Date of Award

2010

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Dr. Ambrose Furey

Second Advisor

Dr. Martin Danaher

Abstract

The QuEChERS (quick, easy, cheap, effective, rugged and safe) sample preparation approach was evaluated for the extraction of anthelmintic residues from bovine milk and tissues. Liquid chromatography coupled to tandem mass spectrom.etry (LC- MS/MS) was used for detection and quantification of residues, which included the benzimidazoles (BZs), macrocyclic lactones (MLS) and the generally overlooked flukicides.

Initial work focused on the optimisation of a QuEChERS method to effectively extract anthelmintic residues from milk and liver. The method involves the extraction of residues with acetonitrile (MeCN) in the presence of salts (MgS04 and NaCl) to induce phase separation. After shaking and centrifugation, a portion of supernatant undergoes dispersive solid-phase extraction (d-SPE). The purified extract was analysed by LC-MS/MS using two 15 min injection to cover the positively and negatively ionised compounds. The method was successfully validated at the maximum residue level (MRL) according to the 2002/657/EC guidelines and met acceptability criteria in all but a few cases.

The previously developed QuEChERS method was improved for the detection and quantification of anthelmintic residues in bovine liver and muscle. Two different protocols of the same method were used to detect residues at the MRL and non-MRL levels. The improved method involves the addition of a concentration step when analysing in the low pg kg'' range. Ultra-high performance liquid chromatography (UHPLC) coupled to MS/MS was used for detection and quantification. All 38 anthelmintic residues could to be detected in <2 >µg kg''. A dual validation approach was proposed to validate the two protocols at MRL and non-MRL levels. The method met acceptability criteria in all but a few cases. The inclusion of 19 internal standards, including 14 isotopically labelled internal standards, improved accuracy, precision, decision limits (CCα) and detection capability (CCβ).

Stability studies on anthelmintic residues were carried out in fortified tissues and sample extracts (MeCN and dimethyl sulphoxide). Samples spiked at two concentrations (2 and 500 µg kg'') were stored for different periods of time and at different temperatures (+4°C and/or -20°C). The majority of residues were found to be stable in tissue samples and extracts. However, DMSO extracts stored at -20°C were not very stable. The stability of solvent standards stored at (+4°C, -20°C and - 30°C over 9 months was also investigated. Solvent standards were found to be most stable when stored in a freezer, although there is no advantage to storing standards at - 30°C compared to -20°C. The stability of 20 anthelmintic residues in incurred bovine muscle, kidney and liver tissue was examined during 6 months storage at -20°C. The majority of residues were found to be stable in all three tissues. No interconversion of the benzimidazoles and their metabolites was observed.

Finally, UHPLC-MS/MS was used for the qualitative and quantitative analysis of the new anthelmintic monepantel and its sulphone metabolite in goat’s milk. Sample preparation was carried out using a modified QuEChERS method, which included a concentration step to enable detection of residues to <1 >µg kg''. The method was successfully validated according to 2002/657/EC.

Access Level

info:eu-repo/semantics/openAccess

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