Detection of glucocorticoid residues in pig liver by high-performance liquid chromatography with on-line electrogenerated [Cu(HIO6)2]5−—luminol chemiluminescence detection

A novel method was developed for the simultaneous determination of glucocorticoid residues such as triamcinolone (TR), prednisolone (PR), hydrocortisone (HC), cortisone (CO), methylprednisolone (MP), dexamethasone (DE) and triamcinolone acetonide (TA) by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection. The procedure was based on the enhancement effect of glucocorticoids on the chemiluminescence reaction between luminol and the complex of trivalent copper and periodate ([Cu(HIO6)2]5−), which was on-line electrogenerated by constant current electrolysis. The HPLC separation used a Nucleosil RP-C18 column (250 mm × 4.6 mm i.d., 5 μm, pore size, 100 Å) with a mobile phase consisting of acetonitrile and 1.0 mmol L−1 ammonium acetate (pH 6.8, 40:60,v/v) at a flow rate of 0.8 mL min−1. The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Liver samples were hydrolyzed with Helix pomatia juice followed by a solid-phase extraction procedure. Under optimum conditions, the limits of detection (LOD) at a signal-to-noise of 3 ranged from 0.08 to 1.0 ng g−1 and the limits of quantification (LOQ) at a signal-to-noise of 10 ranged from 0.27 to 3.33 ng g−1 for seven glucocorticoids. The relative standard deviations (RSD) of intra- and inter-day precision were below 6.8%. The average recoveries for glucocorticoids (spiked at the levels of 5–50 ng g−1) in pig liver ranged from 88 to 106%, and the relative standard deviations of the quantitative results were from 2.0 to 6.9%. The proposed method had been successfully applied to the determination of glucocorticoid residues in pig liver.