SCREENING OF COCCIDIOSTATS IN ANIMAL FEED AND DRINKING WATER USING LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

Coccidiostats are veterinary drugs commonly used in animal husbandry to prevent and control coccidiosis. However, their residues in animal feed and drinking water raise One Health concerns about cross-resistance, food safety issues, and risk to non-target species. This study presents the development and validation of a screening method for detecting coccidiostat residues in animal feed and drinking water, using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The method targets both ionophore (maduramicin ammonium alpha, lasalocid sodium, monensin sodium, narasin, salinomycin sodium, semduramycin sodium) and non-ionophore coccidiostats (diclazuril, halofuginone hydrobromide, decoquinate, nicarbazin, robenidine hydrochloride, amprolium, toltrazuril, clopidol, nequinate, ethopabate). Screening is performed at concentration in compliance with Directive 2002/32/EC, that set two permissible levels of cross contamination: LP1 (1% of the maximum authorized additive content, based on the sensitivity of the intended non-target animal species) and LP2 (3%). For simplification, any analyte detected at or above the limit of quantification (LOQ) - set at 50% of LP1 for regulated compounds and at 0.1 mg/kg for those not authorized or not explicitly listed – is subject to confirmatory analysis. Additionally, for internal quality control purposes, fortified samples at 50% of LP2 were included to assess method rougedness. The method was validated following Regulation (EU) 2021/808, with performance parameters such as selectivity, sensitivity, and ruggedness evaluated. Sensitivity and false positive/negative rates at LP1 and LP2 levels were assessed by analyzing 20 blank feed samples of various types and 20 fortified samples at both LP1 and LP2. To account for matrix variability, the 20 feed samples used for validation were selected to ensure maximum diversity and complexity of matrices, including different animal species and feed formulations. All blank samples were confirmed negative, and all fortified samples returned non-negative results for the target analytes, confirming the method's specificity and sensitivity in the relevant matrix context. Matrix effect evaluation is not required for qualitative screening methods under Regulation (EU) 2021/808 and subsequent amendments. Nevertheless, to enhance method reliability and minimize potential matrix interferences, the screening LOQ was conservatively set at 50% of LP1. Feed samples, including fortified controls, were extracted using a QuEChERS-based protocol with acetonitrile followed by purification at -80°C and LC-MS/MS analysis, requiring only 5.00±0.05 g of material. For drinking water, a simplified procedure involving filtration, dilution, and direct LC-MS/MS analysis was applied. Validation confirmed the method’s capability of detecting coccidiostat residues at the relevant regulatory thresholds, demonstrating its effectiveness for compliance monitoring in both animal feed and water samples. This validated screening method provides a reliable and efficient tool for routine surveillance of coccidiostat residues. By ensuring accurate detection at regulatory limits, the method supports food safety compliance, enhances risk assessment in animal feed production, and strengthens regulatory enforcement in water quality management. Its simplicity, efficiency and ruggedness make it a valuable asset for routine monitoring, helping to safeguard public health and uphold industry standards.