Pub Date : 2024-05-14DOI: 10.1016/j.jchromb.2024.124157
Tiantian Tang , Xianzhang Luo , Na Li , Qiaoqiao Li , Min Zhang , Jin Zeng , Haichi Song , Lixian Li , Wanyi Chen
In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (Vu). The results indicated that the centrifugal force and centrifugal time have an influence on Vu that is significantly positive (P < 0.05). This developed method with good linearity (r2 = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %−106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice.
{"title":"A developed and validated centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method for rapid quantification of unbound lenvatinib in human plasma","authors":"Tiantian Tang , Xianzhang Luo , Na Li , Qiaoqiao Li , Min Zhang , Jin Zeng , Haichi Song , Lixian Li , Wanyi Chen","doi":"10.1016/j.jchromb.2024.124157","DOIUrl":"10.1016/j.jchromb.2024.124157","url":null,"abstract":"<div><p>In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (<em>V</em><sub>u</sub>). The results indicated that the centrifugal force and centrifugal time have an influence on <em>V</em><sub>u</sub> that is significantly positive (<em>P</em> < 0.05). This developed method with good linearity (<em>r</em><sup>2</sup> = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %−106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140955309","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 : 2024-05-10DOI: 10.1016/j.jchromb.2024.124154
Yuan-Yuan Zhang , Hong-Li Guo , Jie Wang , Wei-Jun Wang , Yue Li , Chen-Chao Chu , Chun-Ying Wu , Jian Huang , Ya-Hui Hu , Feng Chen
Cyclosporine A (CsA) is a widely used immunosuppressive drug with a narrow therapeutic index and large individual differences. Its therapeutic and toxic effects are closely related to blood drug concentrations, requiring routine therapeutic drug monitoring (TDM). The current main methods for TDM of CsA are enzyme multiplied immunoassay technique (EMIT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, few study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood CsA concentration in children has been reported. In this study, we developed a simple and sensitive LC-MS/MS assay for the determination of CsA, and 657 cases of CsA concentrations were determined from 197 pediatric patients by a routine EMIT assay and by the validated in-house LC-MS/MS method on the same batch of samples, aimed to address the aforementioned concern. Consistency between the two assays was evaluated using linear regression and Bland-Altman analysis. The linear range of LC-MS/MS was 0.500–2000 ng/mL and that of the EMIT was 40–500 ng/mL, respectively. Overall, the correlation between the two methods was significant (r-value ranging from 0.8842 to 0.9441). Unsatisfactory consistency was observed in the concentrations < 40 ng/mL (r = 0.7325) and 200–500 ng/mL (r = 0.6851). Bland-Altman plot showed a mean bias of −18.0 % (±1.96 SD, −73.8 to 37.8 %) between EMIT and LC-MS/MS. For Passing-Bablok regression between EMIT and LC-MS/MS did not differ significantly (p > 0.05). In conclusion, the two methods were closely correlated, but the CsA concentration by LC-MS/MS assay was slightly higher than that by EMIT method. Switching from the EMIT assay to the LC-MS/MS method was acceptable, and the LC-MS/MS method will receive broader application in clinical settings due to its better analytical capabilities, but the results need to be further verified in different laboratories.
