Razvoj kromatografske metode za določanje sladkorjev s fluorescenčno detekcijo

Erzin, Leja (avtor), Pompe, Matevž (mentor)

V sklopu magistrske naloge sem razvila kromatografsko metodo za kvantifikacijo N-glikanov s fluorescenčno detekcijo. Postopek priprave vzorca (derivatizacija z 2-aminobenzamidom (2-AB), odstranjevanje presežnega označevalca 2-AB z ekstrakcijo na trdo fazo (SPE)) in kromatografsko metodo sem razvijala na vzorcu maltodekstrinov, glukoze in maltoze. Na vzorcu glukoze in maltoze sem izvedla tudi validacijo SPE. Pripravo vzorca sem nato z dodatno stopnjo deglikolizacije aplicirala na realni vzorec glikoproteinov (HUMIRA). Deglikolizacije sem se lotila encimatsko s PNGazo F. Tekom razvoja sem preverila separacijo maltodekstrinov na dveh kolonah z mešanimi separacijskimi režimi. Prva, ki sem jo preverila, je bila Thermo Scientific, GlycanPac AXR-1. 2,1 x 150 mm, 3 µm, ki separira glede na gostoto naboja in hidrofoben značaj spojin (šibko anionsko-izmenjevalni in reverzno-fazni princip). Druga pa Thermo Scientific, GlycanPac AXH-1. 2,1 x 150 mm, 3 µm, ki separira glede na gostoto naboja in hidrofilnost (šibko anionsko-izmenjevalni in HILIC princip). Preverila sem tudi separacijo na koloni Waters, Acquity UPLC (R) BEH AMIDE 1,7 µm, 2,1 x 100 mm, ki za separacijo uporablja HILIC princip. Na njej se dobro separirajo zelo polarni analiti, ki se ne zadržujejo na reverzno-fazni koloni. Najbolj optimalno separacijo maltodekstrinov sem dobila na Thermo Fisher koloni, GlycanPac AXH-1. Na tej koloni sem zato razvila in validirala kromatografsko metodo za kvantifikacijo N-glikanov, označenih z 2-AB. V sklopu validacije sem preverila ponovljivost, BIAS, selektivnost, izkoristek SPE, linearno območje, mejo detekcije in kvantifikacije ter robustnost. Ugotovila sem, da je razvita metoda ustrezna za kvantifikacijo N-glikanov, zato sem na koncu analizirala še realni vzorec HUMIRA 40 mg/0,4 mL.

• Jezik: Slovenski jezik
• Ključne besede: kromatografija z mešanimi separacijskimi režimi, N-glikani, SPE, derivatizacija z 2-AB, deglikolizacija glikoproteinov
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-124440
• COBISS.SI-ID: 48290563
• Datum objave v RUL: 21.01.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of a chromatographic method for the determination of sugars by fluorescence detection

Izvleček

As part of master's thesis, I developed a chromatographic method for quantifying N-glycanes by fluorescence detection. The sample preparation process (derivatisation with 2-aminobenzamide (2-AB), removal of excess marker 2-AB with solid-phase extraction (SPE)) and chromatographic method were developed on maltodextrins, glucose and maltose. Validation of SPE was also carried out on glucose and maltose. The preparation of the sample was then applied to a sample of glycoproteins (HUMIRA) with an additional stage - deglycolysation. We did encimatic deglycosylation with PNGase F. During development I checked the separation of maltodextins on two columns with a mixed mode stacionary phase. The first one was Thermo Scientific, GlycanPac AXR-1. 2,1 x 150 mm, 3 μm, which separates analytes according the charge density and hydrophobic nature of the compounds (weak anion-exchange and revrse-phase principle). The other was Thermo Scientific, GlycanPac AXH-1. 2,1 x 150 mm, 3 μm, which separates according to the charge density and hydrophilicity (weak anion-exchange and HILC principle). I also checked the separation on the Waters column, Acquity UPLC (R) BEH AMIDE 1,7 μm, 2.1 x 100 mm, which uses the HILIC principle for separation. There can be separated very polar analytes, which can't be on a reverse-phase column. The most optimal separation of maltodextins was obtained on the Thermo Fisher column, GlycanPac AXH-1. On this column, I therefore developed and validated a cromatographyc method for the quantification of N-glycanes, taged with 2-AB. As part of the validation, I checked repeatability, BIAS, selectivity, SPE efficiency, linear range, limit of detection and quantification and robustness of method. The developed method was suitable for the quantification of N-glycanes, therefore I also analyzed a real sample HUMIRA 40 mg/0,4 mL

• Ključne besede: mixed mode chromatography, N-glycanes, SPE, derivatisation with 2-AB, deglycolation of glycoproteins