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Barely three months into the new year and we are happy to announce a monumental milestone reached - 150 million downloads.
\n\nThis achievement solidifies IntechOpen’s place as a pioneer in Open Access publishing and the home to some of the most relevant scientific research available through Open Access.
\n\nWe are so proud to have worked with so many bright minds throughout the years who have helped us spread knowledge through the power of Open Access and we look forward to continuing to support some of the greatest thinkers of our day.
\n\nThank you for making IntechOpen your place of learning, sharing, and discovery, and here’s to 150 million more!
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\r\n\tRapid urbanization is one of the most important global change problems that land-use planners are facing worldwide. With increased population growth and urbanization, cities around the world are becoming more affluent and putting even greater pressures on various land uses. The greatest challenges include managing traffic and transportation, the urban sprawl of cities, and affordable housing in ways that can improve people’s health and social well-being in a city-based framework, keeping in mind qualitative principles of equity, public participation, and sustainability. The proposed book hopes to bring together leading scholars in the field of transportation or engineering, land use planning, affordable housing, and smart cities growth, to discuss contemporary land use issues and challenges facing cities in both developed and developing countries. The book is also intended to serve as important reference material for academics, land use planning professionals, and students around the globe seeking to understand contemporary land-use problems and innovative solutions.
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He also served on the Technical Advisory Team that advised the government of Ghana on the preparation of a 40-year development plan for the country.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"137858",title:"Dr.",name:"Seth",middleName:null,surname:"Appiah-Opoku",slug:"seth-appiah-opoku",fullName:"Seth Appiah-Opoku",profilePictureURL:"https://mts.intechopen.com/storage/users/137858/images/system/137858.jpg",biography:"Dr. Seth Appiah-Opoku is a Professor of Geography at the University of Alabama, Tuscaloosa, AL, USA. He teaches World Regional Geography, Regional Geography of Africa, Environmental Management, Land Use Regulation, Principles of Planning, Regional Planning and Analysis, and also the Ghana Summer Abroad course. He is a member of the American Institute of Certified Planners and the editor of three books - The Need for Indigenous Knowledge in Environmental Impact Assessment: The Case of Ghana (Edwin Mellen Press, NY, June 2005), Environmental Land Use Planning (IntechOpen, 2012), and International Development (IntechOpen, 2017). His research focuses on international development, urban planning, ecotourism, environmental impact assessment, and resource development. He serves on the Editorial Boards of the Journal of Environmental Impact Assessment Review and the Environment and Social Psychology Journal. He also served as the editor of the Journal of African Geographical Review from 2016 to 2018. He has published scholarly articles in several renowned journals including Environmental Management, Society and Natural Resources, Environmental Impact Assessment Review, Journal of Cultural Geography, and Plan Canada. He served on the Technical Advisory Team that advised the government of Ghana on the preparation of a 40-year development plan for the country.",institutionString:"University of Alabama, Tuscaloosa",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"3",institution:{name:"University of Alabama, Tuscaloosa",institutionURL:null,country:{name:"United States of America"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"12",title:"Environmental Sciences",slug:"environmental-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"429339",firstName:"Jelena",lastName:"Vrdoljak",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/429339/images/20012_n.jpg",email:"jelena.v@intechopen.com",biography:"As an Author Service Manager, my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"2358",title:"Environmental Land Use Planning",subtitle:null,isOpenForSubmission:!1,hash:"45c4591d49ed3ff918fe563a30203cb2",slug:"environmental-land-use-planning",bookSignature:"Seth Appiah-Opoku",coverURL:"https://cdn.intechopen.com/books/images_new/2358.jpg",editedByType:"Edited by",editors:[{id:"137858",title:"Dr.",name:"Seth",surname:"Appiah-Opoku",slug:"seth-appiah-opoku",fullName:"Seth Appiah-Opoku"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"5468",title:"International Development",subtitle:null,isOpenForSubmission:!1,hash:"df06431aaa20f810b8d187e0db9b807d",slug:"international-development",bookSignature:"Seth Appiah-Opoku",coverURL:"https://cdn.intechopen.com/books/images_new/5468.jpg",editedByType:"Edited by",editors:[{id:"137858",title:"Dr.",name:"Seth",surname:"Appiah-Opoku",slug:"seth-appiah-opoku",fullName:"Seth Appiah-Opoku"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"8013",title:"Land Use Change and Sustainability",subtitle:null,isOpenForSubmission:!1,hash:"6b3aee3b93d95ecd84c41753486f7a83",slug:"land-use-change-and-sustainability",bookSignature:"Seth Appiah-Opoku",coverURL:"https://cdn.intechopen.com/books/images_new/8013.jpg",editedByType:"Edited by",editors:[{id:"137858",title:"Dr.",name:"Seth",surname:"Appiah-Opoku",slug:"seth-appiah-opoku",fullName:"Seth Appiah-Opoku"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophile",surname:"Theophanides",slug:"theophile-theophanides",fullName:"Theophile Theophanides"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"66821",title:"Extracellular Vesicles in Cancer",doi:"10.5772/intechopen.85117",slug:"extracellular-vesicles-in-cancer",body:'\nExtracellular vesicles (EVs) are cell-derived membranous vesicles (from normal or cancerous cells) bearing packages of information within or on their surface. Their content can influence neighboring or remote cells, and therefore, EVs are considered to play an important role in intercellular communication [1]. Different functional molecules (proteins, mRNA, and microRNAs) are transferred between cells with the aid of EVs. The content of EVs is highly variable and dependent of the cell of origin. The EVs in human blood originate from platelets, leukocytes, erythrocytes, endothelial cells, vascular smooth muscle cells, and cancer cells (for review see [2]). It is now widely accepted that extracellular vesicles also represent a potential resource for biomarkers.
\nThe first study suggesting the existence of extracellular vesicles was carried out in 1946 [3]. In a 1967 report, membrane particles derived from activated platelets, termed “platelet dust,” were commonly considered as a waste product or cellular debris directly budded from the plasma membrane [4]. Both prokaryotes and higher eukaryotes can release EVs. Different terms are used to describe EVs due to varying methods of isolation and due to the biogenesis mechanism. The terminologies of EVs include microvesicles, dexosomes, texosomes, archaeosomes, argosomes, prostasomes, epididymosomes, and oncosomes [5]. Gradually, while building up knowledge about EVs, a need for its classification emerged and the International Society for Extracellular Vesicles (ISEV) was founded [6]. This society provided some criteria to classify EVs into three groups: microvesicles (MVs), exosomes, and apoptotic bodies (for details visit www.isev.org). These vesicles are secreted by both normal cells and cancerous cells as means of cell-to-cell communication. Alternatively, they may be prepared artificially from the engineered artificial lipid vesicles called liposomes in which EVs’ features, components, or cargos are incorporated and are the most likely to be useful for drug delivery [7].
\nEVs are actively involved in cell-to-cell communication, inflammation, chronic disease development and progression, pre-metastatic niche formation, and the metastatic organotropism of different tumor types [8]. Tumor-derived EVs (TEVs) have been reported to play major roles in the onset, progression, and metastasis of cancer, including ovarian [9], breast [10], colorectal [11, 12], prostate cancer [13], and melanoma [14, 15, 16].
\nHere, we review knowledge about EVs in cancer, with a focus on breast and ovarian cancers. We discuss the importance of the content of EVs (e.g., nucleic acids, and proteins) in cancer development, metastasis, and drug resistance.
\nReplacing EVs includes a heterogeneous population of membrane vesicles categorized depending on the mechanism by which they are released from cells. According to their size and mechanisms of biogenesis, EVs can be categorized into three classes: (a) exosomes, (b) ectosomes or shedding microvesicles, and (c) apoptotic bodies [17, 18]. Differentiation criteria are based on their size, content, and by a certain combination of markers (Figure 1 and Table 1). Cancerous cells have been described to release exosomes and ectosomes and some other additional subpopulations of EVs [19].
\nClassification of EVs based on their diameter (expressed in nm) (A) or on their mechanism of biogenesis (B).
\n | Exosomes | \nEctosomes | \nApoptotic bodies | \n
---|---|---|---|
Size | \n30–100 nm | \n100–1000 nm | \n500–4000 nm | \n
Sedimentation rate | \n100,000–120,000×g | \n16,000–20,000×g | \n5000–16,000×g | \n
Biogenesis | \nEndosomal pathway, accumulated within the multivesicular bodies, exocytosis [20] | \nGenerated directly from the plasma membrane by shedding [21] | \nCell fragmentation Blebbing or zeiosis—bulge of membrane by increasing the surface area through tearing [22] | \n
Types of generation | \nConstitutive | \nRegulated | \nRegulated | \n
Filtration | \n20–200 nm | \n>200 nm | \n>1000 nm | \n
Intracellular storage | \nYes | \nNo | \nNo | \n
Marker proteins | \nCD 9, CD63 and CD61, tetraspanins, HSP70, HSP90, Alix, Rab5a/b [23, 24, 25] | \nTyA and C1a, ARF6 and VCAMP3, β1 integrins, selectins, CD40, MMP, lineage markers, and ezrin [26, 27, 28] | \nCalreticulin, TSP and C3b, and histones [29, 30]. | \n
Content | \nProteins, cholesterol, ceramide, noncoding RNA, mRNA, miRNA, and cytosol [31] | \nProteins, phosphatidylserine, cholesterol, mRNA, miRNA, and cytosol [31] | \nProteins, phosphatidylserine, DNA, rRNA, and cytosol [18] | \n
Organelles | \nNo | \nNo | \nYes | \n
Alternative names | \nProstasomes, tolerosomes, dexosomes, nanovesicles, exosome-like vesicles, and others [18, 32] | \nNanoparticles, microparticles, microvesicles, shedding vesicles, shedding bodies, exovesicles, secretory vesicles, and oncosomes [18] | \nApoptotic blebs [18] | \n
Impact on the immune system | \nImmunostimulators | \nImmunosuppressors | \nImmunosuppressors | \n
Classification of EVs based on size and their biogenesis.
Exosomes are EVs with multivesicular endosomal origin released by all cell types [33]. Exosomes are found in physiological fluids such as blood and plasma [34, 35], urine [36], cerebral fluid [37], saliva [38, 39], seminal fluid [40], breast milk [41, 42], and amniotic fluid [43, 44]. The presence of EVs has been reported in interstitial spaces since they are released by B cells [45], T cells [46], dendritic cells [47], platelets [48], Schwann cells [49], tumor cells [50], cardiomyocytes [51], endothelial cells [52], stem cells [50], and telocytes [53, 54, 55]. Exosomes are able to influence cells from the local environment and also distant target cells, thus regulating intercellular signaling [56]. Their size varies between 30 and 100 nm, and as membrane vesicles, they are delineated by a specific lipid bilayer similar to that of the cells they originate from [57]. Studies have shown that while normal human blood contains about 2000 trillion exosomes, the blood of cancer patients contains a double amount, about 4000 trillion exosomes [57]. In noncancerous cells, exosome secretion was suggested to play a role in cellular homeostasis by removing harmful cytoplasmic DNA of normal cells and in preventing viral hijacking of host cells by excreting viral DNA from cells as shown by Takahashi et al. [58].
\nThe plasma of cancer patients contains different types of exosomes, some released by normal cells and others released by cancerous cells, explaining the heterogeneity in size (30–150 nm) of the exosomal population [59]. Exosomes can be isolated from cancer patients’ plasma with a variety of methods [60]. They are based not only on classical techniques such as ultracentrifugation, but also on some modern ones such as size exclusion chromatography [61].
\nTumor cell-derived exosomes are able to promote inflammation and are able to compromise innate immunity by delivering different signals, which affect the proliferation, apoptosis, cytokine production, and reprogramming of T cells [62].
\nEctosomes are a heterogeneous vesicle population, ranging in diameter between 100 and 1000 nm. Discovered in approximately the same time as exosomes, in 1990s, ectosomes did not attract the same interest as the study of exosomes. While the interest in exosomes reached the maximum between 2008 and 2010, the ectosomes had its peak in 2012 [20, 63].