{"title":"LC-MS/MS and EMIT measure the whole blood concentration of cyclosporine A: The two methods yield concordant results within the dynamic range of the latter, but the former shows broader application scenarios","authors":"Yuan-Yuan Zhang , Hong-Li Guo , Jie Wang , Wei-Jun Wang , Yue Li , Chen-Chao Chu , Chun-Ying Wu , Jian Huang , Ya-Hui Hu , Feng Chen","doi":"10.1016/j.jchromb.2024.124154","DOIUrl":"10.1016/j.jchromb.2024.124154","url":null,"abstract":"<div><p>Cyclosporine A (CsA) is a widely used immunosuppressive drug with a narrow therapeutic index and large individual differences. Its therapeutic and toxic effects are closely related to blood drug concentrations, requiring routine therapeutic drug monitoring (TDM). The current main methods for TDM of CsA are enzyme multiplied immunoassay technique (EMIT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, few study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood CsA concentration in children has been reported. In this study, we developed a simple and sensitive LC-MS/MS assay for the determination of CsA, and 657 cases of CsA concentrations were determined from 197 pediatric patients by a routine EMIT assay and by the validated in-house LC-MS/MS method on the same batch of samples, aimed to address the aforementioned concern. Consistency between the two assays was evaluated using linear regression and Bland-Altman analysis. The linear range of LC-MS/MS was 0.500–2000 ng/mL and that of the EMIT was 40–500 ng/mL, respectively. Overall, the correlation between the two methods was significant (r-value ranging from 0.8842 to 0.9441). Unsatisfactory consistency was observed in the concentrations < 40 ng/mL (<em>r</em> = 0.7325) and 200–500 ng/mL (<em>r</em> = 0.6851). Bland-Altman plot showed a mean bias of −18.0 % (±1.96 SD, −73.8 to 37.8 %) between EMIT and LC-MS/MS. For Passing-Bablok regression between EMIT and LC-MS/MS did not differ significantly (<em>p</em> > 0.05). In conclusion, the two methods were closely correlated, but the CsA concentration by LC-MS/MS assay was slightly higher than that by EMIT method. Switching from the EMIT assay to the LC-MS/MS method was acceptable, and the LC-MS/MS method will receive broader application in clinical settings due to its better analytical capabilities, but the results need to be further verified in different laboratories.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140943747","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 : 2024-05-10DOI: 10.1016/j.jchromb.2024.124155
Jin Wang , Meng Jin , Qian Wang , Xiaogang Lu , Runli Gao , Fengxia Sun , Chengxin Pei , Hongmei Wang
Protein adducts are vital targets for exploring organophosphorus nerve agents (OPNAs) exposure and identification, that can be used to characterize the chemical burden and initiate chemical safety measures. However, the use of protein adducts as biomarkers of OPNA exposure has developed slowly. To further promote the development of biomarkers in chemical forensics, it is crucial to expand the range of modified peptides and active sites, and describe the characteristics of OPNA adducts at specific reaction sites. This study utilized multi-species and multi-source albumins as the protein targets. We identified 56 peptides in albumins from various species (including human, horse, rat and pig), that were modified by at least two OPNAs. Diverse modification characteristics were observed in response to certain agents: including (1) multiple sites on the same peptide modified by one or more agents, (2) different reactivities at the same site in homologous albumins, and (3) different preferences at the same active sites associated with differences in the biological matrix during exposure. Our studies provided an empirical reference with rationalized underpinnings supported by estimated conformation energetics through molecular modeling.
We employed different peptide markers for detection of protein adducts, as (one would do) in forensic screening for identification and quantification of chemical damage. Three characteristic peptides were screened and analyzed in human albumin, including Y287ICENQDSISSK, K438VPQVS443TPTLVEVSR, and Y162LY164EIAR. Stable fragment ions with neutral loss were found from their tandem MS/MS spectra, which were used as characteristic ions for identification and extraction of modified peptides in enzymatic digestion mixtures. Coupling these observations with computer simulations, we found that the structural stability of albumin and albumin-adduct complexes (as well as the effective force that promotes stability of different adducts) changes in the interval before and after adduct formation. In pig albumin, five active peptides existed stably in vivo and in vitro. Most of them can be detected within 30 min after OPNA exposure, and the detection window can persist about half a month. These early findings provided the foundation and rationale for utilizing pig albumin as a sampling target for rapid analysis in future forensic work.
