Pablo E. Ramos-Bermúdez, Satomy Pousa, Paulo Carvalho, Rodrigo Soares Caldeira Brant, Michel Batista, Hironobu Hojo, Hilda E. Garay, Abel Roscoe, Alina Rodríguez Mallón, Vladimir Besada, Toshifumi Takao, Luis Javier González
RATIONALE
Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide–thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable.
METHODS
Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography–MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30.
RESULTS
A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker.
CONCLUSIONS
Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody–drug conjugates.
{"title":"A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines","authors":"Pablo E. Ramos-Bermúdez, Satomy Pousa, Paulo Carvalho, Rodrigo Soares Caldeira Brant, Michel Batista, Hironobu Hojo, Hilda E. Garay, Abel Roscoe, Alina Rodríguez Mallón, Vladimir Besada, Toshifumi Takao, Luis Javier González","doi":"10.1002/rcm.9859","DOIUrl":"10.1002/rcm.9859","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> RATIONALE</h3>\u0000 \u0000 <p>Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide–thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> METHODS</h3>\u0000 \u0000 <p>Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with <sup>18</sup>O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and <i>S</i>-alkylated, digested with trypsin and analyzed by liquid chromatography–MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> RESULTS</h3>\u0000 \u0000 <p>A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial <sup>18</sup>O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> CONCLUSIONS</h3>\u0000 \u0000 <p>Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody–drug conjugates.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141733034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jun Xiang, Qi Zhang, Qian Fan, Zekun Zhang, Haibo Huang, Aizhi Wu, Li Rong, Yumei Wang, Cuixian Zhang
Rational
Aconiti Lateralis Radix Praeparata (AC) is a traditional Chinese medicine with a long history of use. However, the current research on the material basis of AC and its processed products is still not comprehensive, especially the changes in lipo-diterpenoid alkaloids (LDAs) that can be hydrolyzed into diester-diterpenoid alkaloids in AC before and after processing. This study aimed to provide material basis guidance for the clinical use of AC and its processed products by comprehensively analyzing the changes in substances between AC and its processed products.
Methods
An ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS/MS) approach was optimized to chemical profiling. The MS data were processed using molecular networking combined with the in-house library database to fast characterize the compounds. Multivariate statistical methods were adopted to determine the dissimilarities of components in AC and its processed products.
Results
A total of 310 compounds were tentatively identified from AC, including 109 potential new alkaloids, of which 98 were potential novel LPAs. A metabolomics approach was applied to find the characteristic marker components. As a result, 52 potential chemical markers were selected to distinguish the AC samples of different extraction methods and 42 potential chemical markers for differentiating between AC and its processed products were selected.
Conclusion
The results indicate that UHPLC/Q-TOF-MS/MS and Global Natural Products Social Molecular Networking coupled with multivariate analysis strategies was a powerful tool to rapidly identify and screen the chemical markers of alkaloids between the AC samples and its processed products. These results also indicate that the toxicity of water extracts of AC and its processed products were decreased. This research not only guides the clinical safe use of AC and its processed products, but also extends the application of the molecular networking strategy in traditional herbal medicine.