\nEctosomes are known under different names, which might be misleading (Table 1), while ecto is a prefix that means outwardly, externally, and is therefore suggestive of their way of forming. The mechanism of formation of ectosomes differs greatly from that of exosomes, as well as their cargo molecules. Ectosome formation does not require exocytosis. Ectosomes are formed by direct outward budding of the plasma membrane in specialized microdomains of the plasmalemma, the phenomenon known as microvesicle shedding [29]. They are released both by cells in normal resting state and by cells upon stimulation. Ectosome fusion with the plasma membrane of a recipient cell is followed by changes in antigens, enzymes, and other proteins in a specific site of plasmalemma, while their content release into the cytoplasm can alter the recipients’ cell gene expression [64, 65]. Tumor-derived ectosomes were shown to have immunosuppressive properties by inducing the chemotaxis of granulocytes, lymphocytes, and monocytes due to several chemokines (e.g., particularly IL-8) transported in ectosomes [66].
\nOncosomes are a particular type of ectosomes, excessively large, which can even reach 1000 nm, characteristic to advanced cancers. There is a confusion in the use of these terms in the literature and that is why we thought to treat oncosomes as a particular category of ectosomes. The generic name of ectosomes can include oncosomes, while the name of oncosomes excludes ectosomes released from normal cells. Oncosomes content is adapted to serve cancer metabolism, so they will contain enzymes involved in glucose, glutamine, and amino acid metabolism. Furthermore, oncosomes are enriched in proteins, which have a role in cell migration, angiogenesis, and cancer progression and metastasis [28]. Oncosomes allow intercellular transfer of oncogenes, hence the motivation to be considered as existing biomarkers in the blood or plasma of patients for the detection of cancer [67].
\nApoptotic bodies (ApoBDs) are the largest type of extracellular vesicles (typically 1–5 μm in diameter) visible during an apoptotic process. Kerr in 1972 proposed the term “apoptotic body” [68]. ApoBDs are released as blebs of cells undergoing apoptosis and consist of cytoplasm, organelles with or without a nuclear fragment. It has also been shown that ApoBDs can harbor proteins, lipids, DNA, rRNA, organelles, and cytosol [18]; this is the reason why the disassembly of an apoptotic cell into ApoBDs can mediate intercellular communication and may contribute to the development of various disease states [69]. These bodies are then phagocytosed by macrophages or neoplastic cells and degraded within phagolysosomes. Their formation has been proposed to play an important role in the clearance of apoptotic cells by phagocytes. Different cell types can generate ApoBDs via different mechanisms [70]. These ApoBDs can be classified based on cell-type-specific surface markers and content. Jiang et al. showed that ApoBDs share the same surface markers as their cell of origin; this is the reason why apoptotic bodies are very different and can be divided into specific subclasses [70].
\nApoBD occurs spontaneously in untreated malignant neoplasms, and is implicated in both physiological involution and atrophy of various tissues and organs. Pathological settings include inflammation [71], autoimmunity [72, 73, 74], viral infection [75], and tumorigenesis because they participate in the horizontal transfer of oncogenes due to their nuclear material content from the dying cells [76].
\nTumor masses are composed of cancer cells and stromal cells, in which one include mesenchymal cells, fibroblasts and immune cells, and extracellular matrix (ECM) components. All these cells emit EVs and participate in the creation of a unique tumor nanoenvironment. EVs are capable of horizontal transfer of bioactive content to interact with cells in the tumor microenvironment. These interactions can include fusion of the EV with the plasmalemma of the recipient cell or endocytosis of the EVs [77]. EVs represent the bidirectional way of interaction between stromal and cancer cells as a mean to exchange information and modify the tumor microenvironment. Therefore, the content of these vesicles is of great significance in the evolution of the cancer, since it was shown to modulate the complex signaling networks that facilitate tumor progression [78].
\nCirculating DNA can be found in free form or contained in EVs and is thought to be the future in cancer diagnosis and treatment monitoring. This will be possible because the DNA fragments contained in EVs are relatively intact (average 15 kbp) by comparison with the circulating cell-free one (average 130 bp) due to the protection offered by the lipid bilayer [79]. Vagner et al. showed in a very recent study that the majority of the extracellular DNA is contained in large oncosomes, rather than in exosomes, both in vitro and in the patients’ plasma and has all cancer-specific genomic alterations [80]. The majority of the DNA contained in tumor-derived exosomes is double stranded and represent the whole genomic DNA, suggesting its usefulness in identifying mutations present in parental tumor cells, as it was indicated by Thakur et al. [81]. Wyatt et al. showed that cancer-derived DNA is sufficient to identify the DNA alterations from metastatic tissue and is very important because it integrates somatic information from more than one metastatic lesion [82].
\nThe presence of retrotransposons, cDNAs, and ncRNAs has also been reported in EVs and appears to be a unique feature of tumor cells [83]. Several studies reported a correlation between increased retrotransposon activity and tumorigenesis [84]. For example, LINE-1 hypomethylation in various human cancers was intensively studied since it is considered to be an early event in tumorigenesis and to be linked with the induction of proto-oncogenes [85]. Loss of LINE-1 methylation was found to associate with more aggressive progression of colorectal cancer [86]. Moreover, LINE-1 hypomethylation level can be considered as an important epigenetic process, which became a potential prognostic factor for ovarian multistep carcinogenesis [87].
\nmiRNAs represent potential candidates responsible for influencing the tumor microenvironment; however, little is known about the mechanism by which they produce changes in the transcriptome of target cells [88].
\nProteins exported in EVs are signaling molecules that interfere in a whole series of processes such as cell metabolism, cell invasion and growth, angiogenesis, and mRNA processing [89]. Among these, it is worth mentioning that the epidermal growth factor receptor vIII (EGFRvIII), mutant Ras family members, or c-Met have been proposed as cancer biomarkers [90, 91]. Other proangiogenic regulators, such as VEGF and bFGF, are harbored in EVs shed from cancerous cells promoting new blood vessel formation [92].
\nMoreover, EVs also transfer proteases such as MMP-2, MMP-9, and MT1-MMP and become responsible for the partial degradation of the extracellular matrix [93]. The content is released in an acidic environment after the vesicle is stabilized in the extracellular matrix with the aid of β1 integrin adhesion molecules [94].
\nNaturally, EVs also contain a lipid component, which consists of the main membrane lipids: sphingomyelin, phosphatidylserine, and glycosphingolipids and cholesterol, but they also carry polyunsaturated fatty acids PUFAs, mainly arachidonic acid and linoleic acid [95, 96]. Sphingomyelin, as a component of the EVS, was firstly reported by Kim et al. who described its angiogenic properties [97] . In addition, it has been shown that the lipid content of exosomes suppresses critical cancer survival pathways such as notch leading to cancer cell death of human pancreatic tumoral SOJ-6 cells [98]. Moreover, other important signaling mediators, such as prostaglandins, arachidonic acid, phospholipase A2, and phospholipase C and D, are also found in EVs [99]. The prostaglandins found in breast-cancer-derived exosomes, such as PGE2, are responsible for promoting tumor growth by inducing the release of pro-inflammatory cytokines such as IL-6 and VEGF, which induce the accumulation of myeloid-derived suppressor cells capable to differentiate into macrophages in the tumor microenvironment [100, 101]. PGE2 indeed makes the connection between cancer and macrophages and can promote tumorigenesis by enhancing the expression of programmed cell death protein ligand 1 (PD-L1) responsible for tumor escape from immune system during cancer progression [102].
\nCancer stem cells (CSCs) are held directly responsible to promote cancer initiation and progression. Also, there are several studies showing their importance in therapy resistance, recurrence, and metastasis [103]. CSCs themselves do not exist as a static population, their stemness being supported by the mesenchymal stem cells, endothelial cells, fibroblasts, or immune cells by paracrine signaling [104]. For example, in breast cancer, the overexpression of the chemokines CXCL14 and CXCL12 in myoepithelial cells and myofibroblasts favors the metastasis [105]. Cancer-associated fibroblasts (CAF) release exosomes, which induce the stemness of breast cancer cell lines, developing an aggressive cancer cell phenotype [106]. Also, the ECM molecules are relevant for breast cancer colonization, which contribute to the control of CSC. In this sense, tenascin C, a protein in the ECM, contributes in the formation of the stem niche by protecting CSC from immune surveillance [107]. In breast cancer, high levels of tenascin C are associated with poor clinical outcome in breast cancer due to lung cancer metastasis [108].
\nThe role of EVs in tumorigenesis was described in various types of cancer, including ovarian [109, 110, 111] and breast cancers [112, 113]. EVs undergo several alterations in tumorigenesis, including changes in their biogenesis, release rate and/or protein content, incorporation of oncogenic and mutant macromolecules, mediated release of genomic DNA, and uptake of tumoral cells [114]. The transfer of DNA between apoptotic tumoral cells and other cells is important in tumorigenesis. In vitro, it was shown that apoptotic bodies derived from cancer cells are responsible for triggering the expression of oncogenes in fibroblasts due to the information contained in tumor-derived EVs [76].
\nIn ovarian or breast cancer, investigating the content of EVs might give important informations on tumorigenesis. To detail, exosomes released by IGROV1 ovarian cancer cells (with high content of RNA-binding proteins, such as LIN28A or LIN28B), but not by OV420 ovarian cancer cells, were taken up by HEK293 cells, contributing to the tumor development [109]. Moreover, in hypoxia conditions, SKOV3 human epithelial ovarian cancer cells release exosomes with high content of miR-940 that stimulate the M2 macrophage phenotype, and in turn, M2 subtype macrophages stimulate the tumor cell migration and proliferation [111]. The majority of circulating miRNA, packed in EVs, can be used as biomarkers in ovarian cancer, but their use is not only limited for diagnosing the existence of the cancer, but also being reliable markers for monitoring the tumor histology, stage, or the patient outcome [110].
\nThe content of EVs released from two human breast cancer cell lines, MCF-7 (less invasive) and MDA-MB-231 (more invasive), was analyzed, and approximately, 270 proteins were identified [113]. In circulating EVs, epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3) are the extracellular matrix (ECM) protein that was described to play a critical role in tumorigenesis by the activation of integrin-focal adhesion kinase (FAK) signaling cascade [113]. Breast-cancer-derived EVs (e.g., exosomes) present a cell-independent microRNA biogenesis from pre-miRNAs (like Dicer, AGO2, or TRBP) to mature miRNAs [112]. In particular, exosomes detected in the cells and sera of patients affected by breast cancer were shown to stimulate tumorigenesis in nontumoral epithelial cells by a Dicer-dependent mechanism [112]. It was also demonstrated that in the breast tumor microenvironment, there is a downregulation of the tumor suppressor p85α, which is clinically relevant in tumorigenesis, and the mechanism involves the loss of p85α expression in stromal fibroblasts promoting breast cancer progression by the epithelial-to-mesenchymal transition [111].
\nTumor microenvironment was described to undergo series of molecular and cellular changes to form the metastatic-designated sites, called pre-metastatic niche [115, 116]. The formation of pre-metastatic niche requires the cross talk between primary tumor-derived components, and the microenvironment of the host stromal components and of the tumor-mobilized bone-marrow-derived cells [117].
\nInterestingly, the role of EVs in metastatic niches can be exploited in novel therapeutic approaches. Indeed, technologies based on exosomes, separated from the ascitic fluid of ovarian cancer patients, embedded in a 3D scaffold metastatic trap, were successfully tested in murine models of ovarian metastasis in order to improve survival [118]. Numerous studies indicated that tumor-derived exosomes might play a role in promoting angiogenesis and modulation of the immune system [119, 120]. Moreover, exosomes derived from cancerous tumor are capable of remodeling the surrounding parenchyma, thus supporting tumor progression and the generation of the pre-metastatic niche [121, 122].
