Determination of nifursol metabolites in poultry muscle and liver tissue. Development and validation of a confirmatory method

Abstract

A method is described for the identification and quantitative determination of 3,5-dinitrosalicylic acid hydrazide (DSH), the marker residue of nifursol metabolites in poultry (turkey, broiler) muscle and liver tissue. The method is based on the acid-catalysed hydrolysis of tissue-bound metabolites to free DSH and in situ derivatisation with 2-nitrobenzaldehyde to the corresponding nitrophenyl derivative NPDSH. A structural analogue of DSH, 4-hydroxy-3,5-dinitrobenzoic acid hydrazide (HBH) was synthesised to serve as an internal standard. The analytes were isolated from the matrix by liquid-liquid extraction with ethyl acetate. Determination was performed by LC-MS/MS with negative electrospray ionisation. The [M - H](+) ions of NPDSH and NPHBH at m/z 374 were fragmented by collision induced dissociation (CID) producing transition ions at m/z 182, 183 and 226. The transition ions at m/z 182 and 226 were selected for monitoring of NPDSH while the transition ion at m/z 183 was selected for NPHBH. The method has been validated according to the EU criteria of Commission Decision 2002/657/EC at 0.5, 1.0 and 1.5 microg kg(-1) in muscle and liver tissue. A decision limit (CC(alpha)) was obtained of 0.04 and 0.025 microg kg(-1) in muscle and liver, respectively. Similarly a detection capability (CC(beta)) was obtained of 0.10 and 0.05 microg kg(-1) in muscle and liver, respectively. The introduction of HBH as an internal standard did not lead to a significant improvement of the quantitative performance of the method. In fact for liver better performance characteristics were obtained when the IS was not taken into account. Nevertheless, as a qualitative marker for recovery, HBH could still be very useful in the analysis of unknown samples.

Design and Synthesis of Immunoconjugates and Development of an Indirect ELISA for Rapid Detection of 3, 5-Dinitrosalicyclic Acid Hydrazide

“…As nifursol was rapidly metabolized to form the metabolic marker 3,5-dinitrosalicyclic acid hydrazide (DNSH, Figure 1 ) which can persist for a long time in vivo , several laboratories have focused on development of DNSH detection methods [ 2 , 3 , 4 ]. At present, the detection of DNSH is mainly at the μg/kg level by HPLC-MS methods, which are based on the analysis of the DNSH derivative 2-hydroxy-3,5-dinitro- N '-(2-nitrobenzylidene)benzohydrazide (NPDNSH, Figure 1 ) [ 2 , 3 , 4 ]. Disadvantageously, these analytical methods not only require a time-consuming and troublesome derivatization step and expensive apparatus but also have low sample throughput.…”

Monoclonal antibody-based fluorescence polarisation immunoassay for nifursol in feed

“…The European Commission's Scientific Committee on Animal Nutrition (SCAN) has recently published an opinion on the compound, concluding that as both the acceptable daily intake (ADI) and the human exposure to nifursol residues (including metabolites) could not be established, the safety of nifursol for the human consumer could not be ensured (McEvoy, 2002). Because of the rapid metabolism of nifurol in vivo, many laboratories had been focused on detecting the metabolic marker 3,5-dinitrosalicyclic acid hydrazide (DNSH; Figure 1) in animal products for monitoring nifursol abuse (Mulder et al, 2005;Verdon, Couedor, & Sanders, 2007). However, confirming the sample preparation is necessary in monitoring the metabolite.…”

Scientific Opinion on nitrofurans and their metabolites in food

“…The range of values of the decision limit (CCα), 25 CCβ, LOD and LOQ for the various analytes by these methods are 0.11-0.45, 0.19-0.88, 0.01-0.2 and 0.5 µg/kg, respectively (Finzi et al, 2005;Mottier et al, 2005;Barbosa et al, 2007a;Xia et al, 2008;Ryad et al, 2013). A number of papers have been published on the determination of DNSH in turkey and chicken muscle and liver, with reported CCβ values of ≤ 0.1 µg/kg (Kaufmann et al, 2004;Mulder et al, 2005;Vahl, 2005;Zuidema et al, 2005). A method directed at the analysis of all five marker metabolites (AOZ, AMOZ, AHD, SEM, DNSH) in turkey muscle reported CCα values of 0.08-0.20 µg/kg and CCβ values of 0.10-0.25 µg/kg (Verdon et al, 2007).…”