Pub Date : 2025-01-14DOI: 10.1016/j.jpba.2025.116678
Haiwei Cao , Yuting Jin , Yingwu Wang , Hao Wang , Yijia Qin , Xinhua Guo , Suyan Tian , Jing Huang , Yanyan Li
Accurate measurement of serum parathyroid hormone (PTH) is crucial for diagnosing and managing endocrine and osteological diseases. Conventional immunoassay methods struggle with cross reactivity issues between full-length PTH and truncated fragments or post-translationally modified forms. Both the standardization of PTH assays and the peptide's stability are concerning. This study addresses these issues by establishing an immunocapture coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to precisely quantify PTH1–84. PTH1–84 was isolated from one mL serum samples by immunocapture on a polystyrene bead and eluted from matrix, then quantitated by LC–MS/MS. The results from 268 serum samples were compared to an intact PTH immunoassay. The assay's linear range was 5.0–1000.0 pg/mL. The intra-assay coefficients of variation (CVs) ranged from 3.2 % to 6.8 %, and the inter-assay CVs ranged from 4.6 % to 9.5 %. The extraction efficiencies were 98.0 %–100.5 %, with no significant matrix effects observed after internal standard correction. The correlation coefficient between LC–MS/MS and immunoassay was 0.989, but the bias between the methods was substantial. Nevertheless, the immunocapture purification coupled LC–MS/MS method offers a promising approach for accurate PTH measurement.
{"title":"Quantification and clinical performance of serum parathyroid hormone 1–84 via immunocapture coupled to LC–MS/MS in chronic renal failure","authors":"Haiwei Cao , Yuting Jin , Yingwu Wang , Hao Wang , Yijia Qin , Xinhua Guo , Suyan Tian , Jing Huang , Yanyan Li","doi":"10.1016/j.jpba.2025.116678","DOIUrl":"10.1016/j.jpba.2025.116678","url":null,"abstract":"<div><div>Accurate measurement of serum parathyroid hormone (PTH) is crucial for diagnosing and managing endocrine and osteological diseases. Conventional immunoassay methods struggle with cross reactivity issues between full-length PTH and truncated fragments or post-translationally modified forms. Both the standardization of PTH assays and the peptide's stability are concerning. This study addresses these issues by establishing an immunocapture coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to precisely quantify PTH1–84. PTH1–84 was isolated from one mL serum samples by immunocapture on a polystyrene bead and eluted from matrix, then quantitated by LC–MS/MS. The results from 268 serum samples were compared to an intact PTH immunoassay. The assay's linear range was 5.0–1000.0 pg/mL. The intra-assay coefficients of variation (CVs) ranged from 3.2 % to 6.8 %, and the inter-assay CVs ranged from 4.6 % to 9.5 %. The extraction efficiencies were 98.0 %–100.5 %, with no significant matrix effects observed after internal standard correction. The correlation coefficient between LC–MS/MS and immunoassay was 0.989, but the bias between the methods was substantial. Nevertheless, the immunocapture purification coupled LC–MS/MS method offers a promising approach for accurate PTH measurement.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"256 ","pages":"Article 116678"},"PeriodicalIF":3.1,"publicationDate":"2025-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143023505","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 : 2025-01-13DOI: 10.1016/j.jpba.2025.116677
Meiting Lin , Zhen Zhang , Qun He , Hongyuan Hao , Ping Xiang , Junbo Zhao
Etomidate and its structural analogues, which have anesthetic effects, are classified as controlled psychotropic drugs. Electronic cigarettes (e-cigarettes) have become more and more popular. With the increase of adding etomidate and its analogues to electronic liquids (e-liquids), there is a trend of abuse, which is a tough problem urgently need to be solved. This seriously affects the health and security of the public and the development of society. A simple, rapid and effective screening method is very crucial for their identification. In this study, we applied a newly developed method, probe electrospray ionization quadrupole time-of-flight mass spectrometry (PESI-QTOF-MS) with DPiMS QT ion source to analyze etomidate and its analogues in e-liquids. It allowed identification in 0.3 min with lower sample usage. Isomers can be distinguished by ion abundance ratios at collision energy (CE) 15 eV, which provided possibility for distinguishing more isomers by in-situ mass spectrometry. Limit of detection (LOD) and limit of quantitation (LOQ) of four substances were 20 ng/mL and 50 ng/mL, respectively. Good linear relationships were obtained in the concentration range of 50–5000 ng/mL with little matrix effect. The accuracy, precision, dilution effect and carryover of the method were also validated. Positive specimens (n = 38) were analyzed by both PESI-QTOF-MS and gas chromatography-mass spectrometry (GC-MS). There were five impurities including nicotine, cooling agent and flavorings were investigated by PESI-QTOF-MS, which provided the possibility for tracing the origin of illegal e-liquids. This study will help solve the backlog of cases and improve work efficiency effectively by reducing analysis time. Furthermore, it meets the need of addressing current situation of drug control and can assist forensic laboratories in investigating cases. It also demonstrates the application prospects of rapid screening in new drugs.