理由附子（AC）是一种传统中药，具有悠久的使用历史。然而，目前对阿胶及其加工品的物质基础研究尚不全面，尤其是对阿胶中可水解成二酯二萜生物碱的脂二萜生物碱（LDAs）在加工前后的变化研究尚少。本研究旨在通过全面分析交流及其加工品的物质变化，为交流及其加工品的临床使用提供物质基础指导：方法：优化了超高效液相色谱-四极杆飞行时间质谱（UHPLC/Q-TOF-MS/MS）的化学分析方法。质谱数据经分子网络处理后与内部库数据库相结合，可快速确定化合物的特征。采用多元统计方法确定交流电及其加工产品中成分的相似性：结果：从 AC 中初步鉴定出了 310 种化合物，包括 109 种潜在的新生物碱，其中 98 种是潜在的新型 LPA。采用代谢组学方法寻找特征标记成分。结果，选出了 52 个潜在的化学标记物来区分不同提取方法的 AC 样品，并选出了 42 个潜在的化学标记物来区分 AC 及其加工产品：结果表明，UHPLC/Q-TOF-MS/MS 和全球天然产品社会分子网络以及多元分析策略是快速鉴定和筛选 AC 样品及其加工产品中生物碱化学标记物的有力工具。这些结果还表明，AC 水提取物及其加工产品的毒性均有所降低。这项研究不仅为交流及其加工品的临床安全使用提供了指导，还拓展了分子网络策略在传统草药中的应用。
{"title":"Structural characterization and screening of chemical markers of alkaloids in Aconiti lateralis radix Praeparata and its processed products by UHPLC/Q-TOF-MS/MS and GNPS combining multivariate statistical methods based on the clinic","authors":"Jun Xiang, Qi Zhang, Qian Fan, Zekun Zhang, Haibo Huang, Aizhi Wu, Li Rong, Yumei Wang, Cuixian Zhang","doi":"10.1002/rcm.9857","DOIUrl":"10.1002/rcm.9857","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> Rational</h3>\u0000 \u0000 <p><i>Aconiti Lateralis Radix Praeparata</i> (AC) is a traditional Chinese medicine with a long history of use. However, the current research on the material basis of AC and its processed products is still not comprehensive, especially the changes in lipo-diterpenoid alkaloids (LDAs) that can be hydrolyzed into diester-diterpenoid alkaloids in AC before and after processing. This study aimed to provide material basis guidance for the clinical use of AC and its processed products by comprehensively analyzing the changes in substances between AC and its processed products.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>An ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS/MS) approach was optimized to chemical profiling. The MS data were processed using molecular networking combined with the in-house library database to fast characterize the compounds. Multivariate statistical methods were adopted to determine the dissimilarities of components in AC and its processed products.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>A total of 310 compounds were tentatively identified from AC, including 109 potential new alkaloids, of which 98 were potential novel LPAs. A metabolomics approach was applied to find the characteristic marker components. As a result, 52 potential chemical markers were selected to distinguish the AC samples of different extraction methods and 42 potential chemical markers for differentiating between AC and its processed products were selected.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>The results indicate that UHPLC/Q-TOF-MS/MS and Global Natural Products Social Molecular Networking coupled with multivariate analysis strategies was a powerful tool to rapidly identify and screen the chemical markers of alkaloids between the AC samples and its processed products. These results also indicate that the toxicity of water extracts of AC and its processed products were decreased. This research not only guides the clinical safe use of AC and its processed products, but also extends the application of the molecular networking strategy in traditional herbal medicine.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141632127","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The detection of organic nitrogen compounds in exhaled breath is expected to provide an early warning of diseases such as kidney disease. Detecting these trace disease markers in exhaled breath with complex composition and high moisture content is a challenge. Surface ionization (SI) shows a highly selective ionization of organic nitrogen compounds, and it is a good candidate for breath analysis combined with ion mobility spectrometry (IMS).
Methods
A stepwise SI method of low-temperature adsorption/high-temperature ionization was proposed, and trimethylamine (TMA) was detected when combined with an ion mobility spectrometer. TMA at different concentrations and humidity levels and spiked in human breath was detected to evaluate the method's properties.
Results
TMA with concentrations from 2 to 200 ppb was detected. The peak intensity of the TMA characteristic ions was linearly related to the “e” exponent of the concentration with a curve fit of 0.996. A standard deviation of less than 0.306% was obtained with 10 replicate analyses of 10 ppb TMA. The signal intensity difference between dry and wet (relative humidity > 93%) TMA samples is only 2.7%, and the recovery rate of the sample was 106.819%.
Conclusions
SI-IMS based on the stepwise SI method has the advantages of low ionization temperature, high detection sensitivity, strong resistance to humidity interference, and good repeatability. It is a promising method for detecting organic nitrogen compounds in exhaled breath.
{"title":"A stepwise surface ionization method for ion mobility spectrometry","authors":"Jianhua Lin, Xiaoguang Gao, Jian Jia, Xiuli He","doi":"10.1002/rcm.9862","DOIUrl":"10.1002/rcm.9862","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>The detection of organic nitrogen compounds in exhaled breath is expected to provide an early warning of diseases such as kidney disease. Detecting these trace disease markers in exhaled breath with complex composition and high moisture content is a challenge. Surface ionization (SI) shows a highly selective ionization of organic nitrogen compounds, and it is a good candidate for breath analysis combined with ion mobility spectrometry (IMS).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>A stepwise SI method of low-temperature adsorption/high-temperature ionization was proposed, and trimethylamine (TMA) was detected when combined with an ion mobility spectrometer. TMA at different concentrations and humidity levels and spiked in human breath was detected to evaluate the method's properties.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>TMA with concentrations from 2 to 200 ppb was detected. The peak intensity of the TMA characteristic ions was linearly related to the “e” exponent of the concentration with a curve fit of 0.996. A standard deviation of less than 0.306% was obtained with 10 replicate analyses of 10 ppb TMA. The signal intensity difference between dry and wet (relative humidity > 93%) TMA samples is only 2.7%, and the recovery rate of the sample was 106.819%.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>SI-IMS based on the stepwise SI method has the advantages of low ionization temperature, high detection sensitivity, strong resistance to humidity interference, and good repeatability. It is a promising method for detecting organic nitrogen compounds in exhaled breath.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141615393","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Younes Valadbeigi, Fatemeh Mirzahosseini, Vahideh Ilbeigi, Stefan Matejcik
Rationale
Compounds like caffeine metabolites with more than one proton acceptor site can produce a mixture of isomeric protonated ions (protomers) in electrospray ionization and atmospheric pressure chemical ionization (APCI) ion sources. Discrimination between the protomers is of interest as the charge location influences ion structure and chemical and physical properties.
Methods
Protonation of caffeine in an APCI ion source was studied using ion mobility spectrometry. The hydronium ions, H3O+(H2O)n, are the main reactant ions in the APCI ion source. Different dopant gases including NO2, NH3, and CH3NH2 were used to produce new reactant ions NO+, NH4+, and CH3NH3+, respectively. Density functional theory was employed to explain the experimental results and calculate the energies of the ionization reactions.
Results
The ion mobility spectrum of caffeine showed three peaks. In the presence of NO2 dopant and NO+ reactant ion, caffeine was ionized via charge transfer and formation of M+ ion. As NH3 and CH3NH2 are stronger bases than H2O, the reactant ions NH4+ and CH3NH3+ selectively protonated the more basic site of caffeine, that is, the imidazole nitrogen. Using these dopants, we could attribute the first ion mobility peak to M+ ion, the second peak to the protonation of caffeine at the carbonyl oxygen atom, and the third peak to the protonation of the imidazole nitrogen atom. The calculated collisional cross-sections of M+ and the protomers of caffeine confirmed the peaks' assignment.
Conclusions
The criterion for the selection of an appropriate dopant is that its proton affinity (PA) should be between those of the proton acceptor sites of the molecule studied.
{"title":"Using dopants in the atmospheric pressure chemical ionization ion source to determine the site of protonation by ion mobility spectrometry","authors":"Younes Valadbeigi, Fatemeh Mirzahosseini, Vahideh Ilbeigi, Stefan Matejcik","doi":"10.1002/rcm.9858","DOIUrl":"10.1002/rcm.9858","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>Compounds like caffeine metabolites with more than one proton acceptor site can produce a mixture of isomeric protonated ions (protomers) in electrospray ionization and atmospheric pressure chemical ionization (APCI) ion sources. Discrimination between the protomers is of interest as the charge location influences ion structure and chemical and physical properties.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Protonation of caffeine in an APCI ion source was studied using ion mobility spectrometry. The hydronium ions, H<sub>3</sub>O<sup>+</sup>(H<sub>2</sub>O)<sub><i>n</i></sub>, are the main reactant ions in the APCI ion source. Different dopant gases including NO<sub>2</sub>, NH<sub>3</sub>, and CH<sub>3</sub>NH<sub>2</sub> were used to produce new reactant ions NO<sup>+</sup>, NH<sub>4</sub><sup>+</sup>, and CH<sub>3</sub>NH<sub>3</sub><sup>+</sup>, respectively. Density functional theory was employed to explain the experimental results and calculate the energies of the ionization reactions.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The ion mobility spectrum of caffeine showed three peaks. In the presence of NO<sub>2</sub> dopant and NO<sup>+</sup> reactant ion, caffeine was ionized via charge transfer and formation of M<sup>+</sup> ion. As NH<sub>3</sub> and CH<sub>3</sub>NH<sub>2</sub> are stronger bases than H<sub>2</sub>O, the reactant ions NH<sub>4</sub><sup>+</sup> and CH<sub>3</sub>NH<sub>3</sub><sup>+</sup> selectively protonated the more basic site of caffeine, that is, the imidazole nitrogen. Using these dopants, we could attribute the first ion mobility peak to M<sup>+</sup> ion, the second peak to the protonation of caffeine at the carbonyl oxygen atom, and the third peak to the protonation of the imidazole nitrogen atom. The calculated collisional cross-sections of M<sup>+</sup> and the protomers of caffeine confirmed the peaks' assignment.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The criterion for the selection of an appropriate dopant is that its proton affinity (PA) should be between those of the proton acceptor sites of the molecule studied.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141615395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Irina V. Minenkova, Viacheslav V. Lebedev, Alexey K. Buryak
Rationale
Phosphorotungstic acid (PTA) has many applications, especially in the field of catalysis, due to its structural properties. However, the structure of PTA is studied mainly using theoretical methods. Matrix-assisted laser desorption/ionization (MALDI) has the potential to be an effective method for the experimental study of heteropolyacids. Limitations of MALDI are the high molecular weight of the particles and the complex distribution of isotopic peak intensities. Both problems can be solved by automatically identifying observed signals by generating hypothetical molecular formulas and estimating their isotopic distributions.
Methods
Phosphotungstic acid was studied under conditions of laser desorption/ionization in the absence and in the presence of the matrix. Three types of matrices were used: 2,5-dihydroxybenzoic acid in water, α-cyano-4-hydroxycinnamic acid in acetonitrile, and sinapic acid (SA) in tetrahydrofuran. Part of the peaks in the resulting mass spectra was identified using in-house developed software that implements the automated isotopic distribution brute force.
Results
The most informative mass spectra were obtained using SA as the matrix, which enabled the detection of particles containing PTA dimers for the first time. The compositions of particles incorporating PTA dimers were determined in an automated manner and can be written as [H3PW12O40]2·2H2O (m/z = 5791.2 Da) and [H3PW12O40]2·4H2O (m/z = 5836.5 Da). Other observed species included (WO3)n·PO3−, HPO2·(WO3)n, and WO2·(WO3)n clusters, with the latter containing W in mixed oxidation states.
Conclusions
The combined use of MALDI and an automated identification procedure provided valuable experimental data on the structure and fragmentation of phosphotungstic acid. To the best of our knowledge, this study was the first to report on particles containing phosphotungstic acid dimers.
{"title":"Application of matrix-assisted laser desorption/ionization in the studies of phosphotungstic acid","authors":"Irina V. Minenkova, Viacheslav V. Lebedev, Alexey K. Buryak","doi":"10.1002/rcm.9870","DOIUrl":"10.1002/rcm.9870","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>Phosphorotungstic acid (PTA) has many applications, especially in the field of catalysis, due to its structural properties. However, the structure of PTA is studied mainly using theoretical methods. Matrix-assisted laser desorption/ionization (MALDI) has the potential to be an effective method for the experimental study of heteropolyacids. Limitations of MALDI are the high molecular weight of the particles and the complex distribution of isotopic peak intensities. Both problems can be solved by automatically identifying observed signals by generating hypothetical molecular formulas and estimating their isotopic distributions.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Phosphotungstic acid was studied under conditions of laser desorption/ionization in the absence and in the presence of the matrix. Three types of matrices were used: 2,5-dihydroxybenzoic acid in water, α-cyano-4-hydroxycinnamic acid in acetonitrile, and sinapic acid (SA) in tetrahydrofuran. Part of the peaks in the resulting mass spectra was identified using in-house developed software that implements the automated isotopic distribution brute force.