Advanced method development strategy for derivatization LC/ESI/MS
Kuupäev
2012-06-28
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Aminohapete tundlikuks analüüsiks kasutatakse derivatiseerimise vedelikkromatograafia-elektropihustus-ionisatsioon-massispektromeetriat (LC/ESI/MS). Antud töö raames kontrolliti kõikide metoodika etappide sobivust aminohapete analüüsiks LC-ESI-MSiga. Esiteks töötati välja metoodikad viie derivatiseeriva reagendi kasutamiseks: Fmoc-Cl, DNS, Deemm, TAHS ning laboris disainitud ja sünteesitud uudse fosfaseenil põhineva reagendi (FOSF) jaoks ja võrreldi neid. Kokkuvõtvalt on Deemm parim derivatiseeriv reagent pakkudes kompromissi hea tundlikkuse ja kromatograafilise lahutuse vahel. Käesolevas töös uuriti esmakorselt derivatiseerimisprotseduuride sobivust LC/ESI/MS analüüsiga. Tulemused näitasid, et derivatiseeriva segu puhvri valik mõjutab oluliselt signaali stabiilsust ja intensiivsust. Samuti tehti töö käigus kindlaks reagentidele kõige paremini sobivad kromatograafilised eluendid ja uuriti erinevaid võimalusi tundlikkuse parandamiseks. Teostati ka ionisatsiooni efektiivuste mõõtmisi logIE skaala täiendamiseks ning uuriti põhjalikumalt MS optimeerimist, mis andsid olulist lisainformatsiooni, et meetodiarendus oleks veelgi täielikum ja süstemaatilisem. Praktilised rakendused näitasid derivatiseerimise LC/ESI/MS analüüsi head sobivust tundlikuks aminohapete sisalduse määramiseks meeproovidest ning selenoaminohapete määramiseks vereseerumist ja toidust (sibulast).
Töö tulemusena näidati, et derivatiseerimise LC/ESI/MS meetodiarenduseks on vaja kaasata uusi aspekte, et võimaldada veelgi madalamaid määramispiire. Antud töö koondab kokku olulisemad aspektid ning pakub seega välja parendatud meetodiarenduse strateegia derivatiseerimise LC/ESI/MS jaoks.
For sensitive amino acid analysis derivatization liquid chromatography mass spectrometry (LC/MS) with electrospray ionization (ESI) is used. In current work the compatibility of the method steps with LC-ESI-MS analysis was evaluated. Method development was carried out for five derivatization reagents, Fmoc-Cl, DNS, Deemm, TAHS and in-house designed and synthesized FOSF and these were compared. It was concluded that Deemm is an optimal choice for derivatization reagent since it provides sensitive analysis and better chromatographic separation. Moreover, it was found out that solid phase extraction is most suitable sample preparation technique. In this work, derivatization procedures of the derivatization reagents were evaluated considering LC/ESI/MS compatibility. Results showed that derivatization buffer can have a significant effect on the stability and intensity of the signal. For all reagents, most suitable eluent components were determined and other signal enhancement techniques were sought for. Measurements of ionization efficiency in terms of logIE scale and a thorough MS optimization investigation provided useful additional information for even more complete and systematic method development. Practical applications showed the high performance of the derivatization LC/ESI/MS for analysis of amino acid in honey and selenoamino acid in bloodserum and food (onion). In conclusion, in this work, it is shown that for a sensitive derivatization LC/ESI/MS analysis, new aspects of method development are needed and therefore combines together most important aspects and proposes an advanced method development strategy for derivatization LC/ESI/MS.
For sensitive amino acid analysis derivatization liquid chromatography mass spectrometry (LC/MS) with electrospray ionization (ESI) is used. In current work the compatibility of the method steps with LC-ESI-MS analysis was evaluated. Method development was carried out for five derivatization reagents, Fmoc-Cl, DNS, Deemm, TAHS and in-house designed and synthesized FOSF and these were compared. It was concluded that Deemm is an optimal choice for derivatization reagent since it provides sensitive analysis and better chromatographic separation. Moreover, it was found out that solid phase extraction is most suitable sample preparation technique. In this work, derivatization procedures of the derivatization reagents were evaluated considering LC/ESI/MS compatibility. Results showed that derivatization buffer can have a significant effect on the stability and intensity of the signal. For all reagents, most suitable eluent components were determined and other signal enhancement techniques were sought for. Measurements of ionization efficiency in terms of logIE scale and a thorough MS optimization investigation provided useful additional information for even more complete and systematic method development. Practical applications showed the high performance of the derivatization LC/ESI/MS for analysis of amino acid in honey and selenoamino acid in bloodserum and food (onion). In conclusion, in this work, it is shown that for a sensitive derivatization LC/ESI/MS analysis, new aspects of method development are needed and therefore combines together most important aspects and proposes an advanced method development strategy for derivatization LC/ESI/MS.
Kirjeldus
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Märksõnad
aminohapped, keemiline analüüs, derivatiseerimine, amino acids, chemical analysis, derivatization