Internal report on the "in-house" validation of an analytical method for the determination of semicarbazide in fresh egg and in industrial egg products

Nitrofurans form a group of antibiotics used for the treatment of gastrointestinal and dermatological infections including salmonellosis in cattle, swine, poultry, fish and shrimps. The use of four nitrofurans, namely furazolidone, furaltadone, nitrofurantoin and nitrofurazone, has however been banned within the European Union (Commission regulation 1442/95 and Commission Regulation 2901/93). Nevertheless these antibiotics are still in use in some countries outside the EU. Nitrofuran-type antibiotics are characterised by a very fast metabolism such that they have "in-vivo" half-lives of only some hours. Semicarbazide (SEM) has shown to be a stable side chain metabolite of nitrofurazone (NFZ) and therefore it has been used as a marker of the mentioned antibiotic. SEM has been found in tissue and eggs of poultry fed with NFZ, at concentrations proportional to the amount of NFZ added to the feed. This work contains the standard operational procedure and the "in-house" validation results of a method for the determination of SEM in whole fresh egg and in egg powder. The method is similar to a recently validated one for the determination of SEM in baby food, and consists in hydrolysis of the protein-bound SEM to get it free in solution and simultaneous derivatisation of the free SEM with 2-nitrobenzaldehyde to form the respective hydrazone derivative, clean-up by solid-phase extraction, and final LC-MS/MS determination.