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The most informative mass spectra were obtained using SA as the matrix, which enabled the detection of particles containing PTA dimers for the first time. The compositions of particles incorporating PTA dimers were determined in an automated manner and can be written as [H<sub>3</sub>PW<sub>12</sub>O<sub>40</sub>]<sub>2</sub>·2H<sub>2</sub>O (<i>m/z</i> = 5791.2 Da) and [H<sub>3</sub>PW<sub>12</sub>O<sub>40</sub>]<sub>2</sub>·4H<sub>2</sub>O (<i>m/z</i> = 5836.5 Da). Other observed species included (WO<sub>3</sub>)<sub><i>n</i></sub>·PO<sub>3</sub><sup>−</sup>, HPO<sub>2</sub>·(WO<sub>3</sub>)<sub><i>n</i></sub>, and WO<sub>2</sub>·(WO<sub>3</sub>)<sub><i>n</i></sub> clusters, with the latter containing W in mixed oxidation states.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The combined use of MALDI and an automated identification procedure provided valuable experimental data on the structure and fragmentation of phosphotungstic acid. To the best of our knowledge, this study was the first to report on particles containing phosphotungstic acid dimers.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141615394","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Juliana F. Gonzalez, Noela Sánchez-Carnero, Esteban Frere, Pablo Yorio, Javier E. Ciancio
Rationale
Understanding the migration of marine animals is hindered by the limitations of traditional tracking methods. It is therefore crucial to develop alternative methods. Stable isotope-based tracking has proven useful for this task, although it requires detailed isoscapes in the focal area. Here, we present predator-based isoscapes of the coastal zone of the Patagonian Shelf Large Marine Ecosystem (PSLME), which offers a novel tool for geolocation.
Methods
Whole-blood samples from breeding Magellanic penguins nesting at 11 colonies were used to create δ15N and δ13C isoscapes. Isotopic values were assigned to random positions inside their corresponding foraging area. Spatial analysis and data interpolation resulted in δ15N and δ13C isoscapes for the coastal zone of the PSLME, which were validated through cross-validation.
Results
The isoscapes mean standard error ranged from 0.05 to 0.41 for δ15N and from 0.07 to 0.3 for δ13C, similar to the error range of the mass spectrometer used for measuring isotope ratios. Predictive surfaces reflected the latitudinal trends, with δ13C and δ15N values increasing northwards. δ13C values showed a strong latitudinal gradient, while δ15N values had two distinct domains, with higher values in the north. The error surface indicated the highest certainty within 130 km from the shore and within the reported Magellanic penguin foraging areas.
Conclusions
Both isoscapes revealed strong spatial variation. The δ13C isoscape showed a latitudinal gradient, consistent with patterns in other oceans. The δ15N isoscape clearly separated northern and southern colonies, likely influenced by nitrogen sources. The error obtained fell within the measurement error ranges, adding credibility to the models.
{"title":"Developing δ15N and δ13C isoscapes using whole blood from Magellanic penguins, Spheniscus magellanicus","authors":"Juliana F. Gonzalez, Noela Sánchez-Carnero, Esteban Frere, Pablo Yorio, Javier E. Ciancio","doi":"10.1002/rcm.9860","DOIUrl":"10.1002/rcm.9860","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>Understanding the migration of marine animals is hindered by the limitations of traditional tracking methods. It is therefore crucial to develop alternative methods. Stable isotope-based tracking has proven useful for this task, although it requires detailed isoscapes in the focal area. Here, we present predator-based isoscapes of the coastal zone of the Patagonian Shelf Large Marine Ecosystem (PSLME), which offers a novel tool for geolocation.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Whole-blood samples from breeding Magellanic penguins nesting at 11 colonies were used to create δ<sup>15</sup>N and δ<sup>13</sup>C isoscapes. Isotopic values were assigned to random positions inside their corresponding foraging area. Spatial analysis and data interpolation resulted in δ<sup>15</sup>N and δ<sup>13</sup>C isoscapes for the coastal zone of the PSLME, which were validated through cross-validation.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The isoscapes mean standard error ranged from 0.05 to 0.41 for δ<sup>15</sup>N and from 0.07 to 0.3 for δ<sup>13</sup>C, similar to the error range of the mass spectrometer used for measuring isotope ratios. Predictive surfaces reflected the latitudinal trends, with δ<sup>13</sup>C and δ<sup>15</sup>N values increasing northwards. δ<sup>13</sup>C values showed a strong latitudinal gradient, while δ<sup>15</sup>N values had two distinct domains, with higher values in the north. The error surface indicated the highest certainty within 130 km from the shore and within the reported Magellanic penguin foraging areas.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>Both isoscapes revealed strong spatial variation. The δ<sup>13</sup>C isoscape showed a latitudinal gradient, consistent with patterns in other oceans. The δ<sup>15</sup>N isoscape clearly separated northern and southern colonies, likely influenced by nitrogen sources. The error obtained fell within the measurement error ranges, adding credibility to the models.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mégane Moreau, Serge Auger, Pierre Picard, Jean Lacoursière, Pedro A. Segura