{"title":"Rapid determination of etomidate and its structural analogues in e-liquid by probe electrospray ionization quadrupole time-of-flight mass spectrometry","authors":"Meiting Lin , Zhen Zhang , Qun He , Hongyuan Hao , Ping Xiang , Junbo Zhao","doi":"10.1016/j.jpba.2025.116677","DOIUrl":"10.1016/j.jpba.2025.116677","url":null,"abstract":"<div><div>Etomidate and its structural analogues, which have anesthetic effects, are classified as controlled psychotropic drugs. Electronic cigarettes (e-cigarettes) have become more and more popular. With the increase of adding etomidate and its analogues to electronic liquids (e-liquids), there is a trend of abuse, which is a tough problem urgently need to be solved. This seriously affects the health and security of the public and the development of society. A simple, rapid and effective screening method is very crucial for their identification. In this study, we applied a newly developed method, probe electrospray ionization quadrupole time-of-flight mass spectrometry (PESI-QTOF-MS) with DPiMS QT ion source to analyze etomidate and its analogues in e-liquids. It allowed identification in 0.3 min with lower sample usage. Isomers can be distinguished by ion abundance ratios at collision energy (CE) 15 eV, which provided possibility for distinguishing more isomers by in-situ mass spectrometry. Limit of detection (LOD) and limit of quantitation (LOQ) of four substances were 20 ng/mL and 50 ng/mL, respectively. Good linear relationships were obtained in the concentration range of 50–5000 ng/mL with little matrix effect. The accuracy, precision, dilution effect and carryover of the method were also validated. Positive specimens (n = 38) were analyzed by both PESI-QTOF-MS and gas chromatography-mass spectrometry (GC-MS). There were five impurities including nicotine, cooling agent and flavorings were investigated by PESI-QTOF-MS, which provided the possibility for tracing the origin of illegal e-liquids. This study will help solve the backlog of cases and improve work efficiency effectively by reducing analysis time. Furthermore, it meets the need of addressing current situation of drug control and can assist forensic laboratories in investigating cases. It also demonstrates the application prospects of rapid screening in new drugs.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"256 ","pages":"Article 116677"},"PeriodicalIF":3.1,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006831","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 study aimed to determine the chromatographic retention and dissociation/protonation constant (pKa) values of lapatinib and tamoxifen, key drugs used in metastatic breast cancer treatment, at 37°C using both conventional and green high-performance liquid chromatography (HPLC) methods. Qualitative analysis was conducted on an XTerra C18 column (250 ×4.6 mm I.D., 5 μm particle size) at a flow rate of 1 mL/min. Hydroorganic mixtures with 45 %, 50 %, 55 %, and 60 % (v/v) organic modifiers were used to evaluate the retention times of the compounds. The compatibility of pKa values of the compounds in water-organic solvent mixtures obtained from these studies, which were carried out without any significant change in liquid chromatography performance, with the values obtained by the conventional method is remarkable. The values determined in this study were correlated with the macroscopic parameters of acetonitrile, methanol, ethanol and the pKa (values of lapatinib and tamoxifen in water were calculated. The values calculated from these studies are compatible with each other and with the literature values. The environmental impact of the study, which was carried out using the green method and the conventional RPLC method, was evaluated using the Green Solvent Selection Tool (GSST), Green Analytical Procedures Index (GAPI), and Analytical Greenness Metric Approach (AGREE).
