Determination of Salbutamol, Clenbuterol, and Brombuterol in Urine by a Highly Sensitive Chemiluminescence Enzyme Immunoassay

Abstract

A salbutamol-bovine serum albumin and a salbutamol-ovalbumin coating antigen were synthesized, and six New Zealand white rabbits were treated with the immunogen to obtain polyclonal antibodies to develop a rapid, sensitive, and indirect chemiluminescent enzyme immunoassay (ciCLEIA) for the analysis of swine and bovine urine. The prepared antibodies showed high cross-reactivities with clenbuterol (139.6%) and brombuterol (225%). Under the optimized conditions, the linear dynamic range and the limit of detection (LOD) for salbutamol were from 0.007 to 0.17 µg L⁻¹ and 0.003 µg L⁻¹, with a correlation coefficient of 0.9965 and a half maximum inhibition concentration of 0.028 µg L⁻¹. Recoveries for salbutamol, clenbuterol, and brombuterol were from 78.8% to 119.0% with intra-assay and inter-assay coefficients of variation less than 13.9% and 19.7%, respectively. The reported ciCLEIA was about 10-fold more sensitive for salbutamol and 20-fold more sensitive for clenbuterol compared to conventional methods. This study showed that ciCLEIA was a reliable, convenient, and sensitive method for the simultaneous determination of salbutamol, clenbuterol, and brombuterol in swine and bovine urine.

Keywords:

• Brombuterol
• Chemiluminescent enzyme immunoassay
• Clenbuterol
• Polyclonal antibody
• Salbutamol
• Urine

Notes

^a Intra-assay variation was determined by six replicates on a single day.

^b Inter-assay variation was determined by three replicates on three different days.

^c Coefficient of variation.

^a IC₅₀ value was the competitor concentrations by indirect competitive ELISA. Data was obtained from three separate experiments run on three different days.

^b Not determined.

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