Abstract:

A comprehensive analytical method based on ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS) with negative mode has been developed for the simultaneous determination of twelve glucocorticoids residues in milk. The multi-reaction monitoring mode was employed for the determination. Milk samples were extracted by sonication in a methanol/acetate buffer (pH 5.20) solution, and then defatted with n-hexane. Sample concentration and purification were performed using Oasis HLB, Sep-pak silica and Sep-pak amino-propyl solid phase extraction cartridges. The separation was performed on a Waters ACQUITY UPLCTM BEH C18 column (100 mm×1.0 mm i.d., 1.7 μm) with gradient elution using methanol and water (containing 0.1% formic acid) at a flow rate of 0.1 mL/min. Identification of the glucocorticoids was done using retention times and the distribution of diagnostic ion pairs. Quantification of the glucocorticoids was based on the peak areas of the parent ion and a fragment ion with a higher signal. The limits of detection (LOD) of the method were from 0.02 to 0.38 μg/kg and the limits of quantification (LOQ) ranged from 0.07 to 1.27 μg/kg. Average recoveries for the twelve glucocorticoids (spiked at the levels of 2 and 0.4 μg/kg) ranged from 69.3% to 94.3%, with relative standard deviations between 3.5% and 16.7%. Routine tests showed that the method is fast, sensitive and specific for the determination of glucocorticoids residues in milk.

Key words: glucocorticoids, milk
, residues, solid-phase extraction, ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS)