The use of ELISA tests and immunoaffinity chromatography combined with reversed-phase high-performance liquid chromatography for dexamethasone detection in equine urine

Abstract

Dexamethasone is a corticosteroid drug widely used in racehorses because of its anti-inflammatory effect. It is, therefore, frequently detected in antidoping tests. A method for the antidoping control of dexamethasone in equine urine using screening by ELISA and confirmation by immunoaffinity chromatography combined with reversed-phase high-performance liquid chromatography-diode array detection (HPLC-DAD) is described. The ELISA test is frequently used in antidoping tests for its sensitivity, relative speed, and low cost. The test showed linearity in the range of 4-500 ng/mL of urine, and the intra-assay and interassay imprecision were 9.4 and 9.7%, respectively. The confirmation method showed a limit of detection of 4 ng/mL for dexamethasone. The intra-assay and interassay imprecisions were 10.3 and 14.4%, respectively. The HPLC-DAD showed a limit of detection of 5 ng and linearity in the range of 25-500 ng of dexamethasone. The absolute method recovery was 56.4%. The proposed method detected dexamethasone up to 52 h after administration and proved to be adequate for the antidoping control.