Determination of melamine in pet food by enzyme immunoassay, high-performance liquid chromatography with diode array detection, and ultra-performance liquid chromatography with tandem mass spectrometry.

Melamine in pet food (fortified or originally contaminated) was determined by enzyme immunoassay (EIA), high-performance liquid chromatography with diode array detection (HPLC-DAD), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The limits of detection (LOD) for EIA and HPLC-DAD were 0.02 and 0.1 μg/mL, respectively. The linear ranges of the calibration curves for EIA and HPLC-DAD were 0.02-0.5 and 0.1-500 μg/mL, respectively. The coefficient of determinations (r2) of the standard curves for EIA and HPLC were 0.9991 and 0.9999, respectively. Coefficient of variations from both inter- and intra-assay were <9.31%, and recovery range for all concentrations was between 71 and 105%. The r2 values between the EIA and HPLC-DAD methods for melamine analysis of the fortified and originally contaminated samples were 0.9973 and 0.9885. The r 2 values for UPLC-MS/MS with HPLC-DAD and with EIA were 0.9566 and 0.9489, respectively.