Rationale
Rivaroxaban is an anticoagulant prescribed to patients who are at risk of medical conditions such as deep-vein thrombosis, pulmonary embolisms, and strokes caused by blood clots. The administration of this drug is monitored to adjust the dosage and evaluate patients' blood concentration. Rapid quantification of this drug in plasma could make it possible to ensure that the dose present in the blood of patients does not represent a danger for the medical intervention to be carried out.
Methods
Liquid chromatography–tandem mass spectrometry is usually employed to quantify rivaroxaban in blood, plasma, and serum. Here, an alternative method of analysis based on laser diode thermal desorption–triple quadrupole mass spectrometry (LDTD-QqQMS) was developed and comprehensively validated. This new method allows the quantification of rivaroxaban in less than 13 s from sample to sample. The extraction of rivaroxaban in human serum was done by a salting-out liquid–liquid extraction with acetonitrile and a saturated sodium chloride solution.
Results
The proposed method allows the quantification of rivaroxaban in less than 13 s from sample to sample. During validation, all criteria were respected. The accuracy was <15% of the nominal value, the precision was <15%CV, and the recovery was ≥89.9%. There were no observed carryover or matrix effects. Analysis of the extracted samples established the stability of dry (24 h) and wet samples (1 week) when samples cannot be analyzed immediately, a considerable advantage in a clinical setting.
Conclusions
This method improves sample throughput by more than 1200% compared to liquid chromatography–tandem mass spectrometry methods of analysis of rivaroxaban and decreases analysis costs by reducing solvent consumption and instrument time.
{"title":"Development and validation of an ultrafast method of quantification of rivaroxaban in human serum using laser diode thermal desorption coupled to triple quadrupole mass spectrometry","authors":"Mégane Moreau, Serge Auger, Pierre Picard, Jean Lacoursière, Pedro A. Segura","doi":"10.1002/rcm.9855","DOIUrl":"10.1002/rcm.9855","url":null,"abstract":"<div>\u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>Rivaroxaban is an anticoagulant prescribed to patients who are at risk of medical conditions such as deep-vein thrombosis, pulmonary embolisms, and strokes caused by blood clots. The administration of this drug is monitored to adjust the dosage and evaluate patients' blood concentration. Rapid quantification of this drug in plasma could make it possible to ensure that the dose present in the blood of patients does not represent a danger for the medical intervention to be carried out.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Liquid chromatography–tandem mass spectrometry is usually employed to quantify rivaroxaban in blood, plasma, and serum. Here, an alternative method of analysis based on laser diode thermal desorption–triple quadrupole mass spectrometry (LDTD-QqQMS) was developed and comprehensively validated. This new method allows the quantification of rivaroxaban in less than 13 s from sample to sample. The extraction of rivaroxaban in human serum was done by a salting-out liquid–liquid extraction with acetonitrile and a saturated sodium chloride solution.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The proposed method allows the quantification of rivaroxaban in less than 13 s from sample to sample. During validation, all criteria were respected. The accuracy was <15% of the nominal value, the precision was <15%CV, and the recovery was ≥89.9%. There were no observed carryover or matrix effects. Analysis of the extracted samples established the stability of dry (24 h) and wet samples (1 week) when samples cannot be analyzed immediately, a considerable advantage in a clinical setting.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>This method improves sample throughput by more than 1200% compared to liquid chromatography–tandem mass spectrometry methods of analysis of rivaroxaban and decreases analysis costs by reducing solvent consumption and instrument time.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 17","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/rcm.9855","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The application of infliximab (IFX) to immune-mediated disease is limited by the significant individual variability and associated clinical nonresponse, emphasizing the importance of therapeutic drug monitoring (TDM). Because of the cross-reactivity, limited linear range, and high costs, the clinical application of the previous reported methods was limited. Here, an improved high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to address the issues.