{"title":"Chromatographic analysis and pKa evaluation of active pharmaceutical ingredients in anti-metastatic breast cancer: Green vs. conventional RPLC","authors":"Fatmanur Kalkir , Ebru Çubuk Demi̇ralay , Yaşar Doğan Daldal , Hülya Yilmaz","doi":"10.1016/j.jpba.2025.116671","DOIUrl":"10.1016/j.jpba.2025.116671","url":null,"abstract":"<div><div>This study aimed to determine the chromatographic retention and dissociation/protonation constant (pK<sub>a</sub>) values of lapatinib and tamoxifen, key drugs used in metastatic breast cancer treatment, at 37°C using both conventional and green high-performance liquid chromatography (HPLC) methods. Qualitative analysis was conducted on an XTerra C18 column (250 ×4.6 mm I.D., 5 μm particle size) at a flow rate of 1 mL/min. Hydroorganic mixtures with 45 %, 50 %, 55 %, and 60 % (v/v) organic modifiers were used to evaluate the retention times of the compounds. The compatibility of pK<sub>a</sub> values <span><math><mrow><mfenced><mrow><mmultiscripts><mrow><mi>pK</mi></mrow><mrow><mi>a</mi></mrow><none></none><mprescripts></mprescripts><mrow><mi>s</mi></mrow><mrow><mi>s</mi></mrow></mmultiscripts></mrow></mfenced></mrow></math></span> of the compounds in water-organic solvent mixtures obtained from these studies, which were carried out without any significant change in liquid chromatography performance, with the values obtained by the conventional method is remarkable. The <span><math><mmultiscripts><mrow><mi>pK</mi></mrow><mrow><mi>a</mi></mrow><none></none><mprescripts></mprescripts><mrow><mi>s</mi></mrow><mrow><mi>s</mi></mrow></mmultiscripts></math></span> values determined in this study were correlated with the macroscopic parameters of acetonitrile, methanol, ethanol and the pK<sub>a</sub> (<span><math><mrow><mmultiscripts><mrow><mi>pK</mi></mrow><mrow><mi>a</mi></mrow><none></none><mprescripts></mprescripts><mrow><mi>w</mi></mrow><mrow><mi>w</mi></mrow></mmultiscripts><mrow><mo>)</mo><mspace></mspace></mrow></mrow></math></span>values of lapatinib and tamoxifen in water were calculated. The <span><math><mmultiscripts><mrow><mi>pK</mi></mrow><mrow><mi>a</mi></mrow><none></none><mprescripts></mprescripts><mrow><mi>w</mi></mrow><mrow><mi>w</mi></mrow></mmultiscripts></math></span> values calculated from these studies are compatible with each other and with the literature values. The environmental impact of the study, which was carried out using the green method and the conventional RPLC method, was evaluated using the Green Solvent Selection Tool (GSST), Green Analytical Procedures Index (GAPI), and Analytical Greenness Metric Approach (AGREE).</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"256 ","pages":"Article 116671"},"PeriodicalIF":3.1,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143007221","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 : 2025-01-09DOI: 10.1016/j.jpba.2025.116670
Hanyang Wang , Yue Du , Chunwei Zhou , Yan Wang , Xin Wang
Radix Rehmanniae (RR) is a widely used herb in traditional Chinese Medicine with properties of tonifying the kidneys and nourishing the blood. Both raw and processed RR are effective for the treatment of diabetes in clinical practice. Oligosaccharides and iridoid glycosides are the primary active components responsible for the anti-diabetic effects of RR. In this study, a rapid and sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC−MS/MS) method was developed for simultaneous determination of oligosaccharides (raffinose, manninotriose and stachyose) and iridoid glycosides (catalpol and ajugol) in rat plasma. Significant analytical challenges were encountered during method development, including distinct retention behaviors of oligosaccharides and iridoid glycosides, low ionization and extraction efficiency of oligosaccharides, thermal instability of catalpol and reduced column performance. The strategies to overcome these challenges were presented by optimizing chromatographic separation, mass spectrometric detection and sample preparation. The best separation was achieved using an Accucore-150-Amide-HILIC column (100 mm × 2.1 mm, 2.6 μm) at 50 °C with mobile phase consisted of acetonitrile and ammonium acetate (2.5 mM) under gradient elution. Ammonium adduct ions produced by positive electrospray ionization were chosen as precursor ions for multiple reaction monitoring transitions. The established HILIC−MS/MS method exhibited good linearity (r > 0.9937) with the lower limits of quantification of 0.01–0.2 μg/mL using only 50 µL of plasma sample. The method was successfully applied to pharmacokinetic characterization of oligosaccharides and iridoid glycosides in normal and type 2 diabetic rats following intragastric administration of raw and processed RR extracts.
