Characterization of amino acid derivatization reagents for LC-MS analysis
Date
2017-07-06
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Aminohapped on valkude „ehituskivideks“, neil on oluline roll ainevahetuses ja seega ka haiguste diagnoosimisel ja toiduainetes. Seetõttu on olulised ka aminohapete madalad sisaldused, näiteks kaasasündinud ainevahetushäirete tuvastamisel vastsündinute verest.
Madala sisaldusega ainete uurimiseks keerulistes proovides (nt aminohapped toiduainetes või veres) kasutatakse tänapäeval vedelikkromatograafia-massispektromeetria (LC-MS) meetodit, mis lahutab segud komponentideks (LC) ja tuvastab need (MS). LC-MS analüüsiks peavad analüüsitavad ained kromatograafiliselt lahutuma ja efektiivselt ioniseeruma. Selle saavutamiseks on mõnel juhul, sh aminohapete analüüsil, vaja ained keemiliselt muundada sobivasse vormi, mida nimetatakse derivatiseerimiseks.
LC-MS analüüsi olulisimaks nõrkuseks on proovi muude komponentide poolt põhjustatud ebamäärasus analüüsitulemustes, mida nimetatakse maatriksiefektiks. Derivatiseerimisega LC-MS analüüsil esinevaid maatriksiefekte on vähe uuritud.
Käesolevas töös uuriti maatriksiefekte, mis esinevad derivatiseeritud aminohapete LC-MS analüüsil. Selleks kasutati kolme meetodit, nelja juba kasutusel olevat ja kahte Tartu Ülikoolis väljatöötatud derivatiseerivat reagenti ning ühe rakendusena määrati 22 aminohappe sisaldused kaheksas kodumaises taimeekstraktis. Leiti, et erinevate derivatiseerivate reagentide korral on maatriksiefektid erinevad. Maatriksiefekte põhjustasid üldjuhul proovide teised komponendid, kuid mõnel juhul ka derivatiseerimisel kasutatud abistavad ained. Leiti, et tavalisest erineva MS ioonallika (APCI) ja detekteerimisrežiimi (negatiivsete ioonide ESI) kasutamine aminohapete derivaatide analüüsimiseks võimaldab märkimisväärselt vähendada maatriksiefekte. Töö tulemused võimaldavad konkreetse analüütilise probleemi lahendamiseks valida sobiva derivatiseeriva reagendi koos vastava detekteerimisrežiimiga. Samuti aitavad töö tulemused kaasa uudsete derivatiseerivate reagentide arendamisele ja disainile
Amino acids are „building blocks“ of proteins, they have important role in metabolism and are therefore also important when diagnosing diseases and analyzing food. Even low concentrations of amino acids are important, e.g. diagnosing inherited metabolic disorders of newborn children. The most widely applied analytical method for low concentrations in complex matrices is the liquid chromatography mass spectrometry (LC-MS), which separates mixture into components (LC) and detects them (MS). LC-MS analysis requires that the analyzed components are separated chromatographically and are efficiently ionized. In order to achieve this in some cases, including for analysis of amino acids, the analyte is converted into a suitable form. The process is called derivatization. One of the main weaknesses of LC-MS analysis is the influence of other sample components on the analysis results, called matrix effects. The matrix effects of analyses involving derivatization have not been thoroughly investigated. The present work focused on the study of matrix effects which affect LC-MS analysis of derivatized amino acids. Matrix effects were investigated using three methods, six derivatization reagents (four previously used and two developed at the University of Tartu) and, as one application, the concentrations of 22 amino acids were determined in eight Estonian herbal extracts. The results showed that different derivatization reagents are affected differently by the matrix effects. Matrix effects are caused by other sample components or by reagents used for the derivatization reaction. The application of less common ionization source (APCI) and ion mode (negative ion mode ESI) for analysis of derivatized amino acids reduces matrix effects substantially. The results of this work enable to choose a suitable derivatization reagent with respective detection mode for a specific analytical problem. Moreover, the conclusions contribute to the development and design of novel derivatization reagents
Amino acids are „building blocks“ of proteins, they have important role in metabolism and are therefore also important when diagnosing diseases and analyzing food. Even low concentrations of amino acids are important, e.g. diagnosing inherited metabolic disorders of newborn children. The most widely applied analytical method for low concentrations in complex matrices is the liquid chromatography mass spectrometry (LC-MS), which separates mixture into components (LC) and detects them (MS). LC-MS analysis requires that the analyzed components are separated chromatographically and are efficiently ionized. In order to achieve this in some cases, including for analysis of amino acids, the analyte is converted into a suitable form. The process is called derivatization. One of the main weaknesses of LC-MS analysis is the influence of other sample components on the analysis results, called matrix effects. The matrix effects of analyses involving derivatization have not been thoroughly investigated. The present work focused on the study of matrix effects which affect LC-MS analysis of derivatized amino acids. Matrix effects were investigated using three methods, six derivatization reagents (four previously used and two developed at the University of Tartu) and, as one application, the concentrations of 22 amino acids were determined in eight Estonian herbal extracts. The results showed that different derivatization reagents are affected differently by the matrix effects. Matrix effects are caused by other sample components or by reagents used for the derivatization reaction. The application of less common ionization source (APCI) and ion mode (negative ion mode ESI) for analysis of derivatized amino acids reduces matrix effects substantially. The results of this work enable to choose a suitable derivatization reagent with respective detection mode for a specific analytical problem. Moreover, the conclusions contribute to the development and design of novel derivatization reagents
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
aminohapped, analüüs, derivaadid (keem.), vedelikukromotograafia, massispektromeetria, amino acids, analysis, derivatives (chem.), mass spectrometry