{"title":"Study on phosphonylation and modification characteristics of organophosphorus nerve agents on multi-species and multi-source albumins","authors":"Jin Wang , Meng Jin , Qian Wang , Xiaogang Lu , Runli Gao , Fengxia Sun , Chengxin Pei , Hongmei Wang","doi":"10.1016/j.jchromb.2024.124155","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124155","url":null,"abstract":"<div><p>Protein adducts are vital targets for exploring organophosphorus nerve agents (OPNAs) exposure and identification, that can be used to characterize the chemical burden and initiate chemical safety measures. However, the use of protein adducts as biomarkers of OPNA exposure has developed slowly. To further promote the development of biomarkers in chemical forensics, it is crucial to expand the range of modified peptides and active sites, and describe the characteristics of OPNA adducts at specific reaction sites. This study utilized multi-species and multi-source albumins as the protein targets. We identified 56 peptides in albumins from various species (including human, horse, rat and pig), that were modified by at least two OPNAs. Diverse modification characteristics were observed in response to certain agents: including (1) multiple sites on the same peptide modified by one or more agents, (2) different reactivities at the same site in homologous albumins, and (3) different preferences at the same active sites associated with differences in the biological matrix during exposure. Our studies provided an empirical reference with rationalized underpinnings supported by estimated conformation energetics through molecular modeling.</p><p>We employed different peptide markers for detection of protein adducts, as (one would do) in forensic screening for identification and quantification of chemical damage. Three characteristic peptides were screened and analyzed in human albumin, including Y<sub>287</sub>ICENQDSISSK, K<sub>438</sub>VPQVS<sub>443</sub>TPTLVEVSR, and Y<sub>162</sub>LY<sub>164</sub>EIAR. Stable fragment ions with neutral loss were found from their tandem MS/MS spectra, which were used as characteristic ions for identification and extraction of modified peptides in enzymatic digestion mixtures. Coupling these observations with computer simulations, we found that the structural stability of albumin and albumin-adduct complexes (as well as the effective force that promotes stability of different adducts) changes in the interval before and after adduct formation. In pig albumin, five active peptides existed stably <em>in vivo</em> and <em>in vitro</em>. Most of them can be detected within 30 min after OPNA exposure, and the detection window can persist about half a month. These early findings provided the foundation and rationale for utilizing pig albumin as a sampling target for rapid analysis in future forensic work.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140909742","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 : 2024-05-10DOI: 10.1016/j.jchromb.2024.124143
Mariam M. Abady , Ji-Seon Jeong , Ha-Jeong Kwon
This study aims to establish an LC-MS/MS method to simultaneously analyze 11 antiepileptic drugs with a particular focus on maintaining accuracy while reducing the number of isotope-labeled internal standards employed for cost-effectiveness. By applying a water/acetonitrile gradient elution containing 0.1 % formic acid and 2 mM ammonium formate as the mobile phase, optimal sensitivity for the target drugs could be obtained in positive ESI mode in LC-MS/MS. After optimizing various extraction techniques, extraction with 70 % acetonitrile was selected as it provided good recoveries (>93 %) for all targets without matrix effects. Accuracies within 3 % were achieved from the combination of six internal standards, while accuracies of 5 % and 10 % were obtained by reducing the number of internal standards to four and two, respectively, for more economical analysis. The accuracy of the established method was maintained in hyperglycemia, hyperlipidemia, and hyperalbuminemia sera, suggesting that it can be successfully applied to individual serum samples with various properties.
