Utility of the Redox Cycle of Nitrofurantoin for the Development of a New Chemiluminescence Method for Its Analysis in Milk Samples

Nitrofurantoin is utilized in various industries, including dairy, livestock, poultry, and aquaculture, as a growth promoter and antibacterial agent. Because prolonged use can cause mutagenesis and other side effects, many countries have prohibited its use in food-producing animals. In this work, we introduce a simple, rapid, and highly sensitive chemiluminescence (CL) approach for the quantitation of nitrofurantoin using its redox cycle activity. Nitrofurantoin is reduced to nitrofurantoin radicals by the reducing agent dithiothreitol, and reactive oxygen species (ROS) formed during the reoxidation process (superoxide anion radical) are detected by luminol CL. The CL conditions were optimized, including types of solvents, CL and reducing reagents, and their concentrations. The method was validated as per International Council for Harmonization (ICHQ2(R2)) guidelines, regarding linearity, detection and quantitation limits, accuracy, and precision. A good linearity with <i>r</i> = 0.9992 was obtained between the CL intensity and the nitrofurantoin concentration in the range of 4.0–400.0 ng/mL with a high sensitivity down to 1.15 ng/mL. The method was utilized to determine nitrofurantoin in milk samples, and a good recovery range was obtained (97.5–103.1%; RSD ≤ 4.4%); the results were comparable to the reported method, demonstrating the method’s reliability. Finally, the method demonstrated good practicality using a recently developed assessment tool.