Pub Date : 2024-06-14DOI: 10.1016/j.jchromb.2024.124174
An efficient technique for quantitative analysis of tetrodotoxin (TTX) in human plasma and urine has been developed, which combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online MCX solid phase extraction (SPE) cleanup. Sample preparation, including extraction with acetonitrile containing 0.5 % acetate acid, centrifugation, and filtration, was followed by online SPE cleanup. The whole run-time was less than 15 min, including online cleanup, chromatographic separation, and re-equilibration of the online SPE − LC-MS/MS system. The parameters of sample extraction, purification, separation, and detection were optimized. The matrix-matched internal standard calibration standard curves with linear regression coefficients larger than 0.9990 were established for quantification. The LOD and LOQ for this approach were determined to be 0.1 ng/mL and 0.3 ng/mL, respectively. The recoveries for varied concentrations of TTX in human plasma and urine were 84.9–104.2 % and 89.2–109.6 %, respectively. The matrix effects of TTX in human plasma and urine matrices were 85.5 % and 74.3 %, respectively, and both the inter- and intra-day precision values were less than 9.5 %. This analytical method was successfully employed for detecting TTX in biological samples from a poisoned patient who accidentally ingested the nassarius glans.
{"title":"Determination of tetrodotoxin in human plasma and urine using online MCX SPE column cleanup coupled with liquid chromatography-tandem mass spectrometry","authors":"","doi":"10.1016/j.jchromb.2024.124174","DOIUrl":"10.1016/j.jchromb.2024.124174","url":null,"abstract":"<div><p>An efficient technique for quantitative analysis of tetrodotoxin (TTX) in human plasma and urine has been developed, which combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online MCX solid phase extraction (SPE) cleanup. Sample preparation, including extraction with acetonitrile containing 0.5 % acetate acid, centrifugation, and filtration, was followed by online SPE cleanup. The whole run-time was less than 15 min, including online cleanup, chromatographic separation, and re-equilibration of the online SPE − LC-MS/MS system. The parameters of sample extraction, purification, separation, and detection were optimized. The matrix-matched internal standard calibration standard curves with linear regression coefficients larger than 0.9990 were established for quantification. The LOD and LOQ for this approach were determined to be 0.1 ng/mL and 0.3 ng/mL, respectively. The recoveries for varied concentrations of TTX in human plasma and urine were 84.9–104.2 % and 89.2–109.6 %, respectively. The matrix effects of TTX in human plasma and urine matrices were 85.5 % and 74.3 %, respectively, and both the inter- and intra-day precision values were less than 9.5 %. This analytical method was successfully employed for detecting TTX in biological samples from a poisoned patient who accidentally ingested the nassarius glans.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141397866","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 : 2024-06-14DOI: 10.1016/j.jchromb.2024.124209
Nadeen Anabtawi , Thomas Drabison , Yan Jin, Eric D. Eisenmann, Alex Sparreboom, Rajgopal Govindarajan, Sharyn D. Baker, Eman Ahmed
Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine.
地西他滨是一种 DNA 甲基转移酶抑制剂,用于治疗急性髓性白血病和骨髓增生异常综合征。目前正在进行的试验正在探索地西他滨与或不与胞苷脱氨酶抑制剂西达脲苷以及其他抗白血病药物的联合使用,这就需要全面了解药代动力学特性并评估药物相互作用的可能性。我们在此报告了一种灵敏的超高效液相色谱-质谱/质谱法的开发和验证情况,该方法可用于小鼠血浆中地西他滨的定量分析。该方法包括一步蛋白质沉淀提取,以及在 XBridge HILIC 色谱柱上使用梯度洗脱进行色谱分离。该方法稳健、准确、精确,灵敏度高(定量下限为 0.4 纳克/毫升),可测定口服或静脉注射地西他滨的小鼠微量血浆中地西他滨的浓度。
{"title":"Pharmacokinetic assessment of low dose decitabine in combination therapies: Development and validation of a sensitive UHPLC-MS/MS method for murine plasma analysis","authors":"Nadeen Anabtawi , Thomas Drabison , Yan Jin, Eric D. Eisenmann, Alex Sparreboom, Rajgopal Govindarajan, Sharyn D. Baker, Eman Ahmed","doi":"10.1016/j.jchromb.2024.124209","DOIUrl":"10.1016/j.jchromb.2024.124209","url":null,"abstract":"<div><p>Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570023224002186/pdfft?md5=6638712045435504b77de0a762e23046&pid=1-s2.0-S1570023224002186-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141403773","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 : 2024-06-14DOI: 10.1016/j.jchromb.2024.124187
N. Arbouche , A. Blanchot , J.S. Raul , P. Kintz
The use of semaglutide, also known by its trade name Ozempic®, has been increasing worldwide in recent years due to its benefits in treating type II diabetes. Thanks to its effects on appetite regulation, in many countries it is also used to treat obesity. However, due to its promotion by social media and celebrities as a weight-loss treatment, semaglutide is misused by a non-diabetic and non-obese population and by a young public, which is the main target of these media. Following the alert by the ANSM (Agence nationale de sécurité du médicament) in France and the FDA (Food and Drug Administration) in the United States, which imposed the addition of fatal effects to the list of side effects, the misuse of semaglutide seems to be becoming a public health problem. For this reason, it seems important that a toxicology laboratory has the capacity to test for semaglutide in blood.
In this study, the authors have developed and validated a method for the identification and quantification of semaglutide in whole blood using a LC-HRMS.
After the addition of the internal standard (bovine insulin), the blood was subjected to protein precipitation using a mix of acetonitrile/methanol (70:30,v:v). The validation procedure demonstrated an acceptable linearity between 2 and 500 ng/mL. LOD and LOQ were 1 and 2 ng/mL, respectively. Intra and inter-day precision were below 20 % at three concentrations. The method was successfully applied to the blood samples of 3 diabetic patients under treatment of semaglutide. The samples tested positive with concentrations ranging from 31 to 70 ng/mL which fall within the limits of therapeutic blood concentrations described in the literature.
{"title":"Semaglutide and health risk: Development and validation of a LC-HRMS method for testing semaglutide in whole blood and application to real cases","authors":"N. Arbouche , A. Blanchot , J.S. Raul , P. Kintz","doi":"10.1016/j.jchromb.2024.124187","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124187","url":null,"abstract":"<div><p>The use of semaglutide, also known by its trade name Ozempic®, has been increasing worldwide in recent years due to its benefits in treating type II diabetes. Thanks to its effects on appetite regulation, in many countries it is also used to treat obesity. However, due to its promotion by social media and celebrities as a weight-loss treatment, semaglutide is misused by a non-diabetic and non-obese population and by a young public, which is the main target of these media. Following the alert by the ANSM (Agence nationale de sécurité du médicament) in France and the FDA (Food and Drug Administration) in the United States, which imposed the addition of fatal effects to the list of side effects, the misuse of semaglutide seems to be becoming a public health problem. For this reason, it seems important that a toxicology laboratory has the capacity to test for semaglutide in blood.</p><p>In this study, the authors have developed and validated a method for the identification and quantification of semaglutide in whole blood using a LC-HRMS.</p><p>After the addition of the internal standard (bovine insulin), the blood was subjected to protein precipitation using a mix of acetonitrile/methanol (70:30,v:v). The validation procedure demonstrated an acceptable linearity between 2 and 500 ng/mL. LOD and LOQ were 1 and 2 ng/mL, respectively. Intra and inter-day precision were below 20 % at three concentrations. The method was successfully applied to the blood samples of 3 diabetic patients under treatment of semaglutide. The samples tested positive with concentrations ranging from 31 to 70 ng/mL which fall within the limits of therapeutic blood concentrations described in the literature.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141328908","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 : 2024-06-13DOI: 10.1016/j.jchromb.2024.124208
Yuanyuan Ji , Yulemni Morel , Anh Q. Tran , Marta M. Lipinski , Chinmoy Sarkar , Jace W. Jones
Reactive aldehydes are a class of electrophilic low molecular weight compounds that play an essential role in physiological function and lipid peroxidation. These molecules are implicated in many diseases, especially cardiovascular and neurodegenerative diseases, and are potential endogenous markers of lipid peroxidation. However, there are limited options to accurately quantify multiple reactive aldehydes in brain tissue. This study developed and validated a 3-nitrophenylhydrazine derivatization-based LC-MS/MS method to quantify four reactive aldehydes: malondialdehyde, acrolein, 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal. Method development involved comparing the sensitivity of detection between widely used derivatization reagents: 2,4-dinitrophenylhydrazine and 3-nitrophenylhydrazine. Our data showed that 3-nitrophenylhydrazine resulted in greater sensitivity. Additional method development included evaluation of hydrolysis sample pretreatment, selection of protein precipitation reagent, and optimization of derivatization conditions. The optimized conditions included no hydrolysis and use of 20 % trichloroacetic acid as the protein precipitation reagent. The optimized derivatization condition was 25 mM 3-nitrophenylhydrazine reacted at 20 °C for 30 min. The chromatographic conditions were optimized to reduce matrix effects, ion suppression, and efficient analysis time (<7-minute analytical run). The four aldehyde species were accurately quantified in brain tissue using stable-labeled internal standards. Application of this assay to a traumatic brain injury mouse model revealed significant accumulation of acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal at 28 days post injury. Overall, a validated method was developed to rapidly quantify the most prominent reactive aldehydes associated with lipid peroxidation during injury progression following acute brain trauma.
反应性醛是一类亲电性低分子量化合物,在生理功能和脂质过氧化过程中发挥着重要作用。这些分子与许多疾病(尤其是心血管和神经退行性疾病)有关,是潜在的脂质过氧化内源性标志物。然而,目前能准确量化脑组织中多种活性醛的方法还很有限。本研究开发并验证了一种基于 3-硝基苯肼衍生化的 LC-MS/MS 方法来定量检测四种活性醛:丙二醛、丙烯醛、4-羟基-2-己烯醛和 4-羟基-2-壬烯醛。方法开发包括比较广泛使用的衍生试剂的检测灵敏度:2,4-二硝基苯肼和 3-硝基苯肼。数据显示,3-硝基苯肼的灵敏度更高。其他方法开发包括水解样品预处理评估、蛋白质沉淀试剂的选择和衍生条件的优化。优化条件包括不水解和使用 20% 的三氯乙酸作为蛋白质沉淀试剂。优化的衍生条件为 25 mM 3-硝基苯肼在 20 °C 下反应 30 分钟。对色谱条件进行了优化,以减少基质效应、离子抑制和有效的分析时间 (
{"title":"Development and evaluation of a liquid chromatography-tandem mass spectrometry method for simultaneous measurement of toxic aldehydes from brain tissue","authors":"Yuanyuan Ji , Yulemni Morel , Anh Q. Tran , Marta M. Lipinski , Chinmoy Sarkar , Jace W. Jones","doi":"10.1016/j.jchromb.2024.124208","DOIUrl":"10.1016/j.jchromb.2024.124208","url":null,"abstract":"<div><p>Reactive aldehydes are a class of electrophilic low molecular weight compounds that play an essential role in physiological function and lipid peroxidation. These molecules are implicated in many diseases, especially cardiovascular and neurodegenerative diseases, and are potential endogenous markers of lipid peroxidation. However, there are limited options to accurately quantify multiple reactive aldehydes in brain tissue. This study developed and validated a 3-nitrophenylhydrazine derivatization-based LC-MS/MS method to quantify four reactive aldehydes: malondialdehyde, acrolein, 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal. Method development involved comparing the sensitivity of detection between widely used derivatization reagents: 2,4-dinitrophenylhydrazine and 3-nitrophenylhydrazine. Our data showed that 3-nitrophenylhydrazine resulted in greater sensitivity. Additional method development included evaluation of hydrolysis sample pretreatment, selection of protein precipitation reagent, and optimization of derivatization conditions. The optimized conditions included no hydrolysis and use of 20 % trichloroacetic acid as the protein precipitation reagent. The optimized derivatization condition was 25 mM 3-nitrophenylhydrazine reacted at 20 °C for 30 min. The chromatographic conditions were optimized to reduce matrix effects, ion suppression, and efficient analysis time (<7-minute analytical run). The four aldehyde species were accurately quantified in brain tissue using stable-labeled internal standards. Application of this assay to a traumatic brain injury mouse model revealed significant accumulation of acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal at 28 days post injury. Overall, a validated method was developed to rapidly quantify the most prominent reactive aldehydes associated with lipid peroxidation during injury progression following acute brain trauma.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141329962","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 : 2024-06-13DOI: 10.1016/j.jchromb.2024.124207
Omar Khaled, Lamia Ryad, Nermine Gad
A reliable liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS) method was developed for the simultaneous identification and quantification of 13 β-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg. Dispersive-solid phase extraction (d-SPE) using acetonitrile (ACN) was used to prepare the samples. The analyte in the extracts was separated on a reversed-phase Accucore aQ (50 mm × 2.1 mm, 2.6 μm) using a mobile phase of an aqueous solution containing 2 mM ammonium acetate and acetonitrile (ACN) 0.1 % formic acid. The method was validated in accordance with Commission Implementing Regulation (CIR) EU 2021/808 at six different concentrations ranging from 0.1 to 5 μg/kg. The mean recoveries ranged from 65 to 94 %, while repeatability and reproducibility values were all below 13 %. The linearity, as correlation coefficients (R2) ranged from 0.9955 to 0.9999. The decision limit (CCα) and detection capability (CCβ) ranges were 0.11–0.13 µg/kg and 0.12–0.15 µg/kg, respectively. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.004–0.048 μg/kg and 0.010–0.075 μg/kg, respectively. Of the 180 samples that were collected from local markets in Egypt, 21.11 % had β-agonist residues. The mean concentration (µg/kg) and detection frequency (%) of the most frequently found β-agonist in the samples were as follows: terbutaline (2.63 µg/kg and 90 %), ractopamine (5.14 µg/kg and 23.3 %). The method's applicability was verified by successfully completing two rounds of proficiency testing (PT).
