specific ultra-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (UPLC-APCI-MS/MS) technique featuring a lower limit of quantitation (LLOQ) as low as 5 pg/ml, has been established and validated for the rapid and accurate quantification of eldecalcitol in human plasma samples. Plasma samples were extracted by solid phase extraction (SPE). Stable isotope-labeled compound eldecalcitol-d6 was used as an internal standard (SIL-IS). The detection process was carried out utilizing Multi-Reaction Monitoring (MRM) mode, employing an atmospheric pressure chemical ionization (APCI) source operating in the positive ion modality. The target fragment ion pairs for eldecalcitol and SIL-IS were identified as m/z 508.6 transitioning to 397.4, and m/z 514.6 transitioning to 403.3, respectively. This method was validated regarding selectivity, LLOQ, linearity, accuracy and precision, recovery, matrix effects, dilution reliability, stability, carryover test, and incurred sample reanalysis (ISR). This methodology had been successfully employed to assess the pharmacokinetics (PK) profile of eldecalcitol in a clinical investigation.
{"title":"Determination of eldecalcitol in human plasma by SIL-IS UPLC-APCI-MS/MS method for pharmacokinetics study","authors":"Wanyu Wang , Zhichao Yin , Pengfei Du , Jianbang Wu , Minhui Wang , Yaqin Wang , Ping Wu , Xiaocui Xia , Lin Zhang , Yamin Liu , Zhenyue Gao , Jie Shen , Yuanwei Jia","doi":"10.1016/j.jchromb.2025.124468","DOIUrl":"10.1016/j.jchromb.2025.124468","url":null,"abstract":"<div><div>Eldecalcitol is a novel analog of 1α,25-dihydroxyvitamin D<sub>3</sub> [1,25(OH)<sub>2</sub>D<sub>3</sub>] for the treatment of patients with osteoporosis. A highly sensitive and specific ultra-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (UPLC-APCI-MS/MS) technique featuring a lower limit of quantitation (LLOQ) as low as 5 pg/ml, has been established and validated for the rapid and accurate quantification of eldecalcitol in human plasma samples. Plasma samples were extracted by solid phase extraction (SPE). Stable isotope-labeled compound eldecalcitol-d6 was used as an internal standard (SIL-IS). The detection process was carried out utilizing Multi-Reaction Monitoring (MRM) mode, employing an atmospheric pressure chemical ionization (APCI) source operating in the positive ion modality. The target fragment ion pairs for eldecalcitol and SIL-IS were identified as <em>m</em>/<em>z</em> 508.6 transitioning to 397.4, and <em>m</em>/<em>z</em> 514.6 transitioning to 403.3, respectively. This method was validated regarding selectivity, LLOQ, linearity, accuracy and precision, recovery, matrix effects, dilution reliability, stability, carryover test, and incurred sample reanalysis (ISR). This methodology had been successfully employed to assess the pharmacokinetics (PK) profile of eldecalcitol in a clinical investigation.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124468"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142997686","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}
Human exposure to volatile organic compounds (VOCs) poses significant health risks, contributing to cardiovascular disease, pulmonary disease, and cancer. Measurement of VOC metabolites (VOCm) in urine by liquid chromatography-mass spectrometry (LC-MS) is a preferred method for VOCm analysis; however, existing methods encounter challenges related to sensitivity, throughput, and analyte coverage. In addition to VOCm, the measurement of tobacco alkaloids (TAm) is critical to account for tobacco use in population-based studies. A method is needed that is highly sensitive, offers higher throughput, and can analyze VOCm and TAm in a single run. Herein, we present a robust dilute-and-shoot method aimed at overcoming these analytical challenges and expanding the targeted analysis to include 35 urinary VOCm and TAm and their metabolites. By leveraging high-speed polarity switching and optimized chromatographic parameters, our method achieved comprehensive analyte coverage and enhanced sensitivity, enabling reliable individual level VOC exposure assessment. Validation demonstrates robust linearity, sensitivity, accuracy, and precision, with minimal matrix effects. This high-throughput UPLC-MS/MS method significantly enhances VOC exposure assessment by enabling simultaneous measurement of 35 urinary VOC and TAm with high sensitivity and efficiency. Multiple metabolites from single parent xenobiotics are included in one run, expanding biomarker specificity. Our data indicate the method effectively accounts for tobacco consumption as a confounder in population-based studies, ensuring accurate VOC exposure assessment.