\nEVs have been described to play an essential role in the local and distant communication between cancer cells and their environment and in contributing to the progression of metastasis [123]. Although the function of EVs in metastasis is not completely understood, studies show that miRNAs isolated from EVs are actively involved in complex metastatic processes, including local invasion, angiogenesis, immune modulation, metastatic niche preparation, colonization, and dormancy [123].
\nEVs play an essential role in the tumor metastasis by ensuring the cross talk between tumor and the adipose tissue, and obesity was described to influence the metastatic behavior of tumors, especially in melanoma, breast, and ovarian cancers [124].
\nIn breast cancer, metastatic exosomes creating a facilitating local environment for metastasis was demonstrated, and annexin II contained in these exosomes contributes to this process by promoting angiogenesis [125].
\nNawaz et al. have recently done an extensive review on the role of EVs in ovarian cancer and concluded that the gaining of new insights into these mechanisms would contribute to the identification of new biomarkers among the ovarian-cancer-derived EVs and to the development of efficient EVs-based immunotherapies [126]. Proteomic analysis of exosomes derived from two human ovarian cancer cell lines (i.e., OVCAR-3 and IGROV1) indicated the presence of 2230 proteins, 1017 proteins being common for both cell lines, 380 proteins being newly reported compared to the ExoCarta database, and some of them being associated with tumorigenesis and metastasis and might represent promising biomarkers [127].
\nAdditionally, matrix metalloproteinase-1 (MMP1) might be a very good biomarker for the ovarian cancer due to its overexpression in ascites-derived EVs in correlation with the degree of malignancy and the low prognosis for the ovarian cancer patients [128]. Moreover, the peritoneal dissemination of ovarian cancer is facilitated by malignant EVs containing MMP1 derived from the ascites of patients, and EVs were demonstrated to induce apoptosis in mesothelial cells [128].
\nThe mechanisms of drug resistance development also involve the release of EVs from ovarian cancer cells upon exposure to drug (i.e., cisplatin) and induce invasiveness [129].
\nIn breast cancer, EVs can play two essential roles “diagnosis biomarkers” or “therapeutic targets.” Thus, breast cancer induces the release of exosomes from salivary glands, being potential markers for early diagnosis [130]. Interestingly, EVs serve as a cargo not only for nucleic acids and proteins, but also for anticancer drugs. Considering the critical contribution of EVs in facilitating tumorigenesis, metastasis, and drug resistance [130], they could be considered as potential therapeutic targets in breast cancer.
\nMoreover, the analysis of EVs can help to distinguish the “degree of aggressiveness” in breast cancer. To detail, EVs derived from the aggressive human breast cancer MDA-MB-231 cell line, but not from the less invasive human breast cancer MCF-7 cell line, were demonstrated to induce platelet activation and aggregation by tissue factor-independent and tissue factor-dependent procoagulant activities [131]. EVs have been demonstrated to be involved in the cross talk between neighboring cancer cells and to transmit phenotypic aggressive traits from one cell to another. To date, EVs released by Hs578Ts(i)8 triple-negative breast cancer cells were able to increase the invasion, proliferation, and migration characteristics of Hs578T cells [132].
\nIn different body fluids, especially plasma and serum, EVs biomarkers have been detected with great clinical value in various types of cancer, Table 2.
\nBiomarkers of EVs | \nSample | \nTypes of cancer | \nReference | \n
---|---|---|---|
TGF-beta1, MAGE3/6, and Claudin-4 | \nPlasma | \nOvarian cancer | \n[119, 133] | \n
EpCAM and annexin A3 | \nSerum | \n[134, 135, 136] | \n|
Alpha-1-antitrypsin and haptoglobin precursors | \nSerum | \nBreast cancer | \n[137] | \n
miR-21, miR-939, miR-373, and miR-1246 | \nPlasma | \n[58, 138, 139] | \n|
miR-1290 and miR-375 Survivin, CD9+, CD63+, and alpha-1-antitrypsin | \nPlasma | \nProstate cancer | \n[140, 141, 142, 143, 144] | \n
IL-8 and TGF-beta mRNAs | \nPlasma | \nGlioma | \n[60] | \n
miR-21 | \nCSF | \n[145] | \n|
miR-1246, miR-4644, miR-3976, and miR-4306 CD44v6, Tspan8, EpCAM, and CD104 | \nSerum | \nPancreatic cancer | \n[146] | \n
Alpha-1-antitrypsin, and histone H2B1K | \nUrine | \nUrothelial carcinoma | \n[147] | \n
long coding RNA CRNDE-h | \nSerum | \nLymph node and distant metastasis of colorectal cancer | \n[148] | \n
miR-21 | \nPlasma | \nEsophageal cancer | \n[149] | \n
miR-19a l | \nSerum | \nColorectal cancer | \n[150] | \n
Biomarkers contained in EVs relevant in different types of cancer.
The protein content of the EVs can be potentially used in the early detection of cancer as suggested in a pilot study by Smalley et al. [151]. The plasma levels of exosomal proteins represents an important biomarker that discriminates between ovarian cancer patients and normal ones, and their values correlate with the stage of the disease [119]. Among exosomal proteins, TGF-β1 and MAGE3/6 can be used as reliable biomarkers to discriminate between benign and malignant ovarian tumors, or to ascertain the efficacy of chemotherapy [119]. Although epithelial cell adhesion molecule (EpCAM) was demonstrated to promote epithelial-mesenchymal transition in advanced stages of endometrial cancer [152], studies indicated that EpCAM is not a robust biomarker to classify exosomes derived from benign and malignant ovarian tumors [134] or to detect early stages of the pathology [153]. Besides EpCAM, several exosomal proteins were identified to be overexpressed in ovarian cancer, including proliferation cell nuclear antigen (PCNA), tubulin beta-3 chain (TUBB3), epidermal growth factor receptor (EGFR), apolipoprotein E (APOE), claudin 3 (CLDN3), fatty acid synthase (FASN), ERBB2, and L1CAM (CD171) [127]. Additionally, claudin-4, but not claudin-3, is a valuable biomarker in the peripheral blood of ovarian cancer patients with almost 98% specificity [133]. Exosomal proteins can also represent important biomarkers for the evaluation of efficacy of therapies. Thus, annexin A3 can be employed for early detection of the resistance to platinum-based therapy in ovarian cancer patients [135, 136].
\nIn breast cancer, several studies identified various exosomal miRNAs as potential biomarkers correlated with tumor malignancy degree and prognosis. Indeed, exosomal miR-21 and miR-1246 had higher levels in plasma of breast cancer patients compared to control patients [138]. Additionally, high levels of exosomal miR-939 were associated with low outcome in patients with triple-negative breast cancer [139], and high levels of exosomal miR-373 were identified in triple-negative, estrogen–receptor- and progesterone-receptor-negative, breast cancer patients [58]. Moreover, an extensive proteomics analysis identified alpha1-antitrypsin and haptoglobin precursors as novel biomarkers in the serum of patients with infiltrating ductal breast carcinomas [137].
\nThe release of EVs has a calcium-dependent mechanism, and alterations in calcium signaling have been described in tumorigenesis, metastasis, or drug resistance in various types of cancer, including breast and ovarian cancers [154, 155]. Therefore, more attention should be paid to the calcium-dependent signaling cascades in different cancer stages in direct relationship with the cell-to-cell communication mechanisms of EVs in order to identify novel specific and reliable biomarkers.
\nEVs have a big potential for cancer therapy monitoring (Table 3). These are described as secreted lipid bilayer-enclosed lumens and are claimed to be valuable reservoirs of liquid biopsy biomarker [156]. EVs (mainly EVs-associated proteins and microRNAs) are proved to be the biomarkers in breast cancer diagnosis [157, 158].
\nSource of EVs | \nTherapeutic effect | \nReference | \n
---|---|---|
Tumor peptide-loaded dendriticcells-derived exosomes | \nImmunotherapy—because they suppress tumor growth | \n[159] | \n
EVs from the rat pancreatic adenocarcinoma cell line BSp73ASML | \nAdjuvant therapy in immunotherapy | \n[160] | \n
Tumor antigen containing EVs | \nActivates an antitumor response against OVA-transfected BL6–10 melanoma cells | \n[160] | \n
EV vaccine derived from colorectal cancer (NB4 cell—a human acute promyelocytic leukemia cell line) | \nActivates CTLs through self-derived dendritic cell activation | \n[161] | \n
EVs from self-derived dendritic cells | \nImmunotherapy in cases of unresectable nonsmall-cell lung cancer | \n[162] | \n
EVs from autologous self-derived dendritic cells | \nMetastatic melanoma patients | \n[163] | \n
EVs from ascites in combination with granulocyte macrophage colony-stimulating factor | \nImmunotherapy in colorectal cancer | \n[164] | \n
miR-9 in mesenchymal stem cell-derived EVs | \nChemosensitive in glioblastoma multiforme cells | \n[165] | \n
iRGD-Exos-doxorubicin | \nSuppressed breast tumor growth in an MDA-MB-231 tumor-bearing nude mouse model | \n[166] | \n
Curcumin-primed EVs from a mouse brain endothelial cell line | \nTreating endothelial cell dysfunction during hyperhomocysteinemia in vitro | \n[167] | \n
EVs and their role in therapeutic approaches in cancer.
EVs play an essential role in cellular communication both in physiological and pathological conditions. In pathological conditions, EVs have been implicated in cancer, spreading of viruses or other pathogens, altered immune response, development of neurodegenerative diseases, etc. In cancer, EVs ensure the cross talk between tumoral cells or between tumoral cells and nontumoral cells, and enable the development of multiple processes, including tumorigenesis, pre-metastatic niche formation, metastasis, and drug resistance. In ovarian and breast cancers, the involvement of EVs in all these processes of tumor evolution has been described and the analysis of EVs content is particularly useful for identifying biomarkers of the disease per se and, moreover, for the stage of the pathology evolution. However, there are still technical limitations for separation and/or analysis of EVs, and in clinical practice, the standardization of EVs-based reproductible protocols is required urgently. Considering the presence of EVs in such a variety of body fluids and tissues, an important conclusion is to consider EVs both as biomarkers and potential therapeutic targets (especially for immunotherapies) and to exploit them in the next future to improve the outcome of cancer patients.
\nTwo specific trends can be noticed in modern chemical analysis. One is the continuous demand for more sensitive and accurate analytical methods. The other is the desire for simpler methods that require as little as possible human intervention. One of the various procedures to make the analytical methods more sensitive and accurate is the use of specific chemical changes (e.g., derivatization) applied on the analytes or even on the whole sample. However, these changes frequently involve more human intervention than the direct use of advanced instrumentation. For this reason, the methods involving chemical changes such as derivatizations are not necessarily the first choice when selecting an analytical method. Nevertheless, in many cases, the benefits of derivatization are more important than the disadvantage of requiring human intervention, and for this reason, derivatization is still frequently used in the analytical practice. Also, modern GC, GC/MS (or GC/MS/MS) instrumentation may offer autosampling with the capability of adding reagents to the sample, as well as stirring, heating, and injecting the sample at specific time intervals in the GC system. This type of instrumentation may reduce significantly the human handling involved in derivatization.
\nVarious chemical changes can be performed on an analyte in order to make it suitable for a specific method of analysis. The most common is derivatization, but other chemical changes can be utilized, for example, pyrolytic decomposition and, in the case of polymers, polymer fragmentation using reagents. The choice depends on the nature of the analyte, the sample matrix, the intended changes in the analyte properties, and the analytical method to be used.
\nThe addition of a reagent on a sample may produce a chemical reaction only with the analytes without affecting the matrix. However, it is also possible that some matrix components are derivatized unintentionally. Usually, it is preferable to have only the analytes derivatized since in this way a better separation from the matrix is expected. Some derivatizations are used in the sample cleanup or concentration process. Also, the derivatization process may be combined with simultaneous extraction and concentration of the sample or may be followed by a second preparation step before the chromatographic analysis. More frequently, the derivatization is done to change the analyte properties for the core analytical procedure (GC, GC/MS, etc.).
