Development of a lateral flow immunoassay for the simultaneous detection of four dipyrone metabolites in milk

Dipyrone (DIP) is a commonly used non-steroidal anti-inflammatory drug with strong analgesic, antipyretic, and spasmolytic properties. Following oral administration, DIP is rapidly degraded into four major metabolites: 4-methylaminoantipyrine (MAA), 4-formylaminoantipyrine (FAA), 4-aminoantipyrine (AA), and 4-acetylaminoantipyrine (AAA). In this study, we produced a monoclonal antibody (mAb) and developed a lateral flow immunoassay (LFIA) for the simultaneous detection of the four DIP metabolites in milk samples. By comparing four haptens, a highly sensitive and specific mAb (3C7) was produced with 50% inhibitory concentrations of 8.67, 8.08, 0.98, and 0.82 ng mL−1 for AA, MAA, FAA, and AAA, respectively. Based on 3C7, an LFIA was successfully developed for the determination of DIP metabolites in spiked milk samples with visual limits of detection of 5, 5, 1, and 0.5 ng mL−1 for AA, MAA, FAA, and AAA, respectively, with recoveries (mean ± SD) ranging from 83.0 ± 3.3% to 103.8 ± 3.8%. Our findings revealed that the LFIA we developed is a potential method for the rapid and sensitive detection of DIP metabolites.