Development and validation of a GC-FID/MS analytical procedure for fatty acids determination in beechnuts and fat of edible dormouse

Namen magistrskega dela je bil poenostaviti farmakopejski analizni postopek za določanje srednjeverižnih trigliceridov, ki se uporablja v farmacevtski industriji. Farmakopejski postopek je izredno zapleten, zamuden in zahteva veliko laboratorijskega inventarja, ki ga pogosteje najdemo v laboratoriju za organsko sintezo kot pa v klasičnem analitskem laboratoriju. Z magistrsko nalogo smo izvedli razvoj in validacijo poenostavljenega in za izvajalca prijaznejšega analiznega postopka za določitev maščobnih kislin v vzorcih žira in polhove masti z izvedbo estrifikacije. Izvedli smo tudi primerjavo analiznih postopkov in s tem zagotovili enakovrednost optimiranega analiznega postopka z obstoječimi farmakopejskimi. V literaturi smo zasledili, da hranjenje domačih prašičev z žirom povzroča znižanje tališča svinjske masti, hkrati je splošno znano, da je polhova mast, z izjemo ostalih sesalcev, tekoča, zato smo predvidevali, da je vzrok za posebne fizikalne lastnosti polhove masti žir, ki predstavlja pretežno hrano polhov. Analizirali smo dva vzorca, in sicer polhovo mast in žirovo olje, saj nas je zanimala povezava glede na sestavo maščobnih kislin med njima. V primeru dovolj velikega ujemanja sestave polhove masti in maščob pridobljenih iz žira, bi uspeli dokazati, da je vzrok za posebne lastnosti polhove masti hranjenje z žirom. Prvo analizo smo izvedli na plinskem kromatografu z masnim detektorjem z uporabo analiznega postopka evropske farmakopeje in tako določili vrste maščobnih kislin v polhovi masti in žirovem olju. Nato smo izvedli analizo z GC-FID po ameriški farmakopeji (USP29, Fats and fixed oils) in po evropski farmakopeji (PhEur 9.0 Composition of fatty acids by GC). Sledila je še modifikacija in poenostavitev analiznega postopka ter validacija. Z modifikacijo in poenostavitvijo smo skrajšali pripravo standardov in vzorca, kjer smo postopek priprave vzorca bistveno poenostavili. Za pripravo standardov smo uporabili postopek iz evropske farmakopeje in hkrati spremenili kromatografske pogoje (temperaturni program in dodan gradienti program za pretok) z namenom izboljšati ločljivosti med kromatografskimi vrhovi ter skrajšati analizo. Pri validaciji optimiranega analiznega postopka smo izvedli stabilnost standardnih raztopin in vzorca, linearnost, natančnost sistema in metode, mejo zaznavnosti, mejo določljivosti in selektivnost. Ugotovili smo, da med maščobnokislinsko sestavo žirovega olja in polhovo mastjo obstaja zelo velika podobnost. S to ugotovitvijo lahko potrdimo, da je vzrok za posebne fizikalne lastnosti polhove masti žir, ki predstavlja osnovni vir maščob v polhovi masti.One of objectives of this master\u27s thesis was to simplify the pharmacopoeia analytical procedure for determination of medium-chain triglycerides used in pharmaceutical industry. The procedures in EU and US pharmacopeia’s are very complex, time-consuming and they requires a lot of laboratory inventory, which can be found in the laboratory for organic synthesis, rather than in a classical analytical laboratory. With this master\u27s thesis we developed and validated the simplified and user-friendly analytical procedure for determination of fatty acids in samples of beechnuts and edible dormouse with derivatization procedure of sample esterification. We also performed a comparison of all three analytical procedures to prove the equivalence of the optimized analytical procedure with existing pharmacopoeia’s procedures. In the literature we found claims where authors reported that, the fat of domestic pigs, which were fed with beechnuts, showed a significant drop in melting point similar to that of edible dormouse. At the same time, it\u27s generally known that edible dormouse fat, with exception of other mammals, is liquid. Therefore, we assumed that the specific physical properties of the fat are caused by beechnut\u27s oil, which is mostly the food of edible dormice. We analysed two samples: first, edible dormouse fat and second, beechnut\u27s oil. With the comparison of the fatty acids composition between edible dormouse fat and beechnut\u27s oil, we wanted to prove the correlation of fatty acids composition between them. In the case of a good matching in fatty acids composition, we prove the effect of beechnuts in the diet of edible dormice and therefore prove the cause for special properties of edible dormouse fat. The first analysis was made on gas chromatograph with mass detector using the analytical procedure of European pharmacopoeia. This analysis was used for identification of fatty acids of edible dormouse fat and beechnut oil. Then we performed analysis by GC-FID with analytical procedure of American pharmacopoeia (USP29, Fats and fixed oils) and then with European pharmacopoeia (PhEur 9.0 Composition of fatty acids by GC) analytical procedure. Then the analytical procedure was modified, simplified and validated. With optimization of the analytical procedure we shorten the preparation of standards and samples and achieved significantly simplified sample preparation as well. For the preparation of standards in the optimized analytical procedure, we adopted PhEur standard preparation and at the same time we changed chromatographic conditions (temperature program and gradient program for mobile phase flow was added) in order to improve resolution between peaks and shorten the time of chromatographic run and analysis in general. In the validation process we performed tests for: stability of the standard solutions and sample, linearity, precision of the system and the method, determination of detection and quantitation limit and selectivity. We found out that there is a sufficient similarity between the beechnut oil and the dormouse fatty acids composition, and concluded that the consumption of beechnuts causes, the special fatty acids composition of dormouse fat, which consequently causes the special physical properties of dormouse fat