\nDerivatization can be applied before the core chromatographic process or after it. Precolumn derivatization takes place before the separation and postcolumn derivatization after it. In GC precolumn derivatization is much more common and most derivatizations are performed “offline.” There are however derivatizations that can be done “online,” for example, in the injection port of the GC such as some methylations using tetramethyl ammonium hydroxide (TMAH). Postcolumn derivatizations are performed only for enhancing the detectability of the analytes. Typically, they must be done “online” and should be completed in the specific time frame needed by the analyte to reach the detector.
\nA wide variety of derivatization reagents and procedures are described in the literature, with the reagents carrying specific moieties that provide a desired property to the analytes, as well as with specific reactive groups that permit the reaction with the analyte. Multiple step derivatizations as well as derivatizations followed by a second one are known.
\nDerivatization is not always the first step in sample preparation. Sample preparation typically includes other operations, besides derivatization. Some of these steps are more complex such as sample cleanup or concentration and others more simple such as pH adjustments, addition of proton acceptors or donors, change of the medium (from one solvent to another), and addition of catalysts to enhance the derivatization, and these may be necessary for a successful derivatization.
\nAlthough derivatization is performed in order to make possible or to improve the results of a chemical analysis, there are also some disadvantages of using derivatization. Besides the potential need of more manpower for the analysis, the addition of more operations applied on the sample (including the analytes) can be a source of additional errors. In particular the involvement of a chemical reaction that may not be perfectly controlled can bring significant errors in the analytical results. To minimize the potential errors when using derivatization, specific aspects of the derivatization must be considered in its choice, such as the efficiency of the chemical reaction used in the derivatization, the stability of the derivatized analytes, the availability of reagents and necessary equipment, and the time necessary for performing the analysis. For a given analyte or group of analytes, the reaction with the derivatization reagent must be complete or at least close to complete, must take place in a length of time that is not prohibitive, and must have very little loss of the analyte with formation of artifacts or decomposition products. Only when such criteria are satisfied can a specific chosen derivatization be applied successfully.
\nThe application of derivatization in chromatography is the subject of many studies. Numerous derivatizations have been reported in journals (e.g.,
For GC analysis, the effect of derivatization can be beneficial in a variety of circumstances. Some of the most common uses of derivatization for improving the GC separation are the following:
(a) Derivatization that replaces active (polar) hydrogen atoms in the analyte to decrease its boiling point. The active hydrogens in a chemical compound typically enhance the capability to form hydrogen bonds and increase the compound polarity. For this reason, many compounds containing active (polar) hydrogens are not volatile, the volatility being necessary for using GC or GC/MS as a core analytical method. Derivatization can be used to replace active hydrogens from an analyte Y-H (or Y
In reaction (1), the reagent R-X contains an “active” group X and a group R that carries a desired property (e.g., lack of polarity for GC). Group R in the reagent can be a low molecular mass fragment such as CH3 or C2H5, a short-chain fluorinated alkyl in alkylation reactions, Si(CH3)3 or other silyl groups in silylations, COCH3 or short-chain fluorinated acyl groups in acylations, etc. An example of a chromatogram resulting from the GC/MS analysis of a silylated tobacco sample is given in Figure 1. Tobacco contains many hydroxy acids such as malic, trihydroxybutanoic, citric, quinic, glucuronic, and chlorogenic. Also, it contains monosaccharides (e.g., glucose, fructose), disaccharides (e.g., sucrose), and even trisaccharides. None of these compounds are volatile, having numerous active hydrogens. The replacement of these hydrogens with Si(CH3)3 by silylation renders these compounds volatile, and they can be analyzed by GC/MS as seen in Figure 1.
(b) Derivatization for enhancing the separation. Specific moieties added to an analyte may be necessary for enhancing the separation. This is frequently practiced for general GC separations and is also very useful for the separation of chiral molecules (see Section 4). The derivatized analytes may have significantly different properties from each other, for example, regarding polarity and implicitly in their boiling point, allowing separations that are difficult to achieve otherwise. Also, derivatization may generate more significant differences between the analytes and the matrix components.
(c) Derivatization that replaces active hydrogens in the analyte to improve the behavior of the analyte in the chromatographic separation. The chromatographic column (e.g., a capillary column coated with a bonded stationary phase) may display additional capability to interact with polar molecules, besides the intended interactions due to its bonded phase. This may come, for example, from the silica wall of the column. Secondary interactions taking place with only a portion of the molecules of the analyte generate peak tailing. This is exemplified in Figure 2 which shows a hypothetical case of two different types of interaction between the column and a specific molecular species.
(d) Derivatization for the improvement of stability of a compound. This stability may refer to thermal stability, a property which overlaps to a certain extent to what was described at point (a). However, even some volatile compounds may be further thermally stabilized by derivatization. Also, chemical stability can be enhanced by protecting specific groups in the analyte using derivatization. For example, thiols can be protected using derivatization against oxidation by the traces of oxygen in the heated injection port of the GC.
GC/MS chromatogram of a silylated tobacco sample, with separation on a DB-5 MS column from Agilent (Agilent Technologies Inc., Wilmington, DE, USA) (Note: an internal standard I.S. was added to the sample).
Peak tailing due to multiple retention mechanisms.
The choice of the appropriate derivatization is not always a simple task. The replacement of a hydrogen atom with a group of atoms may increase the molecular weight of the derivatized analyte. In such cases, it must be verified that the increase in the molecular weight by derivatization brings no or only a small increase in the boiling point of the analyte. Most of the time, low molecular weight substituents such as CH3 or Si(CH3)3 are preferable for GC analysis to the active hydrogens for achieving the previously described goals. Large substituents may increase the boiling point too much and make the compound not acceptable for GC analysis.
\nBesides replacement of active hydrogens, other derivatization reactions can be utilized. For example, condensation reactions may decrease the boiling point and improve the thermal stability of an analyte. However, the generation of new active hydrogens must be avoided in condensation reactions or must be followed by a second derivatization.
\nThe compounds with structures that are mirror images to each other are indicated as enantiomers, and their molecules are not superimposable, having the property called chirality. Chirality is commonly caused by the existence in the molecule of at least one tetrahedral carbon atom substituted with groups that are different. However, chiral molecules may be generated with a phosphorus or a sulfur chiral atom. Not only chiral centers (such as an asymmetric carbon) generate enantiomers, but a chiral axis or a chiral plane can lead to enantiomers. The chirality in an enantiomer is specified using the symbols R and S based on specific rules. For the assignment of a symbol R or S to a chiral carbon, the substituents are arranged in a sequence a > b > c > d. For the four atoms directly attached to the asymmetric carbon, a higher atomic number outranks the lower, and a higher atomic mass outranks the lower mass. For the same atoms directly attached to the asymmetric carbon, the priorities are assigned at the first point of difference. After the sequence is established, the molecule is oriented in space with the group “d” of the lowest priority behind the asymmetric carbon. When viewed along the C─d bond (from C) and the three substituents a, b, and c are oriented clockwise, the compound contains an R asymmetric carbon, and it contains an S asymmetric carbon for counterclockwise arrangement.
\nMore than one asymmetric carbon can be present in a molecule, as in the case of carbohydrates. The stereoisomers generated by more than one asymmetric carbon can be mirror image one to the other (enantiomers) or may have different steric arrangements being diastereoisomers. These types of molecules are schematically shown in Figure 3.
\nCompounds with two chiral centers.
The (S,S)- and the (R,R)-compounds from Figure 3 are enantiomers, while the (S,R)-compound is a diastereoisomer to both (S,S)- and to (R,R)-compounds (it is an enantiomer to the (R,S)-compound). The gas chromatographic separation of enantiomers can be done only using chromatographic columns having chiral stationary phases. The derivatization of enantiomers with non-chiral reagents generates molecules that remain enantiomers. This type of derivatization may improve the chromatographic separation from other molecules, but the derivatized compounds of remaining enantiomers cannot be separated except on chiral stationary phases. Sometimes, better separation can be obtained even between the enantiomers (on chiral chromatographic columns) after derivatization. One such example is the separation of (R)- and (S)-nornicotine derivatized with isobutyl chloroformate on a chiral Rt-BDEXsm column with separation improved compared to that of underivatized enantiomers [6]. The derivatization reaction is indicated below:
\nDiastereoisomers can be separated on chromatographic columns with non-chiral stationary phases which offer a much wider possibility to select the column. For this reason, an alternative procedure toward the separation of enantiomers is using derivatization with chiral reagents. This type of derivatization generates diastereoisomers which can be separated on non-chiral stationary phases.
\nA discussion on the separation of enantiomers on chiral phases without derivatization is beyond the purpose of this chapter. Numerous publications are dedicated to this subject, including papers published in general chromatography journals or in dedicated journals (e.g.,
The separation after derivatization with a pure enantiomer reagent is based on formation of diastereoisomers that can be separated on regular stationary phases. Depending on the nature of the analyte and of the derivatization, different separation techniques can be applied. A variety of common columns are used for such GC separations. The choice of the column is again dependent on the analyte and the derivatization procedure. For example, α-substituted organic acids such as α-chloropropionic, α-bromocaproic, etc. can be derivatized with a specific enantiomer of an amino acid ester (e.g., ethyl 2-aminopropanoate) in the presence of a peptide coupling reagent (benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate or BOP) in a reaction of the type:
The derivatized acids that are now diastereoisomers (R,S) and (S,S) can be separated on a common capillary column (e.g., a DB-1701 column from Agilent). Another example of derivatization with a chiral reagent is that of methamphetamines with (R)-menthyl chloroformate. This derivatization allows the separation of over-the-counter (R)-methamphetamine from the illicit (S)-methamphetamine. The reaction of the (R)-enantiomer is indicated below [8]:
The separation of the (R,R) and (S,R) derivatives was possible on a non-chiral column for a GC/MS analysis.
\nGas chromatography (not coupled with mass spectrometry, GC/MS being separately presented) used as an analytical technique can involve various detectors. The variety of such detectors is rather large, and several types include the following: thermal conductivity detector (TCD), flame ionization detector (FID), nitrogen-phosphorus detector (NPD), electron capture detector (ECD), flame photometric detector (FPD), photoionization detector (PID), electrolytic conductivity (Hall), sulfur chemiluminescence, nitrogen chemiluminescence, aroyl luminescence detector (ALD), atomic emission detector (AED), helium ionization detector (HID), vacuum ultraviolet (VUV) absorbance, infrared Doppler (IRD) absorption, FID with catalytic conversion of all analytes in CH4 (e.g., Polyarc system [9]), etc. The derivatization with the purpose of improving detectability in GC is determined by the type of detector utilized. Most derivatizations are performed precolumn, even if they are applied only with the purpose of improving detection. However, it is important that the derivatization for improving detection does not deteriorate the separation. Preferably, both the detection and the chromatographic separation are improved by the same derivatization. Some specific postcolumn reactions applied to the analytes are part of certain types of detectors such as chemiluminescence detectors, atomic emission detectors (AED), and FID with catalytic conversion into CH4. Some of these chemical changes in the analytes are not necessarily classified as derivatization reactions.
\nNo specific derivatization is usually recommended to improve sensitivity when using nonselective detectors such as TCD and FID. However, in some cases when the detector is not sensitive to a specific analyte, such as formaldehyde or heavily halogenated compounds, derivatization can be used to enhance detection.
\nIn case of NPD detector, derivatization with nitrogenous compounds can be done, which should give a higher sensitivity. However, this type of derivatization is not very common. An adverse result occurs for the NPD detectors when silylation is performed on the sample. Besides a possible reduction in the NPD response on silylated compounds containing nitrogen, a drastic decrease in the lifetime of the detector may occur, probably due to the excess of silylating reagent that commonly is injected with a derivatized sample and affects the alkali active element of the NPD.
\nThe response of the photoionization detector (PID) depends on the ionization potential of the analyte, and compounds with higher ionization potential are not sensitive in PID, while those with lower ionization potential may have excellent sensitivity, as low as 10−12 mg of sample. A derivatization resulting in lowering the ionization potential of the analyte may be beneficial for PID detection. However, derivatization for enhancing PID response is not frequently used.
\nSome detectors such as electron capture detectors (ECD) may benefit very much from certain derivatization types. ECD (as well as negative chemical ionization mass spectrometry or NCI-MS) can be extremely sensitive, but they are selective to compounds that are able to form more stable negative ions. ECD, for example, can have sensitivity as low as 10−13 mg of analyte in the detector compared to the best sensitivity of FID that can be 10−8 to 10−11 mg of analyte. The efficiency of the process seems to be related to the ease of attaching an electron on the molecule. In ECD this process can be written as follows:
With some exceptions, ECD response can be correlated with the electron affinity of the analyte [4]. In general, the halogen substituents increase the sensitivity in ECD in the order I > Br > Cl > F. Multiple substitutions seem to have a cumulative effect. Besides halogens, nitro groups seem to have an effect similar to chlorine groups. For aromatic compounds, the substituents affect the sensitivity of the ECD according to their electron withdrawing capability. Strong electron withdrawing groups such as NO2 increase the sensitivity of the detection, while electron donating groups reduce it.
\nA variety of substitution groups containing electronegative elements (halogens) or nitro groups can be attached to an analyte. The procedure to attach these groups is in most cases the typical substitution of an active hydrogen in the analyte Y-H with a group R from a reagent R-X that has the appropriate active X group. Some groups used for enhancing ECD (as well as NCI-MS) sensitivity following an alkylation or aryl derivatization reaction are shown in Figure 4, and several substitution groups introduced by acylation, chloroformylation, or sulfonation used for the same purpose are shown in Figure 5. Besides alkylation or aryl derivatization, other derivatization techniques used to replace an active hydrogen are applied to introduce into a molecule as a substituent containing halogens or nitro groups enhancing significantly the detectability of the derivatized analytes by ECD (as well as NCI-MS). Silylation, for example, can be used for this purpose when silyl groups used for derivatization contain halogens. Several silyl groups containing halogens that can be attached to an analyte by silylation with special reagents are given in Figure 6 [4].
\nSubstitution groups used in alkylation and aryl derivatization for enhancing ECD (and NCI-MS) detectability (the masses are considered only for the most abundant isotope.).
Substitution groups used in acylation chloroformation and sulfonation for enhancing ECD (and NCI-MS) detectability.
Substitution groups used in silylation for enhancing ECD (and NCI-MS) detectability.
The most powerful tool used for compound identification purposes is very likely mass spectrometry (spectroscopy). This technique is capable to provide information from very low amounts of material such as that eluting from a chromatographic column and can be easily coupled with a gas chromatograph. Most analyses performed with MS detection (GC/MS or GC/MS/MS) are using EI+ ionization mode with electron impact at 70 eV. The electrons interact with the molecule A to eject an additional electron leaving a positively charged species (with an odd number of electrons) of the type A▪+. The ions also receive energy during electron impact and the excess of energy induces fragmentation. For most molecules, this process can be written as follows:
The fragments Bi+ are commonly but not always with an even number of electrons. The formation of molecular ions takes place with a range of internal energies, and more than one fragmentation path is possible for a given molecule. Also, the fragments can suffer further fragmentations. In general, the most abundant fragment ion results from the fragmentations that form the most stable products (ion and neutral radical). The abundance of a fragment ion is affected by its stability. For this reason, the intensity of the response of a mass spectrometric detector can be very different for different molecular species, and the prediction of this intensity is difficult. As a result, the improvements in the sensitivity in EI + −type mass spectrometry (in GC/MS using EI+ ionization) are not usually sought (but not impossible) through derivatization.
\nDerivatization for enhancing sensitivity is, however, frequently applied in NCI-MS. In this technique, the electrons interact with the molecules of the CI gas which is lowering their energy but without forming ions. The ionization of analyte molecules takes place by interaction with the low-energy electrons or with already formed negative ions by electron capture, dissociative electron capture, ion pair formation, or ion molecule reaction. The ionization process with the formation of negative ions is efficient only for molecules with positive electron affinities. For this reason, the sensitivity in NCI-MS is highly dependent on the electron affinity of the analyte, similarly to the sensitivity in ECD. For enhancing the electron affinity, the derivatization with reagents containing, for example, fluorinated moieties (indicated in Figures 4, 5, 6) is practiced. The sensitivity of the analytical methods where such derivatization is applicable can have very good sensitivity. For example, derivatization with heptafluorobutyric anhydride of aromatic amines that are present at low trace level in cigarette smoke leads to limit of detection (LOD) values as low as 0.05 ng/cig. for compounds such as 4-aminobiphenyl [10, 11].
\nThe fragmentation pattern generated by EI+ ionization mode that generates a specific mass spectrum of a molecule is very likely the most utilized technique for the identification of the molecular species. For this identification, large libraries of mass spectra are available, and computer algorithms are used for automatic searches. The identification of compounds using mass spectroscopy is not a simple process even with the capabilities offered by the electronic searches in the mass spectral libraries. This is particularly true for analysis of complex mixtures or when the analyzed compound is present in traces. Some compounds do not have a very characteristic mass spectrum, or during the chromatographic process, the separation is not achieved, and it is difficult to make an identification due to the spectra overlapping. Also, numerous compounds may have a mass spectrum that matches more than one compound (with a good quality fit). In such cases, a derivatization with the purpose of obtaining a compound that forms more informative fragments in the mass spectrum can be very useful.
\nThe fragments from derivatized compounds can be used for the identification of unknown compounds using library searches and even when the mass spectrum is not available in the libraries. As an example, the derivatization by silylation allowed the identification of a new pentacyclic triterpenoid present in several bioactive botanicals [12]. An unidentified compound with MW = 456.7 was detected by LC/MS/MS in a rosemary extract. The structure of the compound was elucidated after silylation of the plant material based on the comparison of mass spectrum of the unidentified compound with that of silylated betulinic acid. The new compound was identified as (3β)-3-hydroxy-lupa-18,20(29)-dien-28-oic acid (or betul-18-en-oic acid). The mass spectra of the two acids are shown in Figure 7.
\nMass spectrum of silylated betulinic acid and that of silylated betul-18-en-oic acid.
The two mass units difference between different fragments from the mass spectra of the two compounds allowed the identification of the new compound structure. Neither free betulinic acid nor betul-18-en-oic acid are volatile, such that the use of GC/MS for identification was possible only after derivatization.
\nAnother special procedure that may be utilized for compound identification based on mass spectra is the use of two parallel derivatizations, one of them being done with an isotope-labeled reagent. Common labeling isotopes are 2H (deuterium, d), 13C, 15N, etc. One such isotopic labeling can be done, for example, using silylation with d18-N,O-bis(trimethylsilyl)-trifluoroacetamide (d18-BSTFA). Derivatization of an aliquot of sample with regular BSTFA and another with d18-BSTFA provides a pairing chromatogram with peaks at retention times that have only small differences from the first but with spectra differing by a number of units. The comparison of the spectra for corresponding peaks (based on retention time) of a given compound allows the calculation of the number of silyl groups attached to that compound. In addition, the fragmentation in the spectra can be better interpreted allowing easier compound identification.
\nDerivatization in GC/MS analysis may have multiple other utilizations and benefits. For example, quantitative analysis frequently utilizes isotopically labeled internal standards. In an analysis with multiple analytes, addition of an isotopically labeled internal standard for each analyte may become a complex process. When a derivatization is involved in the analysis, this can be done with a non-labeled reagent for the analytes in the sample, while the internal standards are obtained by derivatization of standards with the same reagent but isotopically labeled. Such technique has been proven to be very successful, for example, in the analysis of multiple amino acids (but using an LC/MS/MS procedure [13]).
\nDerivatizations as chemical reactions can be classified as follows: (1) reactions with formation of alkyl or aryl derivatives, (2) silylation reactions, (3) reactions with formation of acyl derivatives, (4) reactions of addition to carbon-hetero multiple bonds, (5) reactions with formation of cyclic compounds, and (6) other reactions specific to a certain analysis. The selection of the derivatization reaction is typically done based on the desired property to be brought to the analyte and its possible reactivity. For this reason, the reagent is selected to have moieties that add the desired property to the analyte and also to have the capability to react with the specific functional group of the analyte. The matrix of the sample also has a role in the choice of a specific derivatization procedure. Initial matrix of the sample is not always suitable for derivatization, and in some cases preliminary sample preparation is necessary to change this matrix. The change can be as simple as drying the initial sample but can also be rather complex [14]. Table 1 gives a simplified view of preferences for the choice of a derivatization reagent for compounds containing active hydrogens [14].
\nDerivatization preferences for compounds containing active hydrogens.
Besides functionalities with active hydrogens, other functionalities can also be derivatized. Compounds containing carbonyls can be derivatized, for example, using condensation reactions. Some analytes may contain multiple functional groups such as the amino acids. Specific derivatization reactions can be selected for such cases.
\nThe formation of alkyl or aryl derivatives is applied to replace the active hydrogens from an analyte with an alkyl (R) or aryl (Ar) group. The replacement can be done in functionalities such as OH, COOH, SH, NH, or CONH. For example, the derivatization with short-chain alkyl bromides or iodides has numerous analytical applications for compounds such as steroids, amino acids, catecholamines, sulfonamides, phenols, barbiturates, organic acids, and mono- and oligosaccharides. A large number of reagents R-X are known, and in a simplified approach, it can be considered that R is carrying a specific property and X a specific reactivity, although the reactivity of a reagent is influenced by both R and X components of the molecule. The type of moiety R and that of reactive group X are guiding the selection process of selecting a reagent for a specific derivatization.
\nIn most alkylation reactions, the analyte acts as a nucleophile (Y
Various reagents and conditions were utilized in the derivatizations for analytical purposes. As reagents R-X for alkylations, one of the most commonly used are the alkyl halides, especially alkyl iodides and alkyl bromides. Because some of the derivatizations can be slow and inefficient depending on the analyte and on the reagent, the reaction rate becomes an important parameter for the analytical applicability. The reaction with an alkyl halide for the preparation of methyl or ethyl substituents, for example, is frequently performed either with a specific methylation reagent, in the presence of a catalyst, or in some instances using a particular solvent. The enhancement of the alkylation efficiency can be achieved using several other procedures. For example, for the analytical alkylation of carboxylic acids, specific cryptands such as crown ethers can be used to solvate the alkali metal portion of an organic acid salts, allowing the anion to be freer and increasing the rate of nucleophilic substitution. One other approach for enhancing the alkylation efficiency is the use of phase transfer alkylation. This approach is based on the formation of a compound easily extractable in an organic phase and on the displacement of the equilibrium in the direction of the formation of the desired product.
\nOne different way of enhancing the alkylation efficiency is the use of different alkylating reagents besides short-chain alkyl bromides or iodides. One example of a halide that is particularly reactive is pentafluorobenzyl bromide. This reagent can be used for the derivatization of a variety of compounds containing active hydrogens. Another reactive halide is 2-bromoacetophenone (phenacyl bromide). This reagent is used mainly for the alkylation of compounds containing more acidic hydrogens such as carboxylic acids. Another example of methylation using a special reagent R-X is applied on carbohydrates [15]. This methylation uses methylsulfinylmethanide anion. The reagent is prepared from dry DMSO and NaH or KH in a reaction as follows:
A polyol or a monosaccharide dissolved in DMSO is easily methylated with methylsulfinyl-methanide anion.
\nOther alkylating reagents are known (different X in R-X), also reacting in a nucleophilic substitution. For example, dimethyl sulfate can be used for alkylations. Alkylfluoromethyl-sulfonates are even more reactive than sulfates, and the reaction may take place with the active hydrogen even from alcohols or amines as follows:
Even tertiary amines, such as pyridine, also react with this type of reagent forming quaternary ammonium salts. The alkylation with alkylfluorosulfonates can be catalyzed as other alkylation reactions for increasing the reaction rate. A catalyst that can be used in this reaction is Hg(CN)2.