{"title":"Simultaneous quantification of 11 antiepileptic drugs using limited isotope-labeled internal standards in LC-MS/MS: An accuracy assessment","authors":"Mariam M. Abady , Ji-Seon Jeong , Ha-Jeong Kwon","doi":"10.1016/j.jchromb.2024.124143","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124143","url":null,"abstract":"<div><p>This study aims to establish an LC-MS/MS method to simultaneously analyze 11 antiepileptic drugs with a particular focus on maintaining accuracy while reducing the number of isotope-labeled internal standards employed for cost-effectiveness. By applying a water/acetonitrile gradient elution containing 0.1 % formic acid and 2 mM ammonium formate as the mobile phase, optimal sensitivity for the target drugs could be obtained in positive ESI mode in LC-MS/MS. After optimizing various extraction techniques, extraction with 70 % acetonitrile was selected as it provided good recoveries (>93 %) for all targets without matrix effects. Accuracies within 3 % were achieved from the combination of six internal standards, while accuracies of 5 % and 10 % were obtained by reducing the number of internal standards to four and two, respectively, for more economical analysis. The accuracy of the established method was maintained in hyperglycemia, hyperlipidemia, and hyperalbuminemia sera, suggesting that it can be successfully applied to individual serum samples with various properties.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S157002322400151X/pdfft?md5=405067872c30936b43dd89d8d5e3085c&pid=1-s2.0-S157002322400151X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140952178","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 : 2024-05-10DOI: 10.1016/j.jchromb.2024.124156
Guohua Teng , Hezhao Bai , Chen Zhang , Jingyi Yang , Xiaoye Wang , Yu Zhu , Fei Tian
A magnetic composite (Fe3O4@SiO2@PNIPAM-co-NHMA) with high adsorption capacity and recoverability was developed for the enrichment and determination of flavonoids in Scutellaria Radix (SR). A magnetic solid-phase extraction (MSPE) technique using Fe3O4@SiO2@PNIPAM-co-NHMA absorbent in combination with high-performance liquid chromatography (HPLC) was developed for selectively enrichment and determination of the biologically active flavonoids in the aqueous extract of SR, including baicalein, baicalin, wogonoside and wogonin. Under the optimized experimental conditions, the magnetic adsorbent could adsorb up to 77.0 ± 0.98 % − 98.15 ± 0.15 % of four representative flavonoids from SR, with elution rates varying from 55.10 ± 0.25 % to 91.94 ± 1.85 %. The limits of detection (LOD) and limits of quantitation (LOQ) were 0.01–0.35 μg/mL and 0.03–0.98 μg/mL, respectively. In addition, it remained effective after six replicates, demonstrating its potential as a recoverable adsorbent for enriching flavonoids in traditional Chinese medicine.
{"title":"Functionalized magnetic nanomaterials as recyclable adsorbents for efficient flavonoid enrichment in Scutellaria Radix","authors":"Guohua Teng , Hezhao Bai , Chen Zhang , Jingyi Yang , Xiaoye Wang , Yu Zhu , Fei Tian","doi":"10.1016/j.jchromb.2024.124156","DOIUrl":"10.1016/j.jchromb.2024.124156","url":null,"abstract":"<div><p>A magnetic composite (Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>@PNIPAM-co-NHMA) with high adsorption capacity and recoverability was developed for the enrichment and determination of flavonoids in <em>Scutellaria Radix</em> (<em>SR</em>). A magnetic solid-phase extraction (MSPE) technique using Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub>@PNIPAM-co-NHMA absorbent in combination with high-performance liquid chromatography (HPLC) was developed for selectively enrichment and determination of the biologically active flavonoids in the aqueous extract of <em>SR</em>, including baicalein, baicalin, wogonoside and wogonin. Under the optimized experimental conditions, the magnetic adsorbent could adsorb up to 77.0 ± 0.98 % − 98.15 ± 0.15 % of four representative flavonoids from <em>SR</em>, with elution rates varying from 55.10 ± 0.25 % to 91.94 ± 1.85 %. The limits of detection (LOD) and limits of quantitation (LOQ) were 0.01–0.35 μg/mL and 0.03–0.98 μg/mL, respectively. In addition, it remained effective after six replicates, demonstrating its potential as a recoverable adsorbent for enriching flavonoids in traditional Chinese medicine.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140943744","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 : 2024-05-05DOI: 10.1016/j.jchromb.2024.124128
J. Rudolf-Scholik, D. Lilek, M. Maier, T. Reischenböck, C. Maisl, J. Allram, B. Herbinger, J. Rechthaler
Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant.
Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications.
A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant’s Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis.
Our work with Tribolium castaneum larvae demonstrates that sometimes – depending on matrix and research question − more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.
根据不同的研究问题,基于 LC-MS/MS 的自下而上蛋白质组学从最初的生物样本到数据评估都面临着挑战。多维蛋白质提取策略与电泳或色谱离线蛋白质预分馏相结合,提高了从蓖麻鳞虫中分离蛋白质的谱图,减少了纳米LC-MS/MS测量肽段时的共洗脱和离子抑制效应。在综合数据分析中,使用 MaxQuant 进行蛋白质鉴定,使用 R 进行数据评估。对一系列参数进行了评估,以获得可重现、可靠和重要的蛋白质鉴定结果。在提取T.castaneum蛋白质的第一步,使用了含有蛋白酶和磷酸酶抑制剂鸡尾酒的简单磷酸盐缓冲液(pH值为8)和温和提取条件。此外,与色谱分馏法和一锅分析法相比,二维提取法结合提取蛋白质的电泳预分馏和随后的凝胶内消化,使鉴定出的蛋白质几乎增加了100%。额外鉴定出的蛋白质可归属于新的分子功能或细胞分区,强调了自下而上蛋白质组学中扩展样品制备的积极作用。除了后处理过程中的肽段数量,MaxQuant 的 "运行间匹配"(Match between Runs)对鉴定蛋白质的数量也有重要影响。数据分析必须考虑最大 2% 的相对标准偏差。我们用 Tribolium castaneum 幼虫进行的研究表明,有时,根据基质和研究问题的不同,更复杂、更耗时的样品制备有利于在自下而上蛋白质组学中分离和鉴定更多蛋白质。
{"title":"Increasing protein identifications in bottom-up proteomics of T. castaneum − Exploiting synergies of protein biochemistry and bioinformatics","authors":"J. Rudolf-Scholik, D. Lilek, M. Maier, T. Reischenböck, C. Maisl, J. Allram, B. Herbinger, J. Rechthaler","doi":"10.1016/j.jchromb.2024.124128","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124128","url":null,"abstract":"<div><p>Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in <em>Tribolium castaneum</em> by applying free software proteomics platform Max Quant.</p><p>Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from <em>T. castaneum</em> and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications.</p><p>A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for <em>T.castaneum</em> proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant’s Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis.</p><p>Our work with <em>Tribolium castaneum</em> larvae demonstrates that sometimes – depending on matrix and research question − more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570023224001363/pdfft?md5=da8b44c26018e672e114223ad1adb55e&pid=1-s2.0-S1570023224001363-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140952177","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 : 2024-05-04DOI: 10.1016/j.jchromb.2024.124142
Hamid Sinapour , Joar Guterstam , Susan Grosse , Juan Astorga-Wells , Peter Stambeck , Matilda Stambeck , Jesper Winberg , Sigurd Hermansson , Olof Beck
Aerosol microparticles in exhaled breath carry non-volatile compounds from the deeper parts of the lung. When captured and analyzed, these aerosol microparticles constitute a non-invasive and readily available specimen for drugs of abuse testing. The present study aimed to evaluate a simple breath collection device in a clinical setting. The device divides a breath sample into three parallel “collectors” that can be individually analyzed. Urine was used as the reference specimen, and parallel specimens were collected from 99 patients undergoing methadone maintenance treatment. Methadone was used as the primary validation parameter. A sensitive multi-analyte method using tandem liquid chromatography – mass spectrometry was developed and validated as part of the project. The method was successfully validated for 36 analytes with a limit of detection of 1 pg/collector for most compounds. Based on the validation results tetrahydrocannabinol THC), cannabidiol (CBD), and lysergic acid diethylamide (LSD) are suitable for qualitative analysis, but all other analytes can be quantitively assessed by the method. Methadone was positive in urine in 97 cases and detected in exhaled breath in 98 cases. Median methadone concentration was 64 pg/collector. The methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was detected in 90 % of the cases but below 10 pg/collector in most. Amphetamine was also present in the urine in 17 cases and in exhaled breath in 16 cases. Several other substances were detected in the exhaled breath and urine samples, but at a lower frequency. This study concluded that the device provides a specimen from exhaled breath, that is useful for drugs of abuse testing. The results show that high analytical sensitivity is needed to achieve good detectability and detection time after intake.