地黄（RR）是一种广泛使用的中药，具有补肾养血的特性。在临床实践中，生的和加工的红霉素对糖尿病的治疗都是有效的。低聚糖和环烯醚萜苷是RR抗糖尿病作用的主要活性成分。本研究建立了快速、灵敏的亲水相互作用液相色谱-串联质谱（HILIC-MS/MS）同时测定大鼠血浆中低聚糖（棉子糖、甘露糖和水苏糖）和环烯醚萜苷（catalpol和ajugol）的方法。在方法开发过程中遇到了重大的分析挑战，包括低聚糖和环烯醚萜苷的独特保留行为，低聚糖的低电离和提取效率，catalpol的热不稳定性以及色谱柱性能降低。通过优化色谱分离、质谱检测和样品制备，提出了克服这些挑战的策略。Accucore-150-Amide-HILIC色谱柱（100 mm × 2.1 mm, 2.6 μm），温度为50 °C，流动相为乙腈和乙酸铵（2.5 mm），梯度洗脱，分离效果最佳。采用正电喷雾电离产生的铵加合物离子作为前体离子，用于多反应监测跃迁。所建立的HILIC-MS/MS方法具有良好的线性关系（r > 0.9937），定量下限为0.01 ~ 0.2 μg/mL，仅需50 µL血浆样品。该方法成功地应用于正常和2型糖尿病大鼠灌胃RR提取物后低聚糖和环烯醚萜苷的药动学表征。
{"title":"Hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of oligosaccharides and iridoid glycosides in rat plasma: Pharmacokinetic characterization of previously overlooked oligosaccharides from Radix Rehmanniae","authors":"Hanyang Wang , Yue Du , Chunwei Zhou , Yan Wang , Xin Wang","doi":"10.1016/j.jpba.2025.116670","DOIUrl":"10.1016/j.jpba.2025.116670","url":null,"abstract":"<div><div>Radix Rehmanniae (RR) is a widely used herb in traditional Chinese Medicine with properties of tonifying the kidneys and nourishing the blood. Both raw and processed RR are effective for the treatment of diabetes in clinical practice. Oligosaccharides and iridoid glycosides are the primary active components responsible for the anti-diabetic effects of RR. In this study, a rapid and sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC−MS/MS) method was developed for simultaneous determination of oligosaccharides (raffinose, manninotriose and stachyose) and iridoid glycosides (catalpol and ajugol) in rat plasma. Significant analytical challenges were encountered during method development, including distinct retention behaviors of oligosaccharides and iridoid glycosides, low ionization and extraction efficiency of oligosaccharides, thermal instability of catalpol and reduced column performance. The strategies to overcome these challenges were presented by optimizing chromatographic separation, mass spectrometric detection and sample preparation. The best separation was achieved using an Accucore-150-Amide-HILIC column (100 mm × 2.1 mm, 2.6 μm) at 50 °C with mobile phase consisted of acetonitrile and ammonium acetate (2.5 mM) under gradient elution. Ammonium adduct ions produced by positive electrospray ionization were chosen as precursor ions for multiple reaction monitoring transitions. The established HILIC−MS/MS method exhibited good linearity (<em>r</em> > 0.9937) with the lower limits of quantification of 0.01–0.2 μg/mL using only 50 µL of plasma sample. The method was successfully applied to pharmacokinetic characterization of oligosaccharides and iridoid glycosides in normal and type 2 diabetic rats following intragastric administration of raw and processed RR extracts.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"256 ","pages":"Article 116670"},"PeriodicalIF":3.1,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143006647","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 : 2025-01-08DOI: 10.1016/j.jpba.2025.116669
Yangzom Dawa , Yong-Chen Hua , Fang-Di Hu , Juan Chen
Acetylcholinesterase (AChE) is widely recognized as a promising therapeutic target enzyme for Alzheimer's disease (AD). The screening of AChE inhibitors (AChEIs) holds great significance for the treatment of AD. In this study, cellulose filter paper (CFP) -immobilized AChE was prepared and firstly applied to screening AChEIs from 30 % ethanol extract of Phyllanthus emblica L. fruits combined with ultra-high performance liquid chromatography quadrupole time-of-fight mass spectrometry (UHPLC-Q-TOF-MS/MS). Using CFP-immobilized AChE as the bait, AChEIs were harvested and the instantaneous separation characteristics of CFP were utilized to further facilitate the separation of the complex from the inactive components. Ultimately, 27 compounds specifically bound with AChE were screened and identified using UHPLC-Q-TOF-MS/MS. Additionally, molecular docking was employed to explore the binding mechanisms between screened potential inhibitors and AChE. The results show that, most of the screened compounds were found to exhibit higher affinity that of the positive control (huperzine A), and all the compounds expect mucic acid to be well embedded into the active pocket of AChE. To verify the reliability of the screening method and molecular docking, two commercial standards geraniin and ellagic acid were experimented with an AChE inhibition assay in vitro. The results showed that both compounds were found to effectively inhibit AChE with IC50 values of 42.42 ± 7.10 μM, 172.43 ± 9.22 μM. The developed method exhibits the advantages of rapidness and effectiveness in screening of AChEIs from complex herbal extracts.