{"title":"High-throughput UPLC-ESI/MSMS method for simultaneous measurement of the urinary metabolites of volatile organic compounds and tobacco alkaloids","authors":"Shweta Srivastava , Tatiana Krivokhizhina , Rachel Keith , Aruni Bhatnagar , Sanjay Srivastava , Zhengzhi Xie , Pawel Lorkiewicz","doi":"10.1016/j.jchromb.2025.124463","DOIUrl":"10.1016/j.jchromb.2025.124463","url":null,"abstract":"<div><div>Human exposure to volatile organic compounds (VOCs) poses significant health risks, contributing to cardiovascular disease, pulmonary disease, and cancer. Measurement of VOC metabolites (VOCm) in urine by liquid chromatography-mass spectrometry (LC-MS) is a preferred method for VOCm analysis; however, existing methods encounter challenges related to sensitivity, throughput, and analyte coverage. In addition to VOCm, the measurement of tobacco alkaloids (TAm) is critical to account for tobacco use in population-based studies. A method is needed that is highly sensitive, offers higher throughput, and can analyze VOCm and TAm in a single run. Herein, we present a robust dilute-and-shoot method aimed at overcoming these analytical challenges and expanding the targeted analysis to include 35 urinary VOCm and TAm and their metabolites. By leveraging high-speed polarity switching and optimized chromatographic parameters, our method achieved comprehensive analyte coverage and enhanced sensitivity, enabling reliable individual level VOC exposure assessment. Validation demonstrates robust linearity, sensitivity, accuracy, and precision, with minimal matrix effects. This high-throughput UPLC-MS/MS method significantly enhances VOC exposure assessment by enabling simultaneous measurement of 35 urinary VOC and TAm with high sensitivity and efficiency. Multiple metabolites from single parent xenobiotics are included in one run, expanding biomarker specificity. Our data indicate the method effectively accounts for tobacco consumption as a confounder in population-based studies, ensuring accurate VOC exposure assessment.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124463"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142997758","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 : 2025-02-01DOI: 10.1016/j.jchromb.2024.124435
Yan-Long Zhang , Yu-Jun Shi , Bao-Jian Duan , Xian-Hua Wang
For separation of deoxyribonucleic acid (DNA), positively charged amino-modified magnetic nanoparticles (MN) can effectively adsorb negatively charged DNA through electrostatic interaction. However, the reported preparation of amino-modified MN is usually tedious and time-consuming. Therefore, a simple synthesis method of amino-modified MN is necessary for DNA extraction. Herein, a novel polyethyleneimine-coated MN (PMN) was fabricated by one-pot hydrothermal synthesis for high-efficient DNA extraction. The fabricated PMN showed numerous exposed amino groups, which not only could effectively capture DNA through electrostatic interaction, but also limited the aggregation of PMN during application. Under optimized adsorption conditions, the maximum adsorption capacity of PMN for DNA could reach 192.4 μg m g−1. Shigella flexneri (S. flexneri) has the highest mortality rate among Shigella species and has been selected as target model pathogenic bacteria. Based on the optimized extraction conditions, PMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated for detection of S. flexneri. The limit of detection of the proposed strategy was 2.4 × 102 CFU mL−1 and obviously lower than the commercial kit. To prove the practicability, the PMN-based MSPE combined qPCR strategy was successfully used in the determination of S. flexneri in spiked real sample, and the recovery values were in the range of 95.1 % to 102.1 % for apple juice, 60.4 % to 85.7 % for pickled vegetable, 97.8 % to 99.5 % for pig liver and 95.1 % to 102.1 % for pig colon, respectively. Therefore, we believe that the resultant PMN have great potential to become a universal magnetic adsorbent for high-efficient DNA extraction from complex biological samples.
{"title":"One-pot hydrothermal synthesis of polyethyleneimine-coated magnetic nanoparticles for high-efficient DNA extraction of pathogenic bacteria","authors":"Yan-Long Zhang , Yu-Jun Shi , Bao-Jian Duan , Xian-Hua Wang","doi":"10.1016/j.jchromb.2024.124435","DOIUrl":"10.1016/j.jchromb.2024.124435","url":null,"abstract":"<div><div>For separation of deoxyribonucleic acid (DNA), positively charged amino-modified magnetic nanoparticles (MN) can effectively adsorb negatively charged DNA through electrostatic interaction. However, the reported preparation of amino-modified MN is usually tedious and time-consuming. Therefore, a simple synthesis method of amino-modified MN is necessary for DNA extraction. Herein, a novel polyethyleneimine-coated MN (PMN) was fabricated by one-pot hydrothermal synthesis for high-efficient DNA extraction. The fabricated PMN showed numerous exposed amino groups, which not only could effectively capture DNA through electrostatic interaction, but also limited the aggregation of PMN during application. Under optimized adsorption conditions, the maximum adsorption capacity of PMN for DNA could reach 192.4 μg m g<sup>−1</sup>. <em>Shigella flexneri</em> (<em>S</em>. <em>flexneri</em>) has the highest mortality rate among <em>Shigella</em> species and has been selected as target model pathogenic bacteria. Based on the optimized extraction conditions, PMN-based magnetic solid-phase microextraction (MSPE) and quantitative real-time PCR (qPCR) were integrated for detection of <em>S</em>. <em>flexneri</em>. The limit of detection of the proposed strategy was 2.4 × 10<sup>2</sup> CFU mL<sup>−1</sup> and obviously lower than the commercial kit. To prove the practicability, the PMN-based MSPE combined qPCR strategy was successfully used in the determination of <em>S</em>. <em>flexneri</em> in spiked real sample, and the recovery values were in the range of 95.1 % to 102.1 % for apple juice, 60.4 % to 85.7 % for pickled vegetable, 97.8 % to 99.5 % for pig liver and 95.1 % to 102.1 % for pig colon, respectively. Therefore, we believe that the resultant PMN have great potential to become a universal magnetic adsorbent for high-efficient DNA extraction from complex biological samples.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1252 ","pages":"Article 124435"},"PeriodicalIF":2.8,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142918941","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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