{"title":"Validation and application of an automated multitarget LC-MS/MS method for drugs of abuse testing using exhaled breath as specimen","authors":"Hamid Sinapour , Joar Guterstam , Susan Grosse , Juan Astorga-Wells , Peter Stambeck , Matilda Stambeck , Jesper Winberg , Sigurd Hermansson , Olof Beck","doi":"10.1016/j.jchromb.2024.124142","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124142","url":null,"abstract":"<div><p>Aerosol microparticles in exhaled breath carry non-volatile compounds from the deeper parts of the lung. When captured and analyzed, these aerosol microparticles constitute a non-invasive and readily available specimen for drugs of abuse testing. The present study aimed to evaluate a simple breath collection device in a clinical setting. The device divides a breath sample into three parallel “collectors” that can be individually analyzed. Urine was used as the reference specimen, and parallel specimens were collected from 99 patients undergoing methadone maintenance treatment. Methadone was used as the primary validation parameter. A sensitive multi-analyte method using tandem liquid chromatography – mass spectrometry was developed and validated as part of the project. The method was successfully validated for 36 analytes with a limit of detection of 1 pg/collector for most compounds. Based on the validation results tetrahydrocannabinol THC), cannabidiol (CBD), and lysergic acid diethylamide (LSD) are suitable for qualitative analysis, but all other analytes can be quantitively assessed by the method. Methadone was positive in urine in 97 cases and detected in exhaled breath in 98 cases. Median methadone concentration was 64 pg/collector. The methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was detected in 90 % of the cases but below 10 pg/collector in most. Amphetamine was also present in the urine in 17 cases and in exhaled breath in 16 cases. Several other substances were detected in the exhaled breath and urine samples, but at a lower frequency. This study concluded that the device provides a specimen from exhaled breath, that is useful for drugs of abuse testing. The results show that high analytical sensitivity is needed to achieve good detectability and detection time after intake.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570023224001508/pdfft?md5=a2ff45368b6d38fb07aef6a5a14da441&pid=1-s2.0-S1570023224001508-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140880034","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 : 2024-05-04DOI: 10.1016/j.jchromb.2024.124145
Shuting Yu , Xuemei Qin , Zhenyu Li
Farfarae Flos is a traditional herb widely employed for treating coughs, bronchitis, and asthmatic disorders. In the current study, we utilized SWATH and IDA data acquisition modes in combination with multiple data processing techniques to identify Farfarae Flos metabolites in mice serum. A total of 56 compounds were characterized, including 31 phenolic acids, 13 flavonoids, 11 sesquiterpenoids and 1 alkaloid. Further quantitative analysis was conducted on 12 absorbed metabolites, utilizing a newly developed and rigorously validated analytical method. Our approach demonstrated an acceptable level of specificity, accuracy, precision, and stability. When applied to compare the serum of mice treated with FF, all 12 metabolites showed the highest concentration at 0.5 h. Overall, this study presented a novel strategy for unraveling the active compounds of FF via serum pharmacochemistry analysis, which made a foundation for exploring the pharmacodynamic material basis of FF.
