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Separation and identification of synthetic antigens of hexoestrol residue in animal derived food by HPLC-MS

Abstract Hexoestrol and its derivatives, hexoestrol-mono-ether-butyrate-ethyl (HES-MEBE), hexoestrol-di-ether-butyrate-ethyl (HES-DEBE) were separated and identified using liquid chromatography and spectrometry (ESI). The conditions were as follows: Methyl alcohol and water as mobile phase, 0.3 ml/min of flow rate, elution with linear gradient concentrations from 60–100% of methanol for 25 min on a Lichrospher C-18 column. Synthetic antigen was prepared by coupling hexoestrol-mono-caroxyl-propyl-ethyl (HES-MCPE) with carrier protein by the mixed anhydride method, and the conjugation rate, analysed by UV scanning, is 36.