Determination of 20 coccidiostats in milk, duck muscle and non-avian muscle tissue using UHPLC-MS/MS

Abstract

In this paper, methods were developed to measure coccidiostats in bovine milk, duck muscle and non-avian species. The methods were validated to the maximum levels and MRLs laid down in European Union legislation. A simple sample preparation procedure was developed for the isolation of coccidiostat residues from bovine, ovine, equine, porcine and duck muscle tissue, based on solvent extraction with acetonitrile and concentration. An alternative method had to be developed for milk samples based on the QuEChERS sample preparation approach because of the high water content in this matrix. Milk samples were adjusted to basic pH with sodium hydroxide and extracted by using a slurry of acetonitrile, MgSO₄ and NaCl. Purified sample extracts were subsequently analysed by using UHPLC-MS/MS in a 13.2-min chromatographic run. It was found that the use of rapid polarity switching enabled both negatively and positively charged ions to be analysed from a single injection. By using this approach, solvent usage was reduced significantly and sample throughput improved. The method was validated for the analysis of 20 coccidiostats (arprinocid, clopidol, decoquinate, diclazuril, diaveridine, ethopabate, halofuginone, laidlomycin, lasalocid, maduramicin, monensin, narasin, nequinate, nicarbazin, robenidine, salinomycin, semduramicin, toltrazuril, toltrazuril sulphoxide and toltrazuril sulphone) in muscle and milk. The method is quantitative for toltrazurils, but it cannot be used for confirmation because only the precursor ion is monitored. Accuracy values for muscle ranged from 80% to 125%, while CCα ranged from 2.2 µg kg⁻¹ for clopidol to 122 µg kg⁻¹ for toltrazuril sulphoxide. Bovine milk accuracy ranged from 84% to 120% for all analytes except maduramicin, semduramicin and salinomycin, for which the values were higher. CCα values achieved ranged from 1.1 µg kg⁻¹ for arprinocid, nequinate and lasalocid to 27 µg kg⁻¹ for toltrazuril.

Keywords:

• coccidiostats
• anticoccidials
• ionophores
• anti-protozoan drugs
• QuEChERS
• UHPLC-MS/MS
• milk
• non-target muscle
• feed additives

Acknowledgements

This research was funded by the Food for Health Research Initiative (FHRI) administered by the Irish Department of Agriculture, Food and the Marine and the Health Research Board (Contract 7FHRIAFRC5). The authors Lesa Clarke and Mary Moloney contributed equally to the work in this paper.