{"title":"Multicomponent qualitative and quantitative analysis of Farfarae Flos in serum using UHPLC-Q TOF-MS","authors":"Shuting Yu , Xuemei Qin , Zhenyu Li","doi":"10.1016/j.jchromb.2024.124145","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124145","url":null,"abstract":"<div><p>Farfarae Flos is a traditional herb widely employed for treating coughs, bronchitis, and asthmatic disorders. In the current study, we utilized SWATH and IDA data acquisition modes in combination with multiple data processing techniques to identify Farfarae Flos metabolites in mice serum. A total of 56 compounds were characterized, including 31 phenolic acids, 13 flavonoids, 11 sesquiterpenoids and 1 alkaloid. Further quantitative analysis was conducted on 12 absorbed metabolites, utilizing a newly developed and rigorously validated analytical method. Our approach demonstrated an acceptable level of specificity, accuracy, precision, and stability. When applied to compare the serum of mice treated with FF, all 12 metabolites showed the highest concentration at 0.5 h. Overall, this study presented a novel strategy for unraveling the active compounds of FF via serum pharmacochemistry analysis, which made a foundation for exploring the pharmacodynamic material basis of FF.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140918476","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 : 2024-04-30DOI: 10.1016/j.jchromb.2024.124144
Manal E. Alosaimi , Badriyah S. Alotaibi , Maram H Abduljabbar , Reem M. Alnemari , Atiah H. Almalki , Ahmed Serag
This research investigates the effects of the immunotherapeutic agent nivolumab on the metabolism of lung cancer cells (NCI-H1975) using GC–MS metabolomic profiling. Multivariate analysis such as unsupervised PCA and supervised OPLS-DA along with univariate analysis and pathway analysis were employed to explore the metabolomic data and identify altered metabolic pathways induced by nivolumab treatment. The study revealed distinct metabolic alterations in cancer cells, linked to proliferative and survival advantages, such as enhanced glycolysis, increased glutaminolysis, and modified amino acid metabolism. Key findings indicate elevated levels of glycolysis-related metabolites (glycine, alanine, pyruvate, and lactate) and TCA cycle intermediates (succinate, fumarate, malate) in cancer cells, with a significant decrease following nivolumab treatment. Additionally, lower levels of aspartic acid and citrate in cancer cells imply altered nucleotide synthesis and fatty acid production essential for tumor growth. Treatment with nivolumab also reduced oleic acid levels, indicative of its effect on disrupted lipid metabolism. Our research shows nivolumab's potential to modify metabolic pathways involved in lung cancer progression, suggesting its dual role in cancer therapy: as an immune response modulator and a metabolic pathway disruptor.
{"title":"Unveiling the altered metabolic pathways induced by nivolumab in non-small cell lung cancer via GC–MS metabolomics approach coupled with multivariate analysis","authors":"Manal E. Alosaimi , Badriyah S. Alotaibi , Maram H Abduljabbar , Reem M. Alnemari , Atiah H. Almalki , Ahmed Serag","doi":"10.1016/j.jchromb.2024.124144","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124144","url":null,"abstract":"<div><p>This research investigates the effects of the immunotherapeutic agent nivolumab on the metabolism of lung cancer cells (NCI-H1975) using GC–MS metabolomic profiling. Multivariate analysis such as unsupervised PCA and supervised OPLS-DA along with univariate analysis and pathway analysis were employed to explore the metabolomic data and identify altered metabolic pathways induced by nivolumab treatment. The study revealed distinct metabolic alterations in cancer cells, linked to proliferative and survival advantages, such as enhanced glycolysis, increased glutaminolysis, and modified amino acid metabolism. Key findings indicate elevated levels of glycolysis-related metabolites (glycine, alanine, pyruvate, and lactate) and TCA cycle intermediates (succinate, fumarate, malate) in cancer cells, with a significant decrease following nivolumab treatment. Additionally, lower levels of aspartic acid and citrate in cancer cells imply altered nucleotide synthesis and fatty acid production essential for tumor growth. Treatment with nivolumab also reduced oleic acid levels, indicative of its effect on disrupted lipid metabolism. Our research shows nivolumab's potential to modify metabolic pathways involved in lung cancer progression, suggesting its dual role in cancer therapy: as an immune response modulator and a metabolic pathway disruptor.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140824410","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 : 2024-04-28DOI: 10.1016/j.jchromb.2024.124132
