Novel eluent additives for LC-MS based bioanalytical methods
Date
2020-07-14
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Abstract
Käesoleva töö eesmärk oli laiendada teadmisi uudsete eluendilisandite kasutamiseks bioanalüütilises keemias kasutades vedelikkromatograafia-massispektromeetria (LC-MS) analüüsimeetodit. Nende kahe instrumendi: LC ja MS ühendamine laiendab määratavate analüütide hulka, kuigi seab ka piiranguid mobiilfaasi lenduvuse osas. Seejuures mitmed mittelenduvad eluendilisandid, mis aitavad suurendada LC analüüsi efektiivsust ja on küll laialdaselt kasutatud traditsiooniliste UV/Vis detektori puhul, kuid pole kasutatavad MS detektoritele. Uudsete fluoroalkoholide kasutamine eluendilisanditena võimaldab LC-MS analüüsile paljulubavat tulevikku. Fluoroalkoholid kui nõrgad happelised ühendid, millel on ioonpaaride moodustamise võimekus ja mis soodustavad analüütide ioniseerumist MS detekteerimisel, võimaldavad nii keerulistest segudest paremini komponente eraldada kui ka neid paremini detekteetida. Selleks, et uurida mõju ravimite retentsioonile pöördfaas-vedelikkromatograafias, testiti mitmeid paljulubavaid ühendeid aluselise pH juures. Lisandite uurimiseks kasutati erinevaid analüütilisi kolonne - C18, bifenüül ja pentafluorofenüül (PFP) ning uuriti mudel-analüütide ja üldlevinud ravimite ning narkootikumide käitumist. Töö raames leiti, et kasutatud fluoroalkoholid mõjutavad analüüsi vähendades happeliste analüütide retentsiooni ja suurendades aluseliste analüütide retentsiooni. Uudsed eluendilisandid rakendati ka kahes praktilises rakendusnäites. Esiteks kasutati neid uinutite morfiin, klonidiin ja midasolaam (ning nende metaboliitide) ülitundlikuks mõõtmiseks. Teiseks kasutati uudseid lisandeid südame- ja veresoonkonna ravimite milrinooni ja dobutamiini tundlikkuks mõõtmiseks. Neid kahte bioanalüütilist metoodikat kasutati ravimite ja metaboliitide mõõtmiseks laste vereplasmast farmakokineetiliste ja farmakodünaamiliste uuringute tarbeks. Uudsete eluendilisandite mõju analüütide ioniseerumisele võimaldas mõlema metoodikaga saavutada erakordselt madalaid määramispiire, vajades seejuures minimaalset proovikogust, mis oli sobilik just antud patsientide grupile.
The aim of this thesis was to expand knowledge about novel eluent additives used in bioanalytical chemistry using liquid chromatography-mass spectrometry (LC-MS) analysis technique. The combination of the two instruments, an LC and MS expands the limits of analytes that can be detected and quantified. However, it also introduced considerable limitations in terms of mobile phase volatility. Thus, many eluent additives which helped to increase the efficiency of LC analysis and were widely used when UV/Vis was a detector did not pass the criteria anymore. However, a promising perspective in LC-MS analysis is offered by novel fluoroalcoholic eluent additives. They are weakly acidic compounds with ion-pairing capabilities and with a positive influence on analyte ionisation in the MS source. To research the influence on retention patterns of pharmaceuticals, several novel eluent additives were tested in the basic mobile phase pH range. Different analytical column stationary phases - C18, biphenyl and pentafluorphenyl (PFP) - were investigated, both with model analytes and a large set of common toxicology screening compounds. It was observed that the use of these fluoroalcohols influenced the analysis by decreasing retention of acidic analytes and increasing retention for analytes with basic properties. Two practical applications employing novel eluent additives were developed and validated for the pharmaceuticals morphine, clonidine, midazolam, and their metabolites, as well as, for milrinone and dobutamine. The two bioanalytical methods were used to obtain data for pharmacokinetic and pharmacodynamic studies in paediatric patients. Both methods reached exceptionally low limits of quantification, with minimal sample amount used due to the positive influence of novel eluent additives on analyte ionisation
The aim of this thesis was to expand knowledge about novel eluent additives used in bioanalytical chemistry using liquid chromatography-mass spectrometry (LC-MS) analysis technique. The combination of the two instruments, an LC and MS expands the limits of analytes that can be detected and quantified. However, it also introduced considerable limitations in terms of mobile phase volatility. Thus, many eluent additives which helped to increase the efficiency of LC analysis and were widely used when UV/Vis was a detector did not pass the criteria anymore. However, a promising perspective in LC-MS analysis is offered by novel fluoroalcoholic eluent additives. They are weakly acidic compounds with ion-pairing capabilities and with a positive influence on analyte ionisation in the MS source. To research the influence on retention patterns of pharmaceuticals, several novel eluent additives were tested in the basic mobile phase pH range. Different analytical column stationary phases - C18, biphenyl and pentafluorphenyl (PFP) - were investigated, both with model analytes and a large set of common toxicology screening compounds. It was observed that the use of these fluoroalcohols influenced the analysis by decreasing retention of acidic analytes and increasing retention for analytes with basic properties. Two practical applications employing novel eluent additives were developed and validated for the pharmaceuticals morphine, clonidine, midazolam, and their metabolites, as well as, for milrinone and dobutamine. The two bioanalytical methods were used to obtain data for pharmacokinetic and pharmacodynamic studies in paediatric patients. Both methods reached exceptionally low limits of quantification, with minimal sample amount used due to the positive influence of novel eluent additives on analyte ionisation
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
additives, liquid cromatography, mass spectrometry