Dados do Trabalho

Título

BENZOYL CHLORIDE DERIVATIZATION STEP APPLIED FOR p-NITROANILINE DETERMINATION BY LC-MS/MS IN CHICKEN MEAT SAMPLES

Introdução

When administering nicarbazin to broilers, the component 4,4´-dinitrocarbanilide (DNC) is deposited in muscles and organs. Part of the accumulated DNC undergoes degradation when chicken meat is subjected to traditional cooking methods, leading to p-nitroaniline (p-NA) release. This low molecular mass compound (138 Da) is difficult to fragment and analyze by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS), making this technique unfeasible. Therefore, this work aimed at verifying whether the addition of a derivatization step at the end of the sample preparation process, aim at increasing their molecular weight, would make the technique feasible.

Material e Métodos

Chemical reactions using the derivatizing agent benzoyl chloride were carried out, while 104 Da increase in the molecular mass of the analyte of interest was reached (242 Da) and observed in the mass spectrometer by direct infusion in positive mode (m/z 243) and the formation of its daughter ions m/z 105.2 and 77.3. The developed methodology was validated internally, then applied to chicken meat samples subjected to common food preparation methods (grilling, frying, roasting and boiling).

Resultados e Discussão

The best conditions of the derivatization step was achieved with the addition of 50 μL of benzoyl chloride, 450 μL of acetonitrile to 500 μL of the extracted sample at 40 ºC for 15 hours. The obtained MS spectra of the reaction product showed the mass ions expected (m/z 242.9, 105.2 and 77.3). The method showed a linear range of R2 > 0.99, recoveries from 96.6-102.2% in levels between 200 and 2000 ug/kg. The obtained LOD and LOQ were 10 and 30 μg/kg, respectively.

Conclusão

Although triple quadrupole mass spectrometers have a mass analysis range of approximately m/z 20 – 3000 Da, the chemical structure of the molecule of interest also needs to be easy to ionize and capable of generating daughter ions, which was not observed for p-NA. Therefore, derivatization with benzoyl chloride proved to be effective in increasing the desired masses, as well as providing fragmentation sites in the molecule, thus allowing the generation of daughter ions, enabling the identification and quantification of p-NA. The method described herein is applicable to food industries and regulatory labs.