Methods
This study developed an improved bioanalytical HPLC-MS/MS method coupling nanosurface and molecular-orientation limited proteolysis technology. The commercially available compound P14R was selected as the internal standard. This method was developed with fewer volume of reagents and was thoroughly validated. The validated method was applied to TDM in pediatric inflammatory bowel disease (IBD).
Results
Chromatography was performed using a Shim-pack GISS-HP C18 metal-free column (3 μm, 2.1 × 100 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile at 0.4 mL/min. Detection and quantitation were performed using electrospray ionization (ESI) and multiple reaction monitoring in the positive ion mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability. The method exhibited a linear dynamic range of 0.3–100 μg/mL, with intra- and inter-day precision and relative errors below 15%. The recovery and matrix effect were measured as 87.28%–89.72% and 41.98%–67.17%, respectively, which were effectively compensated by the internal standard. A total of 32 samples collected from 24 pediatric patients with IBD were analyzed using the validated method, and only 46.9% achieved the reported targeted trough level.
Conclusion
This study developed an improved HPLC-MS/MS method for the quantitative determination of IFX concentration in human plasma. The accurate, reliable, and cost-effective method was validated and utilized in the analysis of clinical samples. The results confirmed the importance of TDM on IFX and the clinical application prospects of the improved method.
{"title":"Improved high-performance liquid chromatography tandem mass spectrometry method for quantification of infliximab in pediatric plasma and its application in therapeutic drug monitoring","authors":"Boran Yu, Siyao Jin, Jiaqi Han, Xiaona Li, Jiamin Xu, Ning Sun, Liang Sun, Xiaoling Wang, Libo Zhao","doi":"10.1002/rcm.9865","DOIUrl":"10.1002/rcm.9865","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>The application of infliximab (IFX) to immune-mediated disease is limited by the significant individual variability and associated clinical nonresponse, emphasizing the importance of therapeutic drug monitoring (TDM). Because of the cross-reactivity, limited linear range, and high costs, the clinical application of the previous reported methods was limited. Here, an improved high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to address the issues.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>This study developed an improved bioanalytical HPLC-MS/MS method coupling nanosurface and molecular-orientation limited proteolysis technology. The commercially available compound P14R was selected as the internal standard. This method was developed with fewer volume of reagents and was thoroughly validated. The validated method was applied to TDM in pediatric inflammatory bowel disease (IBD).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Chromatography was performed using a Shim-pack GISS-HP C<sub>18</sub> metal-free column (3 μm, 2.1 × 100 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile at 0.4 mL/min. Detection and quantitation were performed using electrospray ionization (ESI) and multiple reaction monitoring in the positive ion mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability. The method exhibited a linear dynamic range of 0.3–100 μg/mL, with intra- and inter-day precision and relative errors below 15%. The recovery and matrix effect were measured as 87.28%–89.72% and 41.98%–67.17%, respectively, which were effectively compensated by the internal standard. A total of 32 samples collected from 24 pediatric patients with IBD were analyzed using the validated method, and only 46.9% achieved the reported targeted trough level.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>This study developed an improved HPLC-MS/MS method for the quantitative determination of IFX concentration in human plasma. The accurate, reliable, and cost-effective method was validated and utilized in the analysis of clinical samples. The results confirmed the importance of TDM on IFX and the clinical application prospects of the improved method.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141562225","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression.
Methods
We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels.
Results
The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes.
Conclusions
The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.
