Razvoj analizne metode za ugotavljanje mikotoksinov v prehranskih vzorcih

Kalan, Jasna (avtor), Roškar, Robert (mentor)

Plesni so že dolgo razširjene v prehrani, uporabljajo se tudi v farmaciji (npr. proizvodnja penicilina). A so tudi neželene, nezahtevne za rast in sčasoma povzročajo razkroj v svoji okolici. V boju za življenjski prostor in hrano izločajo mikotoksine, med katerimi je nekaj zakonsko omejenih v prehranskih izdelkih. Namen magistrske naloge je bil razviti analizno metodo za zaznavo izbranih reguliranih mikotoksinov (aflatoksini B1, G1, B2, G2, ohratoksin A, deoksinivalenol, zearalenon in patulin) v različnih prehranskih vzorcih, in sicer z visoko vsebnostjo škroba, sladkorjev in maščob. Obstoječe metode predpriprave vzorca iz izbranih člankov smo preizkusili, poenostavili in optimizirali z namenom vpeljave poenotenega postopka priprave vzorca. Preverili smo vpliv razmerja vode in različnih ekstrakcijskih topil na učinek ozadja in izkoristek ekstrakcije, vpliv količine in vrste soli za ekstrakcijo, dodatek kisline ter možnosti dodatnega čiščenja vzorcev z disperzno ekstrakcijo na trdni fazi. Najprej smo razvili instrumentalno metodo za sočasno analizo vseh osmih mikotoksinov, ki temelji na tekočinski kromatografiji, sklopljeni z masno spektrometrijo tipa trojni kvadrupol. Ker se patulin zelo razlikuje po fizikalno-kemijskih lastnostih od ostalih mikotoksinov in se tudi specifično pojavlja v vzorcih hrane, smo zanj razvili ločen postopek priprave vzorca. Oba postopka (za patulin in za ostale izbrane mikotoksine) smo po optimizaciji ovrednotili v smislu selektivnosti, linearnosti, ponovljivosti, točnosti, meje določitve, izkoristka ekstrakcije in učinka ozadja. Analizno metodo za patulin smo uspešno validirali v treh vrstah vzorcev, meja določitve pa je bila dovolj nizka, da je metoda primerna tudi za analizo otroške hrane. Metodo za ostale mikotoksine smo tudi validirali na treh vrstah vzorcev hrane, a so se pojavila določena odstopanja, še zlasti za ohratoksin A zaradi nižjega izkoristka ekstrakcije, kar pa je smiselno v prihodnje še prilagoditi. Kljub temu smo uspešno vpeljali analizno metodo, s katero smo sposobni zaznati sedem mikotoksinov na zakonsko predpisanih mejah Analizno metodologijo smo uporabili na več kot 50 izdelkih hrane, za vsako skupino (žitarice, suho sadje, oreški in izdelki iz jabolk) smo zbrali med 10 in 15, tako komercialnih kot tudi domačih vzorcev. Testirali smo tudi s plesnijo kontaminirane vzorce. V nobenem od normalnih izdelkov nismo našli prisotnosti mikotoksinov, uspešno pa smo določili patulin, deoksinivalenol, ohratoksin A in zearalenon v vidno kontaminiranih vzorcih.

• Jezik: Slovenski jezik
• Ključne besede: mikotoksini, prehranski vzorci, določanje, QuEChERS, LC-MS/MS
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2019
• PID: 20.500.12556/RUL-111443
• Datum objave v RUL: 01.10.2019

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of an analytical method for determination of mycotoxins in food samples

Izvleček

Molds are widely spread in food production and are also used in pharmacy (e.g. production of penicillin). On the other hand, they are also unwanted, very modest about growth conditions and tend to cause degradation of their environment. In fight for habitat and food they start producing mycotoxins, some of which are dangerous enough to be regulated by law in food commodities. The main purpose of our work was to develop an analytical method for detection of some of regulated mycotoxins (aflatoxins B1, G1, B2, G2, ochratoxin A, deoxynivalenol, zearalenone and patulin) in different types of food, including starchy samples, samples rich in sugars and lipids. We tested some literature methods of sample preparation, then we simplified and optimised them to make a unified method for preparation of all sample types. We researched the influence of water and organic solvent ratio and the effect of different solvents on matrix effect and extraction efficiency. Furthermore, we inspected the effect of quantity and type of extraction salts, addition of acid and sample cleaning with dispersive solid phase extraction. Firstly, we developed an instrumental method for multiresidue analysis of all 8 mycotoxins based on liquid chromatography tandem mass spectrometry. Because patulin differs from other mycotoxins in physico-chemical properties and also occurs in dissimilar types of food, we developed a separate sample preparation method for patulin. We validated both methods for selectivity, linearity, repeatability, accuracy, limit of quantitation, extraction efficiency and matrix effect. The method for patulin was successfully validated in three types of samples with achieved limit of quantitation low enough to analyse baby food samples. The method for other mycotoxins was also validated showing some deviations, especially for ochratoxin A, mostly because of low extraction yield, which need further development. However, we successfully developed an analytical method for detection of seven mycotoxins in concentration range within their regulation of law. We applied the method to more than 50 food samples, at least 10 for each separate group (grains, dried fruits, nuts and apple products), which we bought in a store or were homemade. None of the store-bought sample contained mycotoxins, but we detected patulin, deoxynivalenol, ochratoxin A and zearalenone in visibly contaminated samples.

• Ključne besede: mycotoxins, food samples, detection, QuEChERS, LC-MS/MS