\nDiazomethane is another common alkylating (methylating) reagent. The alkylation using diazomethane is assumed to take place as follows:
Diazomethane is a gaseous unstable substance, which cannot be stored for long periods of time. It is usually prepared in small quantities and used immediately with or without an intermediate step of dissolution in ether. The preparation can be done from different N-nitroso-N-alkyl compounds in a reaction with a base. A common preparation uses N-nitroso-N-alkyl-p-toluenesulfonamide (Diazald). Methylation with diazomethane may require addition of a Lewis acid catalyst such as BF3. The methylation of partly acetylated sugars and amino sugars using diazomethane and BF3 in ether leads to the methylation of the free OH groups without the migration or substitution of the existent acyl groups.
\nA common alkylation of acidic analytes such as carboxylic acids, phenols, and thiols is performed using another type of alkylating reagent, namely, N,N-dimethylformamide dialkyl acetals. N,N-Dimethylformamide dimethyl acetal (Methyl-8®) is commonly used for methylations. For a compound containing a COOH group, the reaction with this reagent takes place as follows:
The compounds with acidic hydrogens can also be alkylated (methylated) using trimethyl orthoacetate, alkyl-p-tolyltriazenes (R─NH─N═N─C6H4─CH3), and O-alkyl isoureas are also used for the formation of analytes containing acidic hydrogens, imino esters, etc.
\nAlcohols can also act as alkylating reagents in particular when the analyte contains a more acidic hydrogen. Catalyst such as HCl, BF3, CF3 COOH or a cation exchange resin in H+ form is also frequently added to facilitate the reaction. The addition of HCl can be made as a water solution or as gaseous HCl that does not bring additional water to the reaction medium. The formation of alkyl or aryl derivatives of acids is a particularly important reaction known as esterification. Derivatization by esterification has been used with acids as the analyte and the alcohol as the reagent and also with the alcohol as the analyte and the acid the reagent. The esterification can be viewed either as the acid alkylation or as the acylation of the alcohol (see also the esterification mechanism). This reaction is typically catalyzed by strong acids and can be written as follows:
The mechanism of ester formation can be summarized by the following series of reactions:
The esterification efficiency can be improved by removing the water formed in this reaction. This can be done using a chemical reagent or distillation when the compounds of interest boil above 100°C. Among the materials able to eliminate water are desiccants such as anhydrous MgSO4, molecular sieves, or substances that react with water such as CaC2, (CH3)2C(OCH3)2 (2,2-dimethoxypropane), and even an appropriately chosen acid anhydride that reacts faster with water than with the reacting alcohol. The derivatization also may be performed in the presence of SOCl2 (thionyl chloride), which reacts with the water assisting in its removal, and when present in excess, may react with the alcohols forming alkyl chlorides or with the acids forming acyl chlorides. Chloride is a better leaving group in a nucleophilic alkylation reaction, and the efficiency of alkylation increases. Acids also can be esterified using a mixture of an alcohol and an acyl halide.
\nOne procedure for the formation of esters with less active organic acids applies the addition of dicyclohexylcarbodiimide (DCCI) in the derivatization process, to facilitate esterification. The reaction can be performed by adding to the acids that need to be analyzed the appropriate alcohol and DCCI usually in a solvent such as pyridine. Dicyclohexylurea, which is formed in the reaction, is not soluble in pyridine and can be separated. Besides DCCI, other carbodiimides can be used in the reaction of acids and alcohols. Among these are carbonyldiimidazole (CDI), 6-chloro-1-p-chlorobenzensulfonyloxybenzotriazole (CCBBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), etc. Also, 2-chloro-1-methylpyridinium iodide, 2,4,6-triisopropylbenzenesulfonyl chloride, trialkyloxonium fluoroborate, etc. can be used to facilitate esterification.
\nTransesterification is another technique applicable for obtaining certain alkyl derivatives of acids (or acyl derivatives of alcohols). The reaction can be written as follows:
Transesterification can be catalyzed by acids (or Lewis acids) such as HCl, BF3, and H2SO4 or by bases such as CH3OK, CH3ONa, or C4H9ONa. The basic catalysts are commonly used for the methanolysis of triglycerides, followed by the analysis of the fatty acid methyl esters using GC or GC/MS [16].
\nA special alkylation can be achieved online during the heating in the injection port of a gas chromatograph using tertraalkylammonium hydroxides or alkylarylammonium hydroxides. Tetramethylammonium hydroxide (TMAH) is the most common reagents of this type. The reaction takes place as follows (Δ indicates heating):
Numerous other reactive compounds may be used for replacing active hydrogens in specific compounds. For example, epoxides, aziridines, and episulfides react easily with compounds with active hydrogens. Formation of a second group containing an active hydrogen may preclude the use of such reagents for analytical purposes.
\nBesides the desired derivatives, certain unexpected compounds that can be considered artifacts for the particular analysis can also be formed in alkylation reactions. The artifacts may be formed from unexpected interactions of the reagent with the analyte or may be a result of undesired effects of the catalysts or medium used for derivatization. In some cases, the control of the alkylation process may be difficult. Longer or shorter reaction times or intervals between derivatization and analysis may lead to errors, even when an internal standard is used for quantitation.
\nOne common case of artifact formation occurs during the reaction with compounds containing O-acyl or N-acyl groups, such as previously acylated carbohydrates, glycolipids, or glycoproteins, in particular when the reaction is done with short-chain alkyl bromides or iodides. When the OH groups of different sugars or NH2 groups of amino sugars were already protected with acyl groups, it was noted that, depending on the catalyst and the chosen medium, these acyl groups can be replaced by alkyl groups, or they may migrate from one position (such as C1) to other positions.
\nOxidation is another common side reaction when using Ag2O as a catalyst. The oxidation effect of Ag2O can be seen on free sugars as well as when attempting to permethylate peptides. Sulfhydryl groups are particularly sensitive to oxidation with Ag2O as a catalyst. The use of methylsulfinyl carbanion as a methylating reagent may also produce undesired side reactions with certain esters generating methylsulfinylketones. Also, strong alkylating reagents may produce undesired artifacts by unexpected alkylations.
\nThe derivatization with the purpose of obtaining aryl derivatives is similar in many respects to the alkylation reaction. The reaction takes place with compounds containing active hydrogens. Simple aryl halides are generally resistant to be attacked by nucleophiles and do not react similar to alkyl halides. This low reactivity can be significantly increased by changes in the structure of aryl halide or in the reaction conditions. The nucleophilic displacement can become very rapid when the aryl halide is substituted with electron attracting groups such as NO2.
\nSilylation is the chemical reaction of replacing a reactive hydrogen atom in OH, COOH, SH, NH, CONH, POH, SOH, or enolisable carbonyl with a silyl group, most frequently with trimethylsilyl (TMS). A large number of analytical methods involve silylation applied to alcohols including carbohydrates [17], phenols [18], amines, sterols [19], etc. The purpose of silylation in chromatography is mainly to reduce the polarity of the analyte, increase its stability, and improve the GC behavior. The differences in the mass spectra of the silylated compounds as compared to the initial analyte may also be an advantage for detectability. However, the mass spectra of many silylated compounds may not be available in common mass spectral libraries. Also, the silylated compounds plus the commonly present excess of silylating reagent may deteriorate some types of stationary phases such as that of Carbowax (polyethylene glycol)-type columns, and for this reason, their separation cannot be done on such columns.
\nSilylation can be performed on specific analytes or directly on complex samples such as a plant material (see, e.g., [12]). The silylating agent and the solvent can play the double role of extractant and silylating reagent. Many publications describe the use of silylation reactions for analytical purposes (e.g., [1, 5, 20]). The reaction of an analyte Y
The molecular weight for TMS is 73.047 calculated considering in the elemental composition of only the masses of the most abundant isotope. Numerous reagents have been synthesized to be used in silylations. Various aprotic solvents can be used as medium for silylation. The analysis can be focused on one analyte or on a mixture of analytes. The main factors contributing to the increase of the efficiency and the rate of the silylation reaction are the silyl donor ability of the reagent and the ease of silylation of different functional groups in the analyte. The solvent (or mixture of solvents) used as a medium and the compounds present or added in the silylation medium may also play a role for silylation efficiency. The reagent excess is sometimes important for displacing the equilibrium in the desired direction, and usually an excess up to ten times larger than stoichiometrically needed is used for silylation. Temperature also increases reaction rate, as expected, and heating of the sample with the reagents at temperatures around 70°C for 15 to 30 min is common. Some reagents used for trimethylsilylation are shown in Figure 8 [14].
\nSome reagents used for trimethylsilylation.
The approximate order of the increasing silyl donor ability for the reagents shown in Figure 8 is HMDS < TMCS < MSA < TMSA < TMSDEA < TMSDMA < MSTFA < BSA < BSTFA < TMSI. This order may be different on particular substrates where other reagents or reagent mixtures may be more reactive.
\nSilylation reagents can be used pure or in mixtures of two or even three reagents. The reagent mixtures may provide a more efficient silylation for specific compounds. For example, silylation of 3,4-dimethoxyphenylethylamine with BSA leads to the substitution of only one active hydrogen in the NH2 group, while the silylation with BSA in the presence of 5% TMCS produces silylation of both hydrogens in the NH2 [21]. A common silylating mixture is BSTFA with 1% TMCS.
\nOne of the determining factors regarding the silylation efficiency is the nature of the molecule Y
Several functional groups that can be silylated (listed in the approximate order of decreasing ease of silylation).
In general, the silylation of OH and COOH groups takes place with better results than that of NH2, CONH, or NH groups. Excellent results are obtained, for example, for the analysis of phenols after silylation [19]. A chromatogram of a solution containing a mixture of phenols at concentrations between 2.0 and 2.5 μg/mL in DMF, derivatized with BSTFA, separated on a BPX-5 chromatographic column (SGE Anal. Sci.), followed by MS analysis in single-ion monitoring (SIM) mode is shown in Figure 9. Details regarding the analyzed phenols are given in Table 3.
\nChromatogram of a set of phenol standards in DMF with the concentrations between 2.0 and 2.5 μg/mL derivatized with BSTFA, separated on a BPX-5 chromatographic column followed by MS analysis.
No. | \nCompound | \nRet. time | \nm/z | \nAbrrev. | \nNo. | \nCompound | \nRet. time | \nm/z | \nAbrrev. | \n
---|---|---|---|---|---|---|---|---|---|
(1) | \nPhenol | \n6.88 | \n166 | \nPh | \n(14) | \n3,4-Dimethylphenol | \n12.32 | \n194 | \n3,4-diMePh | \n
(2) | \no-Cresol | \n8.57 | \n180 | \no-Cr | \n(15) | \n3-Methoxyphenol | \n13.17 | \n196 | \n3-MeOPh | \n
(3) | \nm-Cresol | \n8.76 | \n180 | \nm-Cr | \n(16) | \n4-Methoxyphenol | \n13.47 | \n196 | \n4-MeOPh | \n
(4) | \np-Cresol | \n9.08 | \n180 | \np-Cr | \n(17) | \nCatechol | \n13.88 | \n254 | \nCa | \n
(5) | \n2-Ethylphenol | \n10.28 | \n194 | \n2-EtPh | \n(18) | \nResorcinol | \n16.05 | \n254 | \nRe | \n
(6) | \n2,5-Dimethylphenol | \n10.70 | \n194 | \n2,5-diMePh | \n(19) | \n4-Methylcatechol | \n16.27 | \n268 | \n4-MeCa | \n
(7) | \n3,5-Dimethylphenol | \n11.07 | \n194 | \n3,5-diMePh | \n(20) | \nHydroquinone | \n16.73 | \n254 | \nHy | \n
(8) | \n2,4-Dimethylphenol | \n11.20 | \n194 | \n2,4 diMePh | \n(21) | \n3-Methylcatechol | \n16.71 | \n268 | \n3-MeCa | \n
(9) | \n2-Methoxyphenol | \n11.28 | \n196 | \n2-MeOPh | \n(22) | \n5-Methylresorcinol | \n18.19 | \n268 | \n5-MeCa | \n
(10) | \n4-Ethylphenol | \n11.59 | \n194 | \n4-EtPh | \n(23) | \n2-Methylresorcinol | \n18.66 | \n268 | \n2-MeRe | \n
(11) | \n4-Chlorophenol | \n11.71 | \n185 | \n4-ClPh | \n(24) | \n4-Ethylresorcinol | \n19.90 | \n282 | \n4-EtRe | \n
(12) | \n2,6-Dimethylphenol | \n11.79 | \n194 | \n2,6-diMePh | \n(25) | \n2,5-Dimethylresorcinol | \n20.18 | \n282 | \n2,5-diMeRe | \n
(13) | \n2,3-Dimethylphenol | \n12.02 | \n194 | \n2,3-dimePh | \n\n | \n | \n | \n | \n |
Details regarding the analyzed phenols with the chromatogram shown in Figure 9.