{"title":"Quick and high-throughput quantification of β-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg using dispersive solid phase extraction","authors":"Omar Khaled, Lamia Ryad, Nermine Gad","doi":"10.1016/j.jchromb.2024.124207","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124207","url":null,"abstract":"<div><p>A reliable liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS) method was developed for the simultaneous identification and quantification of 13 β-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg. Dispersive-solid phase extraction (d-SPE) using acetonitrile (ACN) was used to prepare the samples. The analyte in the extracts was separated on a reversed-phase Accucore aQ (50 mm × 2.1 mm, 2.6 μm) using a mobile phase of an aqueous solution containing 2 mM ammonium acetate and acetonitrile (ACN) 0.1 % formic acid. The method was validated in accordance with Commission Implementing Regulation (CIR) EU 2021/808 at six different concentrations ranging from 0.1 to 5 μg/kg. The mean recoveries ranged from 65 to 94 %, while repeatability and reproducibility values were all below 13 %. The linearity, as correlation coefficients (R<sup>2</sup>) ranged from 0.9955 to 0.9999. The decision limit (CCα) and detection capability (CCβ) ranges were 0.11–0.13 µg/kg and 0.12–0.15 µg/kg, respectively. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.004–0.048 μg/kg and 0.010–0.075 μg/kg, respectively. Of the 180 samples that were collected from local markets in Egypt, 21.11 % had β-agonist residues. The mean concentration (µg/kg) and detection frequency (%) of the most frequently found β-agonist in the samples were as follows: terbutaline (2.63 µg/kg and 90 %), ractopamine (5.14 µg/kg and 23.3 %). The method's applicability was verified by successfully completing two rounds of proficiency testing (PT).</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141328906","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 : 2024-06-13DOI: 10.1016/j.jchromb.2024.124205
Terezie Cernosek , Matthew Dalphin , Nitin Jain , Jason Lor , Noah Richter , Mourad Mellal , Sue Behrens , Peter Wunderli
Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.
{"title":"A case study application of AQbD to the re-development and validation of an affinity chromatography analytical procedure for mAb titer quantitation","authors":"Terezie Cernosek , Matthew Dalphin , Nitin Jain , Jason Lor , Noah Richter , Mourad Mellal , Sue Behrens , Peter Wunderli","doi":"10.1016/j.jchromb.2024.124205","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124205","url":null,"abstract":"<div><p>Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R<sup>2</sup> values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141328907","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 : 2024-06-12DOI: 10.1016/j.jchromb.2024.124186
Zhihui Zhao , Zhiyuan Liu , Qiyao Jiang, Xiaoqing Zhang, Wenfu Ma, Dongran Han
Objective
Optimize the extraction process of earthworm fibrinolytic enzyme.
Methods
Chinese common earthworms underwent a series of purification processes, including grinding, salting out, hydrophobic medium chromatography, ammonium sulfate precipitation, and ion exchange chromatography, to obtain purified earthworm fibrinolytic enzyme.