{"title":"Cellulose filter paper immobilized acetylcholinesterase for rapid screening of enzyme inhibitors in Phyllanthus emblica L.","authors":"Yangzom Dawa , Yong-Chen Hua , Fang-Di Hu , Juan Chen","doi":"10.1016/j.jpba.2025.116669","DOIUrl":"10.1016/j.jpba.2025.116669","url":null,"abstract":"<div><div>Acetylcholinesterase (AChE) is widely recognized as a promising therapeutic target enzyme for Alzheimer's disease (AD). The screening of AChE inhibitors (AChEIs) holds great significance for the treatment of AD. In this study, cellulose filter paper (CFP) -immobilized AChE was prepared and firstly applied to screening AChEIs from 30 % ethanol extract of <em>Phyllanthus emblica L.</em> fruits combined with ultra-high performance liquid chromatography quadrupole time-of-fight mass spectrometry (UHPLC-Q-TOF-MS/MS). Using CFP-immobilized AChE as the bait, AChEIs were harvested and the instantaneous separation characteristics of CFP were utilized to further facilitate the separation of the complex from the inactive components. Ultimately, 27 compounds specifically bound with AChE were screened and identified using UHPLC-Q-TOF-MS/MS. Additionally, molecular docking was employed to explore the binding mechanisms between screened potential inhibitors and AChE. The results show that, most of the screened compounds were found to exhibit higher affinity that of the positive control (huperzine A), and all the compounds expect mucic acid to be well embedded into the active pocket of AChE. To verify the reliability of the screening method and molecular docking, two commercial standards geraniin and ellagic acid were experimented with an AChE inhibition assay in <em>vitro</em>. The results showed that both compounds were found to effectively inhibit AChE with IC<sub>50</sub> values of 42.42 ± 7.10 μM, 172.43 ± 9.22 μM. The developed method exhibits the advantages of rapidness and effectiveness in screening of AChEIs from complex herbal extracts.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"256 ","pages":"Article 116669"},"PeriodicalIF":3.1,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143007233","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 : 2025-01-08DOI: 10.1016/j.jpba.2025.116673
Jianhui Cheng , Qianchuan Lv , Yuanzhao Ji , Chunling Zhou , Jifen Guo , Xinxin Li , Jianzhong Hu
Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is widely used in the biopharmaceutical industry for monitoring purity and analyzing impurities. The accuracy of the method may be compromised by artificial species resulting from sample preparation or electrophoresis separation due to suboptimal conditions. During non-reduced CE-SDS analysis of a multispecific antibody (msAb), named as multispecific antibody C (msAb-C), a cluster of unexpected peaks was observed after the main peak. The corrected peak area ratio of these peaks showed a strong dependence on loaded protein concentration, which affected the accurate assessment of the purity of msAb-C. After investigation, the unexpected peaks were identified as artifacts produced during electrophoresis separation. These artifacts can be mitigated by three different strategies: 1) adding a more hydrophobic surfactant, sodium hexadecyl sulfate (SHS), to the sample and/or sieving gel buffer; 2) reducing the sample loading amount; and 3) increasing the capillary separation temperature to above 40 ℃. We adopted strategy 1) and strategy 3), and successfully developed an optimal non-reduced CE-SDS method for the accurate and reliable purity assessment of msAb-C samples. These strategies of optimizing non-reduced CE-SDS can be used in developing quality control methods for other therapeutic bispecific/multispecific antibodies.
{"title":"Investigation and elimination of noncovalent artificial aggregates during non-reduced capillary electrophoresis-sodium dodecyl sulfate analysis of a multi-specific antibody","authors":"Jianhui Cheng , Qianchuan Lv , Yuanzhao Ji , Chunling Zhou , Jifen Guo , Xinxin Li , Jianzhong Hu","doi":"10.1016/j.jpba.2025.116673","DOIUrl":"10.1016/j.jpba.2025.116673","url":null,"abstract":"<div><div>Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is widely used in the biopharmaceutical industry for monitoring purity and analyzing impurities. The accuracy of the method may be compromised by artificial species resulting from sample preparation or electrophoresis separation due to suboptimal conditions. During non-reduced CE-SDS analysis of a multispecific antibody (msAb), named as multispecific antibody C (msAb-C), a cluster of unexpected peaks was observed after the main peak. The corrected peak area ratio of these peaks showed a strong dependence on loaded protein concentration, which affected the accurate assessment of the purity of msAb-C. After investigation, the unexpected peaks were identified as artifacts produced during electrophoresis separation. These artifacts can be mitigated by three different strategies: 1) adding a more hydrophobic surfactant, sodium hexadecyl sulfate (SHS), to the sample and/or sieving gel buffer; 2) reducing the sample loading amount; and 3) increasing the capillary separation temperature to above 40 ℃. We adopted strategy 1) and strategy 3), and successfully developed an optimal non-reduced CE-SDS method for the accurate and reliable purity assessment of msAb-C samples. These strategies of optimizing non-reduced CE-SDS can be used in developing quality control methods for other therapeutic bispecific/multispecific antibodies.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"255 ","pages":"Article 116673"},"PeriodicalIF":3.1,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142983426","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 : 2025-01-08DOI: 10.1016/j.jpba.2025.116672
Junhuan Lin , Yibo Song , Yangrui Zhang , Tao Ke , Fengting Ou , Kui Zeng , Debo He , Li Li , Lushan Yu
A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 L-amino acids (AAs) in human plasma. Chromatographic separation was achieved on an Agilent AdvanceBio Hilic column within 15 min via gradient elution with an aqueous solution containing 5 mM ammonium formate, 5 mM ammonium acetate and 0.1 % formic acid and an organic mobile phase containing 0.1 % formic acid, 5 mM ammonium formate and 5 mM ammonium acetate acetonitrile-water (90:10, v/v) at the flow rate of 0.25 mL/min. Individual AAs and internal standard were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this LC-MS/MS method as a specific, accurate, and reliable assay. The method was thus utilized to compare the dynamics of individual plasma AAs between healthy females and patients with ovarian tumors. Our results revealed that, in cancer group, plasma 3-Methyl-L-Histidine, L-Proline, L-Phenylalanine and L-Lysine concentrations were significantly increased in patients with malignant ovarian tumors while L-Leucine and L-Isoleucine levels were sharply decreased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for ovarian cancer.
