Development of an analytical method for the determination of β2-agonist residues in animal tissues 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 beta(2)-agonist residues such as terbutaline, salbutamol, and clenbuterol by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection. The procedure was based on the enhancement effect of beta(2)-agonists on the CL 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-C-18 column (250 mm x 4.6 mm i.d., 5 mu m; pore size, 100 A) with a mobile phase consisting of 90% acetonitrile and 10% aqueous ammonium acetate (20 mmol L-1, pH 4.0) at a flow rate of 1.0 mL min(-1). The effects of several parameters on the HPLC resolution and CL emission were studied systematically. Liver samples were hydrolyzed with beta-glucuronidase followed by a solid-phase extraction procedure using Waters OasisMCX cartridges. Under optimum conditions, the limits of detection at a signal-to-noise ratio of 3 ranged from 0.007 to 0.01 ng g(-1) and the limits of quantification at a signal-to-noise ratio of 10 ranged from 0.023 to 0.033 ng g(-1) for three beta(2)-agonists. The relative standard deviations (RSDs) of intra- and interday precision were below 4.5%. The average recoveries for beta(2)-agonists (spiked at the levels of 0.05-5.0 ng g(-1)) in pig liver ranged from 84 to 110%, and the RSDs of the quantitative results were from 1.6 to 7.2%. The proposed method was successfully applied to the determination of beta(2)-agonist residues in pig liver samples.