3,5-Dinitrosalicylic acid hydrazide (DNSAH) is the metabolite of the antibacterial nitrofuran nifursol. A simple and accurate analytical method to determine DNSAH levels in honey by solid-phase extraction–ultraperformance liquid chromatography–tandem mass spectrometry has been established. The honey sample was hydrolyzed under acidic conditions and derivatized with 2-nitrobenzaldehyde in the dark for 16 h, followed by solid-phase extraction and column chromatography. Detection was performed with an electrospray ionization source and multiple reaction monitoring mode and was quantified by the internal standard method. The detection and quantitative limits of DNSAH in honey were 0.1 and 0.3 μg/kg, respectively. Good linear relationships between peak areas and mass concentrations of the analyte were achieved in the range of approximately 0.1 to 200 μg/L, with the correlation coefficient >0.9991. Under the concentration levels of 0.2, 0.5, 1.0, and 2.0 μg/kg, the average recovery rate were between 98.5 and 102.3%, and the relative standard deviations were between 1.1 and 5.4%. The results indicate that the method is simple, rapid, sensitive, and reproducible and can be used to determine DNSAH levels in different types of honey.

  • This study detects 3,5-dinitrosalicylic acid hydrazide in honey by SPE-UPLC-MS/MS.

  • The sample preparation was optimized for good performance.

  • This study analyzed actual samples.

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