{"title":"A reliable LC-MS/MS method for the quantification of natural amino acids in human plasma and its application in clinic","authors":"Junhuan Lin , Yibo Song , Yangrui Zhang , Tao Ke , Fengting Ou , Kui Zeng , Debo He , Li Li , Lushan Yu","doi":"10.1016/j.jpba.2025.116672","DOIUrl":"10.1016/j.jpba.2025.116672","url":null,"abstract":"<div><div>A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 L-amino acids (AAs) in human plasma. Chromatographic separation was achieved on an Agilent AdvanceBio Hilic column within 15 min via gradient elution with an aqueous solution containing 5 mM ammonium formate, 5 mM ammonium acetate and 0.1 % formic acid and an organic mobile phase containing 0.1 % formic acid, 5 mM ammonium formate and 5 mM ammonium acetate acetonitrile-water (90:10, v/v) at the flow rate of 0.25 mL/min. Individual AAs and internal standard were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this LC-MS/MS method as a specific, accurate, and reliable assay. The method was thus utilized to compare the dynamics of individual plasma AAs between healthy females and patients with ovarian tumors. Our results revealed that, in cancer group, plasma 3-Methyl-L-Histidine, L-Proline, L-Phenylalanine and L-Lysine concentrations were significantly increased in patients with malignant ovarian tumors while L-Leucine and L-Isoleucine levels were sharply decreased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for ovarian cancer.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"256 ","pages":"Article 116672"},"PeriodicalIF":3.1,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143007232","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 : 2025-01-06DOI: 10.1016/j.jpba.2025.116667
Zhiyong Wu , Xinhao Wang , Lin Wang , Na Sun , Zihui Yang , Jianguo Zeng
Allocryptopine (ALL), a principal active component of the novel veterinary medicine Bopu Powder®, has gained widespread application in the poultry farming sector for the effective management of Escherichia coli (E. coli) diarrhea. In order to explore the metabolites and the pivotal enzymes associated with ALL, this study was conducted employing an in vitro chicken liver microsomal incubation. The metabolites of ALL were analyzed and identified by combining isotope tracing technology with the application of mass spectrometry fragmentation patterns. The key metabolic enzymes involved in the biotransformation of ALL were explored using the CYP450 recombinant enzyme method, which facilitated the identification of the enzymes contributing to ALL's metabolic pathway. The liver microsomal metabolism investigation revealed a total of five metabolites, with the predominant being M2 (harmol or 3-hydroxy-4-methoxy-6-methyl-5,7,8,15-tetrahydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-g]benzo[c]azecin-14(6 H)-one). The recombinant enzyme analysis conclusively identified CYP2D6 as the pivotal CYP450 isoenzyme that plays a central role in the metabolic pathway of the principal ALL metabolite, M2. This research not only expands our comprehension of the biotransformation process of ALL but also provides significant scientific evidence for the clinical safety of ALL, which was of great importance for guiding the application of ALL in the field of veterinary medicine.