Besides organic active hydrogens, several inorganic compounds with active hydrogens can also react with silylating reagents. Among these are H2O, H2O2, and strong inorganic acids. Also, some salts of the acids may be silylated. The reaction of silylating reagents with water imposes that water should be at the low level in the matrix or the solution of the analytes. The reaction with water takes place as follows:
In many solvents used as medium for derivatization, the trimethylsilanol formed in the reaction with water is separated as a distinct layer of solvent. The formation of two layers impedes a proper sampling of the derivatized material in the GC/MS instrument. In addition to that, the presence of an excess of water suppresses the derivatization of other compounds. The silylation is not recommended on samples with a water content higher than about 10%.
\nThe silylation reaction is commonly performed in a solvent that does not have active hydrogens. The most commonly used solvents as a medium for silylation are dimethylformamide (DMF), pyridine, and acetonitrile. The main role of the solvent is to dissolve the analyte and the reagents. The by-product HX of silylation shown in reaction (17) can be an acid, a base, or a neutral compound. As examples, for TMCS the by-product is HCl, for HMDS the by-product is NH3, for BSTFA the by-product is N-TMS-trifluoroacetamide, and for TMSI the by-product is imidazole. When the silylation reagent generates an acid as a by-product of the reaction, this may interfere with the silylation. For this reason, silylation can be promoted by any acid acceptor used as solvent or present in the solvent. Among such solvents are pyridine, triethylamine, and to a lower extent DMF. They can be used as both solvents and acid acceptors. Mixtures of solvents are commonly used for both enhancing solubility and promoting silylation. For example, formamide in the presence of pyridine may react with an acidic by-product generating CO and an ammonium salt. The addition of basic compounds to the silylation reaction may also influence the efficiency of the silylation. Also, some compounds may act as catalysts for silylation.
\nAlthough the TMS derivatives are by far the most commonly used in the derivatization for analytical purposes, other substituents in the silyl group can be used as reagents. Several such groups are indicated in Figure 10. The groups can be present in a variety of reagents connected to leaving groups “X-” such as Cl-, imidazolyl, F3C-(CO)-N(CH3)-, etc. For example, a common reagent containing
Examples of silyl groups different from TMS used in silylation reagents.
The use of different groups than TMS may serve different purposes. For example, a fluorinated or brominated group may enhance significantly the detection sensitivity when using ECD or NCI-MS. Also, the stability toward hydrolysis of compounds silylated with different groups than TMS may be higher, and such silylation can be advantageous. This is, for example, the case of
As an example, silylation of amino acids with MTBSTFA is commonly used [22, 23], and it is preferred to the silylation generating TMS derivatives. The chromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with MTBSTFA and separated on a DB-5MS chromatographic column (from Agilent) followed by MS analysis is shown in Figure 11. Details regarding the analyzed amino acids are given in Table 4.
\nChromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with MTBSTFA separated on a DB-5MS chromatographic column.
Peak No. | \nAmino acid | \nAbbrev. | \nMW | \nFormula + x TBDMS | \nMW + x TBDMS | \nCharact. ion | \nRet. time | \n
---|---|---|---|---|---|---|---|
(1) | \nα-Alanine | \nα-Ala | \n89.09 | \nC15H35NO2Si2 | \n317 | \n260 | \n31.69 | \n
(2) | \nGlycine | \nGly | \n75.07 | \nC14H33NO2Si2 | \n303 | \n246 | \n32.63 | \n
(3) | \nSarcosine | \nSar | \n89.09 | \nC15H35NO2Si2 | \n317 | \n260 | \n33.85 | \n
(4) | \nα-Amino-n-butyric acid | \nα-ABu | \n103.10 | \nC16H37NO2Si2 | \n331 | \n274 | \n34.36 | \n
(5) | \nβ-Alanine | \nβ-Ala | \n89.09 | \nC15H35NO2Si2 | \n317 | \n260 | \n35.58 | \n
(6) | \nUrea | \n\n | 60.06 | \nC13H32N2OSi2 | \n288 | \n231 | \n36.01 | \n
(7) | \nβ-Aminoisobutyric acid | \nβ-ABu | \n103.10 | \nC16H37NO2Si2 | \n331 | \n274 | \n36.11 | \n
(8) | \nValine | \nVal | \n117.15 | \nC17H39NO2Si2 | \n345 | \n186 | \n36.15 | \n
(9) | \nLeucine | \nLeu | \n131.17 | \nC18H41NO2Si2 | \n359 | \n200 | \n37.71 | \n
(10) | \nNorleucine | \n\n | 131.17 | \nC18H41NO2Si2 | \n359 | \n200 | \n38.8 | \n
(11) | \nIsoleucine | \niLeu | \n131.17 | \nC18H41NO2Si2 | \n359 | \n200 | \n38.8 | \n
(12) | \nγ-Aminobutyric acid | \nγ-ABu | \n103.10 | \nC16H37NO2Si2 | \n331 | \n274 | \n39.79 | \n
(13) | \nProline | \nPro | \n115.13 | \nC17H37NO2Si2 | \n343 | \n184 | \n39.87 | \n
(14) | \n2-Phenylglycine | \nPhGly | \n151.17 | \nC20H37NO2Si2 | \n379 | \n220 | \n46.16 | \n
(15) | \n5-Oxoproline | \noPro | \n129.13 | \nC17H35NO3Si2 | \n357 | \n300 | \n46.18 | \n
(16) | \nMethionine | \nMet | \n149.20 | \nC17H39NO2SSi2 | \n377 | \n320 | \n46.68 | \n
(17) | \nSerine | \nSer | \n105.09 | \nC21H49NO3Si3 | \n447 | \n390 | \n47.52 | \n
(18) | \nThreonine | \nThr | \n119.12 | \nC22H51NO3Si3 | \n461 | \n404 | \n48.43 | \n
(19) | \nPhenylalanine | \nPhe | \n165.19 | \nC21H39NO2Si2 | \n393 | \n336 | \n50.35 | \n
(20) | \nAspartic acid | \nAsp | \n133.10 | \nC22H49NO4Si3 | \n475 | \n418 | \n52.47 | \n
(21) | \nHydroxyproline | \nHyPro | \n131.13 | \nC23H51NO3Si3 | \n473 | \n314 | \n53.23 | \n
(22) | \n3-Methyl-L-histidine | \n3MeHys | \n169.20 | \nC19H39N3O2Si2 | \n397 | \n340 | \n55.15 | \n
(23) | \nGlutamic acid | \nGlu | \n147.13 | \nC23H51NO4Si3 | \n489 | \n432 | \n55.53 | \n
(24) | \nOrnithine | \nOrn | \n132.20 | \nC23H54N2O2Si3 | \n474 | \n286 | \n55.64 | \n
(25) | \n1-Methyl-L-histidine | \n1MeHys | \n169.20 | \nC19H39N3O2Si2 | \n397 | \n302 | \n57.03 | \n
(26) | \nLysine | \nLys | \n146.19 | \nC24H56N2O2Si3 | \n488 | \n300 | \n58.02 | \n
(27) | \nα-Aminoadipic acid | \n\n | 161.20 | \nC24H53NO4Si3 | \n503 | \n446 | \n58.06 | \n
(28) | \nHistidine | \nHys | \n155.16 | \nC24H51N3O2Si3 | \n497 | \n440 | \n62.29 | \n
(29) | \nTyrosine | \nTyr | \n181.19 | \nC27H53NO3Si3 | \n523 | \n302 | \n63.29 | \n
(30) | \nArginine | \nArg | \n174.20 | \nC24H56N4O2Si3 | \n516 | \n144 | \n64.26 | \n
(31) | \nTryptophan | \nTrp | \n204.22 | \nC29H54N2O2Si3 | \n546 | \n244 | \n67.98 | \n
(32) | \nCystine | \nCys | \n240.30 | \nC28H64N2O4S2Si4 | \n668 | \n348 | \n72.65 | \n
(33) | \nHomocystine | \nhCys | \n268.30 | \nC32H72N2O4S2Si4 | \n724 | \n362 | \n76.59 | \n
Details regarding the analyzed amino acids with the chromatogram shown in Figure 11.
In most situations, silylation generates only the desired derivatives. However, there are cases when the expected silylated compound is not formed, and either the silylation is not complete, or some compounds such as aldehydes, ketones, or esters with no obvious active hydrogen generate silylated compounds. Incomplete silylation is usually the result of inappropriate reaction conditions. However, when compounds with multiple functionalities are silylated, it is possible to generate a variety of derivatized compounds, regardless of the intention to obtain fully silylated or partly silylated compounds.
\nIn some cases, artifacts are formed due to the modification of the analyte under the influence of the reagents during derivatization. For example, when the silylation is done in basic or acidic conditions, the analytes that are sensitive to acidic or basic media may suffer unexpected transformations. The most frequent artifacts with compounds not containing obvious active hydrogens occur with aldehydes. Some aldehydes are able to undergo two types of chemical reactions with formation of OH groups, namely, enolization and acetal formation in the presence of water. The OH groups formed in this manner react with different silylating reagents and give the corresponding silylated products. Although the enolization or the acetal formation is negligible for the initial aldehyde, the reactions may be significantly displaced toward the formation of the silylated compounds of the enol or of the acetal. Artifacts can also be generated when the reaction is allowed to continue for an extended period of time. Other uncommon reactions with a specific silylation reagent and analyte may occur. An example of an uncommon reaction is the ring opening of flavanones.
\nThe formation of acyl derivatives is applied for replacing the active hydrogens from an analyte in functionalities such as OH, SH, NH [11, 24], CONH, etc. The acylation is also used for reducing polarity and improving the behavior of the analytes in the chromatographic column. Acylation may confer a better volatility of the analytes, although not as marked as for silylation or methylation. Only the derivatization with acetyl groups or with fluorinated acyl groups (not heavier than heptafluorobutyryl) improves volatility, while other heavier acyl groups are not suitable for this purpose. Acetylation, for example, can be used for compounds such as monosaccharides and amino acids to allow GC analysis. The detectability improvement on the other hand is a very common purpose for acylation. Acylation with fluorinated compounds is frequently used for enhancing detectability in GC with ECD or NCI-MS detection. Other uses of acylation include the enhancement of separation of chiral compounds, etc.
\nMost acylation reactions are nucleophilic substitutions where the analyte is a nucleophile (Y
Some common acyl groups present in acylation reagents are indicated in Table 5.
\nSome common groups present in acylating reagents used in derivatizations for GC analysis [14].
As shown in Table 5, the acyl groups in the reagent can be attached to various “X” groups. One such group is OH and among the acylating reagents are some free acids. When nucleophile is an alcohol, the reaction is known as esterification and has been discussed in Section 7. The acylation with acids can be applied besides alcohols to certain thiols, phenols, amines, etc. and can be written as follows:
The reaction can be displaced toward the formation of the acyl derivatives by eliminating the water using compounds such as anhydrous MgSO4, molecular sieve, or substances that react with water such as CaC2, or (CH3)2C(OCH3)2. Dicyclohexylcarbodiimide (DCCI) also is used for modifying the yield of the desired product. The reaction with reagents containing a carboxylic acid reactive group also can be done in the presence of 2,4,6-trichlorobenzoyl chloride or with various sulfonyl chlorides such as 2,4,6-triisopropyl-benzenesulfonyl chloride or 2,4,6-trimethyl-benzenesulfonyl chloride. The reaction of amines with acids can be displaced toward the formation of the amides using a peptide coupling reagent such as benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), diethyl cyanophosphonate, O-benzotriazol-1-yl-N,N,N′,N′-bis(tetramethylene)uronium hexafluorophosphate, 2,2′-dipyridyl disulfide + triphenylphosphine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), etc.
