RESUMO
In this work the development of a method to determine semicarbazide (SEM) in fresh whole egg and in whole egg and egg white powders as those used in the food industry, by using LC-MS/MS is described. The method is based on a recently validated one for the determination of SEM in baby food. SEM is extracted from egg with 0.2 mol L(-1) hydrochloric acid and derivatised with 2-nitrobenzaldehyde. The extract is neutralised and purified by passing through a solid phase extraction (SPE) cartridge. The 2-nitrobenzaldehyde derivative is eluted with ethyl acetate. The eluate is evaporated to dryness and the residue re-dissolved in methanol:water mobile phase. SEM is determined by reversed-phase LC-MS/MS. The "in-house" validation of this method has been performed taking into account the "Harmonized guidelines for single-laboratory validation of methods of analysis" (IUPAC Technical report) and the Commission Decision (2002/657/EC). The performance characteristics of the method were established by in-house validation procedures employing assays with standard solutions, sample blanks and spiked samples. Linearity, matrix effect, trueness, precision, selectivity, limits of detection and of quantification were determined. The fitness for purpose of this method was assessed based on its performance characteristics. LOD's of 0.15 and 0.4 microg kg(-1) for whole fresh egg and industrial egg powders, respectively, were obtained. The LOQ for fresh whole egg was 0.2 microg kg(-1) and for industrial egg powders 0.8 microg kg(-1). Linear calibration curves were obtained in the ranges 1-100 and 0.8-400 microg kg(-1) for fresh whole egg and the egg powders, respectively.
RESUMO
An interlaboratory validation study was conducted according to harmonized protocols to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in fresh whole egg and in an industrially processed whole egg powder. The sample was extracted with hydrochloric acid and derivatized with 2-nitrobenzaldehyde, with 1,2-[(15)N2(13)C]SEM as the internal standard. The extract was neutralized and purified on a solid-phase extraction cartridge. SEM was determined by reversed-phase LC with detection by MS/MS. Five fresh egg samples, of which 3 were obtained from hens fed nitrofurazone (NFZ), one was spiked with SEM at 50 microg/kg and one was a blank sample, and 5 industrial whole egg powder samples, of which 3 were spiked with fresh whole egg from hens fed NFZ, one was spiked with SEM at 350 microg/kg, and one was a blank sample, were sent to 15 laboratories in 10 different European countries. Results were obtained from 12 participants. Average recoveries of SEM from the fresh egg and the egg powder samples were 105.3 and 121.3%, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.9 to 9.3%, and the relative standard deviation for reproducibility (RSDR) ranged from 22.5 to 38.1%. The method showed acceptable within- and between-laboratory precision for both matrixes, as evidenced by the HorRat values, at the target levels for the determination of SEM.