{"title":"Research on the metabolites and key metabolic enzymes of allocryptopine in chicken liver microsomes via stable isotope tracing technology","authors":"Zhiyong Wu , Xinhao Wang , Lin Wang , Na Sun , Zihui Yang , Jianguo Zeng","doi":"10.1016/j.jpba.2025.116667","DOIUrl":"10.1016/j.jpba.2025.116667","url":null,"abstract":"<div><div>Allocryptopine (ALL), a principal active component of the novel veterinary medicine Bopu Powder®, has gained widespread application in the poultry farming sector for the effective management of <em>Escherichia coli</em> (<em>E. coli</em>) diarrhea. In order to explore the metabolites and the pivotal enzymes associated with ALL, this study was conducted employing an in vitro chicken liver microsomal incubation. The metabolites of ALL were analyzed and identified by combining isotope tracing technology with the application of mass spectrometry fragmentation patterns. The key metabolic enzymes involved in the biotransformation of ALL were explored using the CYP450 recombinant enzyme method, which facilitated the identification of the enzymes contributing to ALL's metabolic pathway. The liver microsomal metabolism investigation revealed a total of five metabolites, with the predominant being M2 (harmol or 3-hydroxy-4-methoxy-6-methyl-5,7,8,15-tetrahydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-<em>g</em>]benzo[<em>c</em>]azecin-14(6 H)-one). The recombinant enzyme analysis conclusively identified CYP2D6 as the pivotal CYP450 isoenzyme that plays a central role in the metabolic pathway of the principal ALL metabolite, M2. This research not only expands our comprehension of the biotransformation process of ALL but also provides significant scientific evidence for the clinical safety of ALL, which was of great importance for guiding the application of ALL in the field of veterinary medicine.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"255 ","pages":"Article 116667"},"PeriodicalIF":3.1,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142978775","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}
Dendrobine is a sesquiterpene alkaloid primarily used in the treatment of inflammatory diseases, immune system disorders, and conditions related to oxidative stress. To understand the possible degradation pathways of dendrobine for its quality control, we conducted an in-depth investigation of its degradation products using forced degradation methods. The separation of dendrobine and its degradation products was achieved on a Shim-pack XR-ODS III (75 mm × 2 mm, 1.6 µm) column with a methanol-water mixture as the mobile phase under isocratic conditions, the isolated compounds were examined in positive ion mode with an ion trap-time of flight mass spectrometer (IT-TOF). In order to obtain in-depth structural information about the degradation products, mass spectrometry was performed using a five-stage fragmentation approach. This method allowed for thorough structural clarification via several rounds of selective fragmentation and high-resolution detection. System control and data acquisition were managed using LCMSsolution 3.81 software. The results showed that dendrobine undergoes significant degradation under oxidative, acidic, hydrolytic and thermal conditions, resulting in the formation of several degradation products with notable structural changes. Under oxidative conditions, dendrobine primarily generates two degradation products with mass increases of 16 Da and 32 Da, indicating mono-oxidation and di-oxidation reactions. Acidic degradation led to the identification of three degradation products, including a novel compound with an 18 Da mass increase, suggesting potential hydrolysis or dehydration reactions. Hydrolytic and thermal conditions resulted in the formation of two and three degradation products, respectively, with structural changes indicating possible molecular cleavage and reorganization mechanisms. In contrast, dendrobine exhibited strong stability under alkaline and photolytic conditions, with no significant degradation products detected. Detailed characterization of the degradation products via multi-stage mass spectrometry revealed key reaction pathways and mechanisms involved in dendrobine's degradation, providing critical insights for assessing its chemical stability and optimizing storage conditions.
石斛碱是一种倍半萜生物碱，主要用于治疗炎症性疾病、免疫系统紊乱和与氧化应激相关的疾病。为了了解石斛石可能的降解途径并进行质量控制，我们采用强制降解方法对石斛石的降解产物进行了深入研究。采用Shim-pack XR-ODS III（75 mm × 2 mm, 1.6 µm）色谱柱，甲醇-水混合物为流动相，等压条件下对石斛碱及其降解产物进行分离，用离子捕获飞行时间质谱仪（IT-TOF）在正离子模式下对分离产物进行检测。为了获得降解产物的深入结构信息，质谱分析采用五段破碎法进行。这种方法允许通过几轮选择性破碎和高分辨率检测来彻底澄清结构。系统控制和数据采集采用LCMSsolution 3.81软件进行管理。结果表明，石斛石在氧化、酸性、水解和热条件下都发生了明显的降解，形成了几种结构变化明显的降解产物。在氧化条件下，石斛石主要生成两种降解产物，质量分别增加16 Da和32 Da，分别为单氧化和双氧化反应。酸性降解鉴定出三种降解产物，包括一种Da质量增加18 的新化合物，表明可能发生水解或脱水反应。水解和热条件下分别形成2种和3种降解产物，其结构变化表明可能的分子裂解和重组机制。相比之下，石斛石在碱性和光解条件下表现出很强的稳定性，没有检测到明显的降解产物。通过多级质谱分析对降解产物进行了详细表征，揭示了石斛降解的关键反应途径和机制，为评估石斛的化学稳定性和优化储存条件提供了重要见解。