Results
Utilizing Pheretima aspergillum as the starting material, we discovered that the specific activity of lumbrokinase extracted via ammonium sulfate precipitation was 58 U/mg, noticeably surpassing that achieved through heat precipitation and ethanol precipitation methods. After undergoing two rounds of chromatographic separations employing hydrophobic affinity chromatography and anion exchange chromatography, the specific activity of the lumbrokinase protein soared to 9267 U/mg, significantly exceeding the 3,178 U/mg specific activity attained through industrial extraction methods.
Discussion
The development of a novel crude extraction method for lumbrokinase protein can significantly boost its activity and purity. The discovery of a high-efficiency purification method and the identification of protein components within highly active lumbrokinase pave the way for further investigations into these proteins.
{"title":"Optimization of High-Activity earthworm fibrinolytic enzyme extraction methods and protein component analysis","authors":"Zhihui Zhao , Zhiyuan Liu , Qiyao Jiang, Xiaoqing Zhang, Wenfu Ma, Dongran Han","doi":"10.1016/j.jchromb.2024.124186","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124186","url":null,"abstract":"<div><h3>Objective</h3><p>Optimize the extraction process of earthworm fibrinolytic enzyme.</p></div><div><h3>Methods</h3><p>Chinese common earthworms underwent a series of purification processes, including grinding, salting out, hydrophobic medium chromatography, ammonium sulfate precipitation, and ion exchange chromatography, to obtain purified earthworm fibrinolytic enzyme.</p></div><div><h3>Results</h3><p>Utilizing Pheretima aspergillum as the starting material, we discovered that the specific activity of lumbrokinase extracted via ammonium sulfate precipitation was 58 U/mg, noticeably surpassing that achieved through heat precipitation and ethanol precipitation methods. After undergoing two rounds of chromatographic separations employing hydrophobic affinity chromatography and anion exchange chromatography, the specific activity of the lumbrokinase protein soared to 9267 U/mg, significantly exceeding the 3,178 U/mg specific activity attained through industrial extraction methods.</p></div><div><h3>Discussion</h3><p>The development of a novel crude extraction method for lumbrokinase protein can significantly boost its activity and purity. The discovery of a high-efficiency purification method and the identification of protein components within highly active lumbrokinase pave the way for further investigations into these proteins.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141324778","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 : 2024-06-11DOI: 10.1016/j.jchromb.2024.124197
Xiangping Meng, Caihong Li, Aichun Gao, Hongjin Wang, Lan Wei, Lixin Sun
Chuanwang xiaoyan capsules (CWXYC) have anti-inflammatory and detoxification effect, are used in the treatment of acute and chronic tonsillitis, pharyngitis and other inflammation-related diseases clinically. However, the anti-inflammatory mechanisms have not been elucidated. This study aimed to investigate the anti-inflammatory mechanisms of CWXYC using cell metabolomics and network pharmacology strategy. Specifically, CWXYC could efficiently reduce the content of nitric oxide (NO), the cytokines Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in LPS-induced RAW264.7 cells. Furthermore, metabolomics was performed to achieve 23 differential metabolites and 9 metabolic pathways containing glutamate metabolism, glutathione metabolism, arginine and proline metabolism, urea cycle, malate-aspartate shuttle, phosphatidylcholine biosynthesis, transfer of acetyl groups into mitochondria, cysteine metabolism and ammonia recycling. The results of network pharmacology showed that CWXYC could treat inflammation through 10 active components, 10 key targets and 55 pathways. Then the results of molecular docking also approved that there existed strong binding energy between the active components and the key targets. Finally, metabolomics and network pharmacology were integrated to get core targets AKT1, SRC and EGFR. Western blot experiments verified that CWXYC could exert anti-inflammatory effect by down-regulating the activated Akt1 and Src proteins. This study demonstrated that CWXYC exerted effects against inflammation, and the potential mechanisms were elucidated. These novel findings will provide an important basis for further mechanism investigations.
{"title":"Integrated metabolomics and network pharmacology approach to exploring the anti-inflammatory mechanisms of Chuanwang xiaoyan capsules","authors":"Xiangping Meng, Caihong Li, Aichun Gao, Hongjin Wang, Lan Wei, Lixin Sun","doi":"10.1016/j.jchromb.2024.124197","DOIUrl":"10.1016/j.jchromb.2024.124197","url":null,"abstract":"<div><p>Chuanwang xiaoyan capsules (CWXYC) have anti-inflammatory and detoxification effect, are used in the treatment of acute and chronic tonsillitis, pharyngitis and other inflammation-related diseases clinically. However, the anti-inflammatory mechanisms have not been elucidated. This study aimed to investigate the anti-inflammatory mechanisms of CWXYC using cell metabolomics and network pharmacology strategy. Specifically, CWXYC could efficiently reduce the content of nitric oxide (NO), the cytokines Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in LPS-induced RAW264.7 cells. Furthermore, metabolomics was performed to achieve 23 differential metabolites and 9 metabolic pathways containing glutamate metabolism, glutathione metabolism, arginine and proline metabolism, urea cycle, malate-aspartate shuttle, phosphatidylcholine biosynthesis, transfer of acetyl groups into mitochondria, cysteine metabolism and ammonia recycling. The results of network pharmacology showed that CWXYC could treat inflammation through 10 active components, 10 key targets and 55 pathways. Then the results of molecular docking also approved that there existed strong binding energy between the active components and the key targets. Finally, metabolomics and network pharmacology were integrated to get core targets AKT1, SRC and EGFR. Western blot experiments verified that CWXYC could exert anti-inflammatory effect by down-regulating the activated Akt1 and Src proteins. This study demonstrated that CWXYC exerted effects against inflammation, and the potential mechanisms were elucidated. These novel findings will provide an important basis for further mechanism investigations.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141409964","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 : 2024-06-10DOI: 10.1016/j.jchromb.2024.124196
Julian Bickel, Hilke Jungen, Alexander Müller, Anne Szewczyk, Benjamin Ondruschka, Stefanie Iwersen-Bergmann
Antiarrhythmic and antihypertensive drugs are frequently encountered in post mortem analysis, and the question may arise as to whether they were administered in therapeutic doses, and if they were taken in accidental, intentional, or suicidal overdose scenarios. Therefore, a novel analytical method was developed and validated for the quantification of 35 drugs with toxicological relevance, including antihypertensive and antiarrhythmic drugs (ajmaline, amlodipine, amiodarone, atenolol, bisoprolol, carvedilol, clonidine, desethylamiodarone, diltiazem, donepezil, doxazosin, dronedarone, esmolol, flecainide, lercanidipine, lidocaine, metoprolol, nebivolol, nimodipine, pindolol, prajmaline, propafenone, propranolol, sotalol, urapidil, and verapamil), as well as other medications commonly found in combination (sildenafil, tadalafil, atorvastatin, clopidogrel, dapoxetine, memantine, pentoxifylline, rivastigmine, and ivabradine). The method enables simultaneous identification and quantification in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Validation exhibited excellent linearity across the concentration range for all analytes. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 9 % and 10 %, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated very high sensitivity, with limits of detection (LOD) as low as 0.01 ng/ml and limits of quantification (LOQ) as low as 0.04 ng/ml. Extraction recovery exceeded 57.5 % for all analytes except atenolol, and matrix effects were <17 % for all analytes except pindolol. Processed sample stability evaluations revealed consistent results with acceptable deviations for all analytes. In addition, the method was specifically tested for the use in post mortem analysis. The applicability of our method was demonstrated by the analysis of two authentic human autopsy blood samples.
{"title":"Toxicologic analysis of 35 drugs in post mortem human blood samples with focus on antihypertensive and antiarrhythmic drugs","authors":"Julian Bickel, Hilke Jungen, Alexander Müller, Anne Szewczyk, Benjamin Ondruschka, Stefanie Iwersen-Bergmann","doi":"10.1016/j.jchromb.2024.124196","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124196","url":null,"abstract":"<div><p>Antiarrhythmic and antihypertensive drugs are frequently encountered in post mortem analysis, and the question may arise as to whether they were administered in therapeutic doses, and if they were taken in accidental, intentional, or suicidal overdose scenarios. Therefore, a novel analytical method was developed and validated for the quantification of 35 drugs with toxicological relevance, including antihypertensive and antiarrhythmic drugs (ajmaline, amlodipine, amiodarone, atenolol, bisoprolol, carvedilol, clonidine, desethylamiodarone, diltiazem, donepezil, doxazosin, dronedarone, esmolol, flecainide, lercanidipine, lidocaine, metoprolol, nebivolol, nimodipine, pindolol, prajmaline, propafenone, propranolol, sotalol, urapidil, and verapamil), as well as other medications commonly found in combination (sildenafil, tadalafil, atorvastatin, clopidogrel, dapoxetine, memantine, pentoxifylline, rivastigmine, and ivabradine). The method enables simultaneous identification and quantification in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS).</p><p>Validation exhibited excellent linearity across the concentration range for all analytes. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 9 % and 10 %, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated very high sensitivity, with limits of detection (LOD) as low as 0.01 ng/ml and limits of quantification (LOQ) as low as 0.04 ng/ml. Extraction recovery exceeded 57.5 % for all analytes except atenolol, and matrix effects were <17 % for all analytes except pindolol. Processed sample stability evaluations revealed consistent results with acceptable deviations for all analytes. In addition, the method was specifically tested for the use in post mortem analysis. The applicability of our method was demonstrated by the analysis of two authentic human autopsy blood samples.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570023224002058/pdfft?md5=e719d8784cd9b7242b4dd8c7ae9e9e2b&pid=1-s2.0-S1570023224002058-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141323652","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 : 2024-06-09DOI: 10.1016/j.jchromb.2024.124193
Xiaoxue Xu , Sheng Guo , Jiangyan Chen , Yuhan Song , Xueli Wu , Feng Liu , Jin-Ao Duan
The Zingiberis Rhizoma − Jujubae Fructus herb pair (ZJHP) is a classic herb pair in traditional Chinese medicine. The herb pair shows the effect of dispelling cold, harmonizing the middle and improving gastrointestinal function, and is widely used for patients with stomach cold syndrome (SCS), stomachache and anemofrigid cold. The gingerols, shogaols, flavonoids and triterpenic acids are the important bioactive ingredients of ZJHP. However, few pharmacokinetic studies have been investigated in vivo for the above compounds. To comprehend the kinetics of active components and promote their curative application, a fast and sensitive ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) method was established for simultaneous determination of 12 analytes in normal and SCS rats in this study. The results showed that the pharmacokinetic parameters (Cmax, Tmax, t1/2z, MRT0-t, AUC0-t and AUC0-∞) in SCS model were significantly different from those in normal rats. In addition, the pharmacokinetics of rats given ZJHP were also varied from single herb oral administration, especially in model condition. These results indicated that the in vivo processes of the above analytes changed under pathological conditions and the compatibility of the herb pair could significantly influence the absorption of active components, which might provide an insight and further supports for the clinical application of ZJHP.
{"title":"Comparative pharmacokinetics of multi-components in normal and stomach cold syndrome rats after oral administration of Zingiberis Rhizoma − Jujubae Fructus herb pair and its single herb extracts by UHPLC-MS/MS","authors":"Xiaoxue Xu , Sheng Guo , Jiangyan Chen , Yuhan Song , Xueli Wu , Feng Liu , Jin-Ao Duan","doi":"10.1016/j.jchromb.2024.124193","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124193","url":null,"abstract":"<div><p>The <em>Zingiberis Rhizoma</em> − <em>Jujubae Fructus</em> herb pair (ZJHP) is a classic herb pair in traditional Chinese medicine. The herb pair shows the effect of dispelling cold, harmonizing the middle and improving gastrointestinal function, and is widely used for patients with stomach cold syndrome (SCS), stomachache and anemofrigid cold. The gingerols, shogaols, flavonoids and triterpenic acids are the important bioactive ingredients of ZJHP. However, few pharmacokinetic studies have been investigated in <em>vivo</em> for the above compounds. To comprehend the kinetics of active components and promote their curative application, a fast and sensitive ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) method was established for simultaneous determination of 12 analytes in normal and SCS rats in this study. The results showed that the pharmacokinetic parameters (C<sub>max</sub>, T<sub>max</sub>, <em>t</em><sub>1/2z</sub>, MRT<sub>0-t</sub>, AUC<sub>0-t</sub> and AUC<sub>0-∞</sub>) in SCS model were significantly different from those in normal rats. In addition, the pharmacokinetics of rats given ZJHP were also varied from single herb oral administration, especially in model condition. These results indicated that the in <em>vivo</em> processes of the above analytes changed under pathological conditions and the compatibility of the herb pair could significantly influence the absorption of active components, which might provide an insight and further supports for the clinical application of ZJHP.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141303585","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}