Xiaoli Ma , Yutong Zou , Jian Zhong , Songlin Yu , Ling Qiu
The lack of individual pure standard has hampered the application of therapeutic drug monitoring (TDM) for multi-component antibiotics in clinical laboratories. Here, we aimed to develop an integrated identification-quantification (ID-Quant) workflow based on ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) to enable the comprehensive determination of all teicoplanin components without needing pure standards. The workflow comprises three steps. First, non-targeted MSE full scanning was used to detect and identify all potential ingredients. Then, characteristic product ions were selected to generate a quantitative time-of-flight multiple reaction monitoring (Tof-MRM) method. Finally, the constituent composition of teicoplanin injection was determined and utilized as an alternative reference standard to monitor the teicoplanin ingredients in human serum samples. As a result, nine teicoplanin analogs were identified from teicoplanin injection (Sanofi-Aventis, France). The overall performance of the Tof-MRM method was satisfactory in terms of linearity, precision, accuracy, and limits of detection. Utilizing the drug as standard, the individual concentrations for each component in patient serum were determined to be 0.120 µg/mL (A3-1), 0.020 µg/mL (N-1), 0.550 µg/mL (N-2), 0.730 µg/mL (A2-1), 4.26 µg/mL (A2-2,3), 4.79 µg/mL (A2-4,5), and 0.290 µg/mL (N-3), respectively. The distribution pattern of teicoplanin components was also discovered to differ from that in the drug injection. Overall, this integrated ID-Quant workflow based on UHPLC-QTOF-MS enables the robust quantitation of all teicoplanin analogs without the need for individual pure standard. This approach could help address the standard unavailability problem in the TDM of multi-component antibiotics.
{"title":"Integrated identification-quantification (ID-Quant) workflow utilizing UPLC-QTOF-MS for the therapeutic drug monitoring of multi-component antibiotics without pure standards: Validation using teicoplanin","authors":"Xiaoli Ma , Yutong Zou , Jian Zhong , Songlin Yu , Ling Qiu","doi":"10.1016/j.jchromb.2024.124132","DOIUrl":"https://doi.org/10.1016/j.jchromb.2024.124132","url":null,"abstract":"<div><p>The lack of individual pure standard has hampered the application of therapeutic drug monitoring (TDM) for multi-component antibiotics in clinical laboratories. Here, we aimed to develop an integrated identification-quantification (ID-Quant) workflow based on ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) to enable the comprehensive determination of all teicoplanin components without needing pure standards. The workflow comprises three steps. First, non-targeted MS<sup>E</sup> full scanning was used to detect and identify all potential ingredients. Then, characteristic product ions were selected to generate a quantitative time-of-flight multiple reaction monitoring (Tof-MRM) method. Finally, the constituent composition of teicoplanin injection was determined and utilized as an alternative reference standard to monitor the teicoplanin ingredients in human serum samples. As a result, nine teicoplanin analogs were identified from teicoplanin injection (Sanofi-Aventis, France). The overall performance of the Tof-MRM method was satisfactory in terms of linearity, precision, accuracy, and limits of detection. Utilizing the drug as standard, the individual concentrations for each component in patient serum were determined to be 0.120 µg/mL (A3-1), 0.020 µg/mL (N-1), 0.550 µg/mL (N-2), 0.730 µg/mL (A2-1), 4.26 µg/mL (A2-2,3), 4.79 µg/mL (A2-4,5), and 0.290 µg/mL (N-3), respectively. The distribution pattern of teicoplanin components was also discovered to differ from that in the drug injection. Overall, this integrated ID-Quant workflow based on UHPLC-QTOF-MS enables the robust quantitation of all teicoplanin analogs without the need for individual pure standard. This approach could help address the standard unavailability problem in the TDM of multi-component antibiotics.</p></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140843831","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}