{"title":"Using peptide barcodes for simultaneous profiling of protein expression from mRNA","authors":"Shun Kumano, Kazuki Tanaka, Rena Akahori, Akiko Yanagiya, Akihiro Nojima","doi":"10.1002/rcm.9867","DOIUrl":"10.1002/rcm.9867","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.</p>\u0000 </section>\u0000 </div>","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141553764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Adamantios Krokos, Amvrosios Orfanidis, Orthodoxia Mastrogianni, Foteini Mitsa, Maria Avgeri, Maria Eboriadou, Georgios Theodoridis, Nikolaos Raikos
<div> <section> <h3> Rationale</h3> <p>Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine's metabolite cotinine (COT) in urine samples, using gas chromatography–mass spectrometry (GC–MS).</p> </section> <section> <h3> Methods</h3> <p>Analysis was performed using a gas chromatograph Agilent Technologies 7890A with an MS 5975C inert XL, EI/CI MSD with Triple-Axis detector. For sample preparation, liquid–liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. Injection of 2 μL was performed using GC–MS, and selected ion monitoring (SIM) analysis was performed with the following ions (<i>m/z</i>): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and 9.743 min for COT.</p> </section> <section> <h3> Results</h3> <p>The method was validated following international principles, assessing characteristics such as absolute recovery, carryover, linearity, specificity, selectivity, accuracy, precision, and stability. The method showed a linear dynamic range from 0.5 to 50 ng/mL, and the limits of detection and quantification were for both NCOT and COT 0.2 and 0.5 ng/mL, respectively. Validation results were found satisfactory. Finally, the method was applied to the analysis of 60 clinical pediatric samples obtained from Aristotle University's pediatric clinic to check for possible exposure to smoke. Concentration levels ranged between 0.5 and 16.2 ng/mL for NCOT and between 1.0 and 25.1 ng/mL for COT.</p> </section> <section> <h3> Conclusions</h3> <p>A rapid, sensitive, accurate, and simple method was developed and used as a tool for the confirmation of passive smoking in children. It is the first method applied to the analysis of such samples belonging to nonsmokers of young age. The total runtime of the GC–MS analysis was short (20 min), and the pretreatment protocol was simple, giving the ability for analysis of a large number of s
{"title":"Gas chromatography–mass spectrometry determination of nicotine and cotinine in urine: A study of the effect of passive smoking","authors":"Adamantios Krokos, Amvrosios Orfanidis, Orthodoxia Mastrogianni, Foteini Mitsa, Maria Avgeri, Maria Eboriadou, Georgios Theodoridis, Nikolaos Raikos","doi":"10.1002/rcm.9864","DOIUrl":"10.1002/rcm.9864","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Rationale</h3>\u0000 \u0000 <p>Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine's metabolite cotinine (COT) in urine samples, using gas chromatography–mass spectrometry (GC–MS).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Analysis was performed using a gas chromatograph Agilent Technologies 7890A with an MS 5975C inert XL, EI/CI MSD with Triple-Axis detector. For sample preparation, liquid–liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. Injection of 2 μL was performed using GC–MS, and selected ion monitoring (SIM) analysis was performed with the following ions (<i>m/z</i>): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and 9.743 min for COT.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The method was validated following international principles, assessing characteristics such as absolute recovery, carryover, linearity, specificity, selectivity, accuracy, precision, and stability. The method showed a linear dynamic range from 0.5 to 50 ng/mL, and the limits of detection and quantification were for both NCOT and COT 0.2 and 0.5 ng/mL, respectively. Validation results were found satisfactory. Finally, the method was applied to the analysis of 60 clinical pediatric samples obtained from Aristotle University's pediatric clinic to check for possible exposure to smoke. Concentration levels ranged between 0.5 and 16.2 ng/mL for NCOT and between 1.0 and 25.1 ng/mL for COT.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>A rapid, sensitive, accurate, and simple method was developed and used as a tool for the confirmation of passive smoking in children. It is the first method applied to the analysis of such samples belonging to nonsmokers of young age. The total runtime of the GC–MS analysis was short (20 min), and the pretreatment protocol was simple, giving the ability for analysis of a large number of s","PeriodicalId":225,"journal":{"name":"Rapid Communications in Mass Spectrometry","volume":"38 18","pages":""},"PeriodicalIF":1.8,"publicationDate":"2024-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141553763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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