\nCommon acylating reagents are acyl halides such as chlorides or bromides, which are reactive compounds suitable for acylation. The reaction of an acyl chloride with an amine, for example, takes place as follows:
Since the reactivity of amides is lower than that of amines, the second hydrogen in the amine is more difficult to replace. Also, steric hindrance may negatively influence the reaction. The generation of a strong acid such as HCl is a disadvantage in the reaction with acyl halides, and usually the acid should be removed either by adding basic compounds such as Na2CO3 or MgCO3 or using pyridine as the reaction medium. The high reactivity of acyl halides is used for the acylation of compounds with less reactive hydrogens. Certain carbonyl cyanides react similarly to acyl chlorides.
\nThe disadvantage of generating a strong inorganic acid in the acylation with acyl halides also can be avoided by having, instead of the acyl halide, an anhydride. The reaction of Y
The acid resulting together with the acylated compound is not a strong acid such as HCl. The anhydrides of trifluoroacetic acid (TFA), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric (HFBA) acids are commonly used for derivatization of alcohols, phenols, amines, etc., with the purpose of enhancing detectability (by ECD or NCI-MS) and also for improving the chromatographic behavior (higher volatility, better thermal stability, better separation). The volatility of fluorinated compounds allows the GC applications. The reactivity of the perfluorinated anhydrides increases in the order HFBA < PFPA < TFA. However, the differences are not significant. Once formed, the heptafluorobutyrates are more stable to hydrolysis than the trifluoroacetates. An inert solvent such as CH2Cl2, ether, ethyl acetate, acetone, tetrahydrofuran or in CH3CN, etc. can be used as a medium for the reaction with perfluoroanhydrides. For the neutralization of the acids formed during derivatization, the basic compounds such as triethylamine, pyridine, or even solid NaHCO3 can be utilized.
\nIn order to avoid the formation of water or of a strong acid in the acylation reaction, certain amides such as N-methyl-bis(trifluoroacetamide), bis(trifluoroacetamide), or 2,2,2-trifluoro-N-methyl-N-(2,2,2-trifluoroacetyl)acetamide (MBTFA) can be used as reagents. Acylation of amines takes place at room temperature. Solvents such as CH3CN, pyridine, DMSO, or THF can be used as a reaction medium:
One other procedure successfully applied to obtain acyl derivatives is the use of acyl imidazoles as reagents. This class of compounds reacts with analytes containing alcohol, primary and secondary amino groups, or thiols. The reaction generates as a by-product imidazole:
Succinimidyl esters also can be used for acylation purposes. Amines and the amino group in amino acids also can be acylated using urethane-protected α-amino acid-N-carboxyanhydrides or oxycarbonyl-amino acid-N-carboxyanhydrides. Alkylketenes and their dimers may be used for acylation.
\nA special type of acylation is that using chloroformates. Carbonic acid, O═C(OH)2, can form amides, esters, halides, etc., due to the presence of two OH groups bonded to the CO group. Carbonic acid ester halides, also called chloroformates or chloroformate esters, with the formula R─O─C(═O)─X, where R is an alkyl or aryl group and X is F, Cl, Br, or I, can react with various compounds containing active hydrogens, such as acids [25], amines, alcohols, thiols, and amino acids. Amino acids, for example, in the presence of an alcohol in water form carbamate esters (urethanes) reacting as follows [26]:
The formation in reaction (25) of the alcohol Ra–OH may lead to traces of a resulting compound with both substituted radicals being Ra. For this reason it is typically recommended to perform the reaction in the presence of an alcohol having the same radical as the chloroformate reagent (Ra = Rb). Chloroformates containing in the alkyl or aryl group halogen substituents are particularly reactive. Even tertiary amines can react with specific chloroformates, such as pentafluorobenzoyl chloroformate or with trichloroethyl chloroformate, by displacing an alkyl group connected to the nitrogen atom and forming the carbamate ester.
\nSimilar in many respects to that of acyl derivatives R–CO–X are the reactions of sulfonyl derivatives R–SO2–X. Sulfonyl halides are in general less reactive than halides of carboxylic acids. The reaction of a sulfonyl derivative may take place with alcohols, phenols, amines, etc. The reactivity toward the sulfonyl sulfur is RNH2 > CH3COOR > H2O > ROH.
\nHigh reactivity toward active hydrogens in alcohols, amines, etc. can also be achieved using reagents with other functionalities. These functionalities include isocyanates, isothiocyanates, carbonyl azides, etc. These reactions can be seen as a replacement of an active hydrogen with a CO-R group or CS-R group as it occurs in other acylations.
\nA variety of other derivatization reactions are reported in the literature (see, e.g., [1]) and used for GC and GC/MS analyses. Among these are the addition to hetero multiple bonds in functional groups such as C═O, C═S, C═N, or C☰N. Many such reactions are additions to multiple bonds. Such reactions are, for example, the additions to the C═O groups in aldehydes and ketones. Reagents containing active hydrogens in groups such as NH2, OH, H2N-NH-, etc. can react, for example, with aldehydes and ketones. Alcohols, for example, form hemiacetals or acetals with aldehydes and ketals with ketones, and although most of such compounds are not stable enough to be suitable for derivatization, cyclic acetals and ketals may be stable and used for analytical purposes. A common reaction of carbonyl compounds is with amines. The initial addition reaction usually continues with water elimination forming a substituted imine or a Schiff base. Similar to the reaction of amines is the reaction with substituted hydroxylamines or hydrazines. A typical reaction of derivatization of carbonyl compounds is that using dinitrophenylhydrazine (DNPH). The derivatized compound can be analyzed either by LC [27] or by GC/MS [28]. The reaction takes place as follows:
The groups Ra and Rb can be H or alkyl or various other substituents.
\nAnother reagent that can be used for ketone derivatization is N-aminopiperidine in the presence of catalytic amounts of acetic acid. The resulting substituted hydrazone can be used in GC analysis:
β-Diketones may react differently with hydrazines generating pyrazole derivatives as shown below:
Several other classes of compounds similar to hydrazines react with the carbonyl compounds. Among these are hydrazones (NH2─N═CR2), hydrazides (NH2NH-COR), and semicarbazide (NH2NH-CONH2). Hydroxylamines also react with carbonyl compounds forming oximes. Hydroxylamine itself, hydroxylamine hydrochloride (STOX® reagent), or derivatives such as H2N-OSO3H in a solvent like pyridine can be used in this reaction:
When the reaction is performed with hydroxylamine, the generated oxime contains an active hydrogen. This can be further derivatized, for example, by silylation in a reaction with a common silylation reagent.
\nFor derivatization purposes other reagents can be used, such as substituted hydroxylamines like methoxyamine hydrochloride NH2OCH3•HCl (MOX® reagent) and O-(pentafluorobenzyl)-hydroxylamine hydrochloride (FLOROX® reagent). The reaction of a ketone or aldehyde with FLOROX is shown below:
The oximes existing in
The transformation of the oximes into nitriles generates one single compound from the two (syn- and anti-) isomers and can be used to simplify the chromatograms of sugars derivatized as oximes.
\nAlcohols, amines, and thiols also can react at other hetero multiple bonds leading to analytical applications. This addition may occur at the isocyanates (─N═C═O), ─C═O group in an amide, at a nitrile, at CS2, or at other groups. One example is the addition under special conditions of alcohols to dimethylformamide. The resulting acetals are very reactive and are used themselves as reagents, as shown previously for N,N-dimethylformamide dimethyl acetal (see reaction 12). Another example is the reaction of CS2 with alcohols in the presence of a base, leading to the formation of xanthates. Amines also react with CS2, and the formed isothiocyanate can be analyzed using GC analysis. The reaction takes place as follows:
Formation of new cycles from noncyclic compounds or replacement of old cycles with new ones that are more stable or have a desired property is also exploited in sample processing using derivatization. Epoxides, for example, can be formed in the reaction of a compound with a carbon–carbon double bond and a peroxy acid. Among the peroxy acids more frequently used for the formation of epoxides are peracetic, performic, perbenzoic, trifluoroperacetic, and 3,5-dinitroperoxybenzoic acids. However, in this reaction a mixture of enantiomers is formed, as shown below for a
The separation of the epoxides may be easier to achieve than that of olefins, and this type of derivatization has been utilized, for example, for better separation of various
Another reaction with formation of new cycles is that of amino acids with phenyl isothiocyanate leading to a thiohydantoin derivative:
This reaction has been successfully used for the analysis of amino acids in proteins [29, 30].
A variety of aromatic cycles can be formed in reactions involving bifunctional compounds. Addition reactions to hetero multiple bonds in bifunctional molecules frequently lead to cyclic compounds. For example, formaldehyde can react with tryptophan or tryptamine generating a β-carboline derivative as follows:
The new compound can be analyzed by GC, usually after further derivatization by silylation of the carboxyl group.
\nA typical reaction leading to pyrazoles is the reaction of hydrazines with diketones such as 2,4-pentandione (acetylacetone). For example, the reaction between hydrazine or methylhydrazine and acetylacetone takes place as follows:
Activated carbonyl groups such as those in hexafluoroacetone are known to react with difunctional compounds. The reaction may take place with an amino acid as follows:
Amino acids can react with an activated anhydride such as trifluoroacetic anhydride (TFAA):
The reaction takes place by heating the amino acids with an excess of TFAA. The reaction mixture is then dissolved in ethyl acetate and analyzed by GC.
\nNumerous other types of derivatization reactions were used for making the analytes suitable for GC and GC/MS analyses. These include formation of various cyclic types of compounds such as azines, siliconides, boronates, etc., that are thermally stable and do not have polar hydrogens such that GC or GC/MS analysis is possible. In addition to reagents that add specific moieties to the analytes, oxidation and reduction were sometimes used for the analyte modification (see, e.g., [4]).
\nSolid-phase reagents are polymeric materials with specific groups that are reactive and can be transferred to the analyte molecule producing derivatization. For an analyte of the form Y
Solid-phase reagents must work analogously to the corresponding small-molecule reagents containing the group R (a tag). Reagents that are insoluble in certain solvents at high concentrations can often provide a high ratio of analyte/substrate in a polymeric microenvironment that yields a high kinetic rate for the heterogeneous reaction.
\nA variety of materials can be used as solid support, such as specifically bound reagents on a silica support (used, e.g., for online derivatization in HPLC analysis), ion exchange resins, as well as other supports [31]. One example of solid-phase support that can produce derivatization is trifluoroacetyl nylon 6,6. This solid-phase reagent can be obtained from poly(hexamethylene adipamide) (nylon 6,6) and trifluoroacetyl anhydride. This solid-phase reagent can be used in amine derivatization in a reaction as follows:
This derivatization of the amine is done by mixing the solid-phase reagent with a solution of amine solution in CH3CN. Following derivatization, the solid-phase reagent is separated by centrifugation, and the solution is concentrated by evaporating part of the solvent and analyzed by GC (an amine internal standard must be used in this procedure). However, some such derivatizations require a long time of interaction between the solid-phase reagent and the analytes and found only limited applications.
\n(Another) alternative of derivatization of specific analytes is using the reaction between the reagent and the analyte both adsorbed on a solid support. This type of derivatization has been used, for example, in connection with a solid-phase microextraction (SPME) technique [32]. In this technique a reagent is initially adsorbed in the SPME fiber, followed by exposure to the analytes. The derivatized analytes are further desorbed in the injection port of the GC and analyzed using a detector such as MS. For example, formaldehyde from air can be analyzed using a polydimethylsiloxane (PDMS) fiber containing
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