{"title":"Characterization of main degradation products from dendrobine under stress conditions by multistage cleavage of UPLC-ESI-IT-TOF","authors":"Hengju Zhou , Meiling Zeng , Keyong Geng , Zaipeng Chen , Zhijia Tang , Jianwei Xu , Xiaoyan Zhang , Wei Zhou","doi":"10.1016/j.jpba.2025.116663","DOIUrl":"10.1016/j.jpba.2025.116663","url":null,"abstract":"<div><div>Dendrobine is a sesquiterpene alkaloid primarily used in the treatment of inflammatory diseases, immune system disorders, and conditions related to oxidative stress. To understand the possible degradation pathways of dendrobine for its quality control, we conducted an in-depth investigation of its degradation products using forced degradation methods. The separation of dendrobine and its degradation products was achieved on a Shim-pack XR-ODS III (75 mm × 2 mm, 1.6 µm) column with a methanol-water mixture as the mobile phase under isocratic conditions, the isolated compounds were examined in positive ion mode with an ion trap-time of flight mass spectrometer (IT-TOF). In order to obtain in-depth structural information about the degradation products, mass spectrometry was performed using a five-stage fragmentation approach. This method allowed for thorough structural clarification via several rounds of selective fragmentation and high-resolution detection. System control and data acquisition were managed using LCMSsolution 3.81 software. The results showed that dendrobine undergoes significant degradation under oxidative, acidic, hydrolytic and thermal conditions, resulting in the formation of several degradation products with notable structural changes. Under oxidative conditions, dendrobine primarily generates two degradation products with mass increases of 16 Da and 32 Da, indicating mono-oxidation and di-oxidation reactions. Acidic degradation led to the identification of three degradation products, including a novel compound with an 18 Da mass increase, suggesting potential hydrolysis or dehydration reactions. Hydrolytic and thermal conditions resulted in the formation of two and three degradation products, respectively, with structural changes indicating possible molecular cleavage and reorganization mechanisms. In contrast, dendrobine exhibited strong stability under alkaline and photolytic conditions, with no significant degradation products detected. Detailed characterization of the degradation products via multi-stage mass spectrometry revealed key reaction pathways and mechanisms involved in dendrobine's degradation, providing critical insights for assessing its chemical stability and optimizing storage conditions.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"255 ","pages":"Article 116663"},"PeriodicalIF":3.1,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142965570","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}
Accurate determination of plasma protein binding (PPB) is crucial in understanding the pharmacokinetics and pharmacodynamics of drugs, particularly for highly bound compounds where traditional methods may fall short. In this study, we present a pioneering approach for the precise determination of PPB that takes advantage of the lipophilicity of highly bound compounds. Twenty four highly bound compounds (with a fraction unbound (fu) from 10−1 to 10−6) were tested with the most commonly used method, i.e., the rapid equilibrium dialysis (RED) method, along with its modifications adapted especially for strongly bound compounds, including dilution, presaturation, competition, and flux-dialysis methods. The results of these methods were compared to data obtained with the modification of RED coupled with extraction to organic phase, and the correlations between them were presented. Comparison studies demonstrate the accuracy of our approach across a set of highly bound compounds within a twofold range. Moreover, PPB values were determined for venetoclax, amiodarone, montelukast, and fulvestrant, for which, to the best of our knowledge and after extensive review of the literature, it has not been possible with the standard RED method until now. In conclusion, our new method can be a big step forward in finding the PPB of strongly plasma protein-bound compounds. It gives us a better understanding of how drugs and proteins interact, which helps us make safer and more effective medicines.
{"title":"Implementation of a novel method for the determination of plasma protein binding of highly bound compounds using the equilibrium dialysis method coupled with extraction to the organic phase and its comparison to known methods","authors":"Dawid Gogola , Sanja Novak Ratajczak , Ewelina Gabor-Worwa , Anna Kowal-Chwast , Nilesh Gaud , Gniewomir Latacz , Krzysztof Brzózka , Kamil Kuś","doi":"10.1016/j.jpba.2025.116665","DOIUrl":"10.1016/j.jpba.2025.116665","url":null,"abstract":"<div><div>Accurate determination of plasma protein binding (PPB) is crucial in understanding the pharmacokinetics and pharmacodynamics of drugs, particularly for highly bound compounds where traditional methods may fall short. In this study, we present a pioneering approach for the precise determination of PPB that takes advantage of the lipophilicity of highly bound compounds. Twenty four highly bound compounds (with a fraction unbound (f<sub>u</sub>) from 10<sup>−1</sup> to 10<sup>−6</sup>) were tested with the most commonly used method, i.e., the rapid equilibrium dialysis (RED) method, along with its modifications adapted especially for strongly bound compounds, including dilution, presaturation, competition, and flux-dialysis methods. The results of these methods were compared to data obtained with the modification of RED coupled with extraction to organic phase, and the correlations between them were presented. Comparison studies demonstrate the accuracy of our approach across a set of highly bound compounds within a twofold range. Moreover, PPB values were determined for venetoclax, amiodarone, montelukast, and fulvestrant, for which, to the best of our knowledge and after extensive review of the literature, it has not been possible with the standard RED method until now. In conclusion, our new method can be a big step forward in finding the PPB of strongly plasma protein-bound compounds. It gives us a better understanding of how drugs and proteins interact, which helps us make safer and more effective medicines.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"255 ","pages":"Article 116665"},"PeriodicalIF":3.1,"publicationDate":"2025-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142965574","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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