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V magistrskem delu obravnavam kromatografsko ločbo protamin sulfata na instrumentu za tekočinsko kromatografijo sklopljeno z masno spektrometrijo (LCMS, angl. Liquid Chromatography Mass Spectrometry). Protamin predstavlja skupino močno bazičnih polipeptidov izoliranih iz semenske tekočine keta lososa. V obliki sulfatne soli se uporablja v medicini, med drugim kot pomožna snov v inzulinskih peresnikih, saj upočasni sproščanje inzulina v človeškem telesu. Sestavljen je iz štirih polipeptidov, katerih večinski delež predstavlja aminokislina arginin. Ker so si polipeptidi kemijsko med seboj zelo podobni, je težko doseči njihovo ločbo. Da bi jo dosegla, sem optimizirala parametre ločbe in sicer stacionarno fazo kolone (z uporabo različnih kolon), pH in koncentracijo mobilne faze ter temperaturo ločbe. Ugotovila sem, da v danih pogojih idealne ločbe ne bom dosegla, zato sem na koncu poizkusila še način s snemanjem izbranih ionov (SIM, angl. Selected Ion Monitoring). Poskus je bil uspešen, metoda je na podlagi razlik v masi uspešno ločila vse štiri polipeptide protamin sulfata. Na koncu sem izbrala najboljšo separacijsko metodo in jo uporabila za določitev protamin sulfata v dveh različnih inzulinskih peresnikih. In my master's thesis, I examine the chromatographic separation of protamine sulphate with a liquid chromatography mass spectrometry instrument, LCMS. Protamine consists of highly basic polypeptides, which are isolated from chum salmon semen. Beside its other medical advantages, protamine regulates insulin release into the human body therefore it is used as a sulphate salt in insulin pens. Protamine is composed of four polypeptides that are high in arginine content. Because of the chemical similarity of those polypeptides, it is very difficult to separate them. To achieve that I had been optimising different separation parameters such as stationary phase (by using different columns), pH and concentration of the mobile phase, temperature of the separation. I concluded that with optimising aforementioned parameters I could not reach the ideal separation, so my last option was to try selected ion monitoring, SIM. This experiment was successful, the method adequately separated all four polypeptides based on their mass difference. In the end, I chose the best separation method and used it on two insulin samples to determinate their protamine content. |
