Development and validation of an indirect competitive lateral flow immunoassay for the detection of acetaminophen (paracetamol) in bovine urine.

Paracetamol (PCM) is a commonly used analgesic and antipyretic agent for humans worldwide. However, PCM overdoses or overuse can cause health issues, such as hepatoxicity. As PCM is also used for the treatment of farm animals, it is essential to monitor these residues in animal-derived matrices at risk-based sites in order to minimize the intake of PCM through the food chain. In the present study, we have developed a novel carbon nanoparticle (CNP)-based indirect competitive lateral flow immunoassay (icLFIA) for the rapid detection of PCM in bovine urine. The developed icLFIA can detect PCM residues within 10 min, and its performance was validated according to Commission Implementing Regulation (EU) 2021/808, i.e., determination of the detection capability (CCβ), specificity, robustness, and stability. The CCβ of the icLFIA for PCM in bovine urine is 5 mg/L and the icLFIA is proven to be selective and specific towards PCM in bovine urine, as no matrix interference and cross-reactivity were observed, except for high concentrations of orthocetamol. The icLFIA for PCM in bovine urine is robust to (small) variations in reading time, but it remains necessary to strictly use a dilution ratio of running buffer/bovine urine of 80/20. Moreover, the produced icLFIAs are stable for at least 56 days under the stored conditions. In conclusion, the developed and validated icLFIA provides a rapid and cost-effective method for on-site monitoring of PCM abuse in cattle.
Competing Interests: Declarations. Ethical approval: An ethical approval was not required for using bovine urine samples because the sampling did not require an animal experiment. The samples used for the development of this assay are anonymized official samples originally taken for residue analysis by the competent authority in the Netherlands on livestock farms. After the official analyses, samples were anonymized and used for the development of this assay. As the purpose of this assay is to be incorporated into the official residue monitoring, it was most appropriate to use these “field samples” for the development of this assay. Conflict of interest: The authors declare no competing interests.
(© 2025. The Author(s).)