Determination of clenbuterol, salbutamol, and cimaterol in bovine retina by electrospray ionization-liquid chromatography-tandem mass spectrometry

An electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI/LC/MS/MS) method was developed for the simultaneous determination of the beta-agonists clenbuterol, salbutamol, and cimaterol in bovine retina. The tissue was homogenized in alkaline buffer and spiked to give 10, 15, and 20 ng/g each of the 3 analytes together with the internal standards d6-salbutamol and d6-clenbuterol. The mixture was incubated with protease enzyme to release any protein-bound analytes and then made alkaline before extraction with isobutanol. The extract was dissolved in water and transferred to a clenbuterol immunoaffinity column. After washing, the analytes were eluted and analyzed by ESI/LC/MS/MS using a C18 column with acetic acid-methanol as mobile phase. No interferences were observed from the spiked retina extract at the various single-reaction monitoring modes. Average recoveries for clenbuterol, salbutamol, and cimaterol were 94, 85, and 87% with coefficients of variation (CVs) of 9.4, 9.9, and 8.6%, respectively. A correlation coefficient of r2 = 0.9999 was obtained for all analytes. The limits of detection for clenbuterol, salbutamol, and cimaterol, determined from 3 times the standard deviation of 7 replicates of the lowest spike, were 2.5, 