Enzyme immunoassay for semicarbazide—The nitrofuran metabolite and food contaminant

Abstract

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed—all SEM testing currently uses expensive LC–MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CC β ) of 0.25 μg kg −1 when a threshold of 0.21 μg kg −1 is applied to the selection of samples for confirmation (lowest observed 0.25 μg kg −1 fortified sample, n = 20), thus satisfying the EU nitrofurans’ minimum required performance limit of 1 μg kg −1. NFZ-incurred muscles (12) containing SEM at 0.5–5.0 μg kg −1 by LC–MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver.