Liquid chromatographic–mass spectrometric analysis of 11 glucocorticoid residues and an optimization of enzymatic hydrolysis conditions in bovine liver Original Research Article

A liquid chromatography combined to mass spectrometry (LC–MS/MS) method to confirm the presence of 11 synthetic glucocorticoids in cattle liver is described. After an enzymatic deconjugation step, liver samples were treated with methanol to extract the glucocorticoids. A further cleanup of the extracts was carried out by SPE. LC separations were carried out as described previously using a column packed with porous graphitic carbon. For the detection positive (ESI+) and negative (ESI−) electrospray ionization were evaluated, but the preference was given to the negative mode for most of the analytes. The complete method was validated according to SANCO/1085/2000, giving decision limits and detection capabilities in 0.08–0.38 μg kg−1 range for the banned glucocorticoids and between 0.1 and 0.5 μg kg−1 above the established maximum residue limits (MRLs) values for betamethasone, dexamethasone, prednisolone and methylprednisolone. Several incurred liver samples of animals treated with different glucocorticoids were analyzed. In order to obtain a maximum amount of unconjugated parent drug, enzymatic hydrolysis conditions were optimized using liver samples of a calf treated simultaneously with beta- and dexamethasone. A central composite experimental design was applied and the estimated response surface plots were used to select optimal conditions of pH, temperature and duration of the preliminary deconjugation step.