Veterinary antibiotics are administered to livestock animals with the aim of treating diseases and undesired residues of such substances could contaminate final products, like milk, with potential adverse toxicological effects on human health, including the transmission of antibiotic resistance. The presence of residues may be the consequence of illicit usage of banned compounds or the failure to adhere to the right withdrawal time before milking. The use of bovine milk with antibiotic residues seems to be a critical issue both for processing (e.g., cheese making) and consumption points of view. Over time, it became necessary to develop sensitive analytical techniques for their control and now public health agencies depend on confirmation by MS techniques to ensure unambiguous results. The present thesis aims to develop a multi-class and multi-residue method based on UHPLC coupled with an Orbitrap HRMS to detect antibiotic residues in bovine raw milk. The work revolves around the determination of the definitive methodology to reach satisfactory recoveries for the selected analytical standards (ampicillin, cefalonium, cloxacillin, danofloxacin, dimetridazole, erythromycin, nitrofuran, penicillin G, sulfadimethoxine, tetracycline, thiamphenicol) and its subsequent validation. The extraction process includes the addition of acetonitrile, an extraction buffer followed by a cleaning step using QuEChERS, and sample concentration. A single injection can detect both positively and negatively charged analytes in a 20 min chromatography run time using full scan MS and polarity switching in electrospray ionization. In the case of dairy cows, mastitis is still a widespread pathology, which could also arise during dry off. Hence, a practical application of the method has been the testing of raw bovine milk to investigate the presence of specific residues, following individuals during and after the administration of specific antibiotics to treat mastitis and dry off.
Gli antibiotici veterinari vengono somministrati agli animali da allevamento con lo scopo di curare malattie e residui indesiderati di tali sostanze potrebbero contaminare i prodotti finali, come il latte, con potenziali effetti tossicologici negativi sulla salute umana, inclusa la trasmissione della resistenza agli antibiotici. La presenza di residui può essere la conseguenza dell'uso illecito di composti vietati o del mancato rispetto del giusto tempo di sospensione. L’utilizzo di latte bovino con residui di antibiotici è un problema rilevante sia dal punto di vista dell’industria casearia specializzata nella produzione di formaggio che del consumo di latte alimentare. Nel corso del tempo è diventato necessario sviluppare tecniche analitiche sensibili per il loro controllo e ora le agenzie di sanità pubblica dipendono dalla conferma delle tecniche di SM per garantire risultati inequivocabili. La presente tesi mirava a sviluppare un metodo basato su UHPLC accoppiato con un HRMS Orbitrap per rilevare residui di antibiotici nel latte crudo bovino ed è destinato ad essere multi-classe e multi-residuo. Il lavoro ruota attorno alla determinazione della metodologia definitiva per raggiungere recuperi soddisfacenti per gli standard analitici selezionati (ampicillina, cefalonio, cloxacillina, danofloxacina, dimetridazolo, eritromicina, nitrofurano, penicillina G, sulfadimetossina, tetraciclina, tiamfenicolo) e la sua successiva validazione. Il processo di estrazione comprende l'aggiunta di acetonitrile e un tampone di estrazione seguito da una fase di pulizia utilizzando QuEChERS e, infine, il campione viene concentrato. Una singola iniezione può rilevare sia analiti caricati positivamente che negativamente in un tempo di esecuzione della cromatografia di 20 minuti utilizzando la MS a scansione completa e la commutazione di polarità nella ionizzazione elettrospray. Nel caso delle vacche da latte la mastite è ancora una patologia diffusa, che potrebbe insorgere anche durante la messa in asciutta. Pertanto, un'applicazione pratica del metodo è stata l'analisi del latte bovino crudo per indagare la presenza di residui specifici, seguendo gli individui durante e dopo la somministrazione di antibiotici specifici per trattare la mastite e l’asciutta.
Development of a multi-class and multi-residue methodology through UHPLC coupled with High-resolution Orbitrap MS for the detection of veterinary antibiotics in bovine raw milk
SABBADIN, SILVIA
2022/2023
Abstract
Veterinary antibiotics are administered to livestock animals with the aim of treating diseases and undesired residues of such substances could contaminate final products, like milk, with potential adverse toxicological effects on human health, including the transmission of antibiotic resistance. The presence of residues may be the consequence of illicit usage of banned compounds or the failure to adhere to the right withdrawal time before milking. The use of bovine milk with antibiotic residues seems to be a critical issue both for processing (e.g., cheese making) and consumption points of view. Over time, it became necessary to develop sensitive analytical techniques for their control and now public health agencies depend on confirmation by MS techniques to ensure unambiguous results. The present thesis aims to develop a multi-class and multi-residue method based on UHPLC coupled with an Orbitrap HRMS to detect antibiotic residues in bovine raw milk. The work revolves around the determination of the definitive methodology to reach satisfactory recoveries for the selected analytical standards (ampicillin, cefalonium, cloxacillin, danofloxacin, dimetridazole, erythromycin, nitrofuran, penicillin G, sulfadimethoxine, tetracycline, thiamphenicol) and its subsequent validation. The extraction process includes the addition of acetonitrile, an extraction buffer followed by a cleaning step using QuEChERS, and sample concentration. A single injection can detect both positively and negatively charged analytes in a 20 min chromatography run time using full scan MS and polarity switching in electrospray ionization. In the case of dairy cows, mastitis is still a widespread pathology, which could also arise during dry off. Hence, a practical application of the method has been the testing of raw bovine milk to investigate the presence of specific residues, following individuals during and after the administration of specific antibiotics to treat mastitis and dry off.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/51947