3,5-Dinitrosalicylic acid hydrazide (DNSAH) is the metabolite of the antibacterial nitrofuran nifursol. A simple and accurate analytical method to determine 3,5-Dinitrosalicylic acid hydrazide in honey by solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) 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 using an electrospray ionization source (ESI - ) and multiple reaction monitoring (MRM) mode, and quantified by internal standard method. It was shown that the detection limit (LOD) and quantitative limit (LOQ) 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 0.1~200 μg/L, with the correlation coefficient (r) greater than 0.9991. Under the concentration levels of 0.2, 0.5, 1.0, 2.0μg/kg, the average recovery rate were between 98.5 ~ 102.3% and the relative standard deviation (RSD) were between 1.1 ~ 5.4%. The results indicate that the method is simple, rapid, sensitive and reproducible, which can be used for determination of DNSAH in different types of honey.
To identify synergistic combinations of different food additives, the antimicrobial effects of thymol and carvacrol against Salmonella Typhimurium were assessed alone and in combination with various other preservatives including EDTA, acetic acid, lactic acid, and citric acid. Overall, growth of Salmonella Typhimurium was significantly inhibited in Mueller-Hinton broth containing thymol, carvacrol, EDTA, acetic acid, lactic acid, or citric acid at concentrations of 400 mg/liter, 400 μl/liter, 300 mg/liter, 0.2% (vol/vol), 0.2% (vol/vol), and 0.2% (wt/vol), respectively. The combination of different antimicrobials such as thymol or carvacrol with EDTA, thymol or carvacrol with acetic acid, and thymol or carvacrol with citric acid all resulted in significantly reduced populations of Salmonella Typhimurium. In samples treated with combinations, these antimicrobials had synergistic effects compared with samples treated with thymol, carvacrol, EDTA, acetic acid, or citric acid alone. However, the combined use of lactic acid with thymol or carvacrol did not produce a synergistic effect against Salmonella Typhimurium. Thus, some chelators or organic acids can be used as food preservatives in combination with thymol and carvacrol to reduce the concentrations needed to produce an adequate antimicrobial effect.