Simultaneous Determination of Twelve Glucocorticoid Residues in Milk by Ultra Performance Liquid Chromatography-Electrospray Tandem Mass Spectrometry

A comprehensive analytical method based on ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS) with negative modes has been developed for the simultaneous determination of twelve glucocorticoid residues in milk. The multireaction-monitoring mode was employed in this determination. Milk samples were extracted by sonication in a methanol/acetate buffer solution (pH 5.2), and then defatted with n-hexane. Sample concentration and purification were performed using Oasis HLB, Sep-pak Silica, and amino-propyl solid-phase extraction cartridges. The separation was performed on a Waters ACQUITY UPLC™ 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. The glucocorticoids were identified 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 12 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 rapid, sensitive, and specific for the determination of glucocorticoid residues in milk.