Validacija analizne metode za določanje makrocikličnih laktonov v mesu

Novak, Melita (avtor), Bajc, Zlatka (mentor), Prosen, Helena (komentor)

Makrociklični laktoni (ML) so endektocidi, ki se uporabljajo za zdravljenje notranjih in zunanjih parazitskih okužb pri živalih in so derivati naravnih fermentacijskih produktov bakterij rodu Streptomyces. Zelo so učinkoviti že pri nizkih dozah, hkrati pa so za sesalce nizko toksični. Mehanizem delovanja ML je zaradi visoke afinitete povezan z od glutamata odvisnimi kloridnimi kanalčki (GluCls), ki so specifični za nekatere parazitske organizme. Zaradi lipofilnih lastnosti ML se njihovi ostanki večinoma akumulirajo v maščobnih tkivih, mesu in mleku. Njihovo vsebnost v živilih živalskega izvora nadzorujejo uradni laboratoriji. Namen mojega magistrskega dela je bil validacija analizne metode za določanje sedmih makrocikličnih laktonov (eprinomektin, moksidektin, emamektin, nemadektin, abamektin, doramektin in ivermektin), ki se bo uporabljala kot presejalna in potrditvena metoda v uradnem laboratoriju. Metoda vključuje ekstrakcijo analitov iz vzorcev mesa v acetonitril in čiščenje ekstrakta s pomočjo ekstrakcije na trdno fazo (SPE). Eluate vzorcev sem derivatizirala z anhidridom trifluoroocetne kisline (TFAA), 1-metilimidazolom (MI) in ocetno kislino v stabilne fluorescenčne derivate, ki sem jih določala z visoko zmogljivo tekočinsko kromatografijo (HPLC) na reverzno-fazni koloni Supelcosil LC-8-DB s fluorescenčno detekcijo (FLD). Pred postopkom validacije sem poskusila med analite vključiti selamektin, vendar pri danih derivatizacijskih in kromatografskih pogojih ni dal ustreznega odziva. Na obogatenih vzorcih govejega mesa sem izvedla popolno validacijo po konvencionalnem postopku v skladu z Izvedbeno uredbo komisije (EU) 2021/808, nato pa metodo razširila na prašičje in perutninsko meso z reducirano validacijo. Povprečen izkoristek metode za goveje meso znaša 89–109 %, v prašičjem mesu 86–114 % in v perutninskem mesu 90–114 %. Metoda ustreza kriterijem za ponovljivost in obnovljivost (CVr in CVW v govejem mesu znašata 3,91–12,8 % oz. 4,57–14,3 %). Ponovljivost metode v prašičjem in perutninskem mesu je prav tako ustrezna (CVr v prašičjem mesu je bila med 2,12 in 6,06 %, v perutnini pa med 3,66 in 15,8 %). Preverjena je bila robustnost metode in stabilnost derivatiziranih analitov v topilu in ekstraktu mesa. Ugotovila sem, da je metoda robustna, derivatizirani analiti pa stabilni najmanj 8 dni pri –20 °C. Validacija je pokazala, da je metoda primerna za določanje eprinomektina, moksidektina, emamektina, nemadektina, abamektina, doramektina in ivermektina v govejem, prašičjem in perutninskem mesu.

• Jezik: Slovenski jezik
• Ključne besede: makrociklični laktoni, avermektini, milbemicini, SPE, HPLC-FLD, validacija analizne metode, derivatizacija
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2026
• PID: 20.500.12556/RUL-183617
• Datum objave v RUL: 16.06.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Validation of analytical method for macrocyclic lactones determination in meat

Izvleček

Macrocyclic lactones (ML) are endectocides, used for treatment of internal and external parasitic infections in animals and are derivatives of natural fermentation products of Streptomyces bacteria. MLs are very effective at very low doses and exhibit low mammal toxicity. The mechanism of action involves glutamate-gated chloride channels (GluCls), specific for certain parasitic organisms, due to high affinity of MLs. Due to their lipophilicity, ML residues are mostly accumulated in adipose tissues, muscle and milk. ML residue levels in food of animal origin is controlled by national residual laboratories. The aim of this thesis was validation of analytical method for determination of seven MLs (eprinomectin, moxidectin, emamectin, nemadectin, abamectin, doramectin, and ivermectin), which will be used as a screening and confirmatory method. Meat samples were extracted in acetonitrile and cleaned-up by SPE C8 cartridges. Sample eluates were derivatized with trifluoracetic acid anhydride (TFAA), 1-methylimidazole (MI) and acetic acid to produce stable fluorescent derivatives and then determined by high-performance liquid chromatography (HPLC) on a reversed phase Supelcosil LC-8-DB column with fluorescence detector (FLD). Prior to validation procedure, an attempt to add selamectin to the scope of method was made, however using the proposed extraction, derivatization and chromatographic method, the response was unsatisfactory. Full validation by conventional procedure according to Commission implementing regulation (EU) 2021/808 was performed on fortified bovine meat samples. The scope of method was then extended on porcine and poultry meat. Average recovery ranged between 89 and 109 % in bovine meat, 86–114 % in porcine meat and in poultry meat 90–114 %. In bovine meat the repeatability (CVr) and within-laboratory reproducibility (CVW) was in the range 3.91–12.8 % and 4.57–14.3 %, respectively. Repeatability of the method in porcine and poultry meat were satisfactory. Robustness of the method and stability of derivatized analytes in solvent and in meat extract was also tested. The method was proven to be robust and derivatized analytes are stable for at least 8 days at –20 °C. Validation proved that the method is suitable for determination of eprinomectin, moxidectin, emamectin, nemadectin, abamectin, doramectin, and ivermectin in bovine, porcine, and poultry meat.

• Ključne besede: macrocyclic lactones, avermectins, milbemycins, SPE, HPLC-FLD, method validation, derivatization