Optimization of analytical conditions and validation of a fluorescence method for the determination of sulfadiazine in milk

This paper describes optimization and validation of a method for sulfadiazine determination in milk samples based on sulfadiazine derivatization with fluorescamine followed by excitation-emission (fluorescence) measurement. For both the optimization and the validation, a comparison between zero-order and first-order signals has been made, showing the advantages of using first-order signals. In the optimization the effects of the temperature of the derivatization reaction, the amount of fluorescamine and the derivatization time on the instrumental signal (maximum intensity or the net analyte signal) are studied by a factorial experimental design, with the optimal values of these factors which give the highest signal being 22 degrees C for the reaction temperature, 50 mu l fluorescamine and 20 min of derivatization time. The validation of the method under the optimal experimental conditions shows that the analytical method is fit-for-purpose, with values of the capability of detection (CC beta) of 4.3 mu g l(-1) at a sulfadiazine concentration of zero and with probabilities of a false positive and a false negative of 5%. Around the permitted limit (established for the sulfonamides at 100 mu g l(-1)), CC beta is 112 mu g l(-1). The precision, as the intermediate reproducibility, was established as 1.2 and 3.3 mu g (-1) around 0 and 100 mu g l(-1), respectively. In the application to milk samples spiked with sulfadiazine a mean recovery of around 90% was obtained with a standard deviation of about 8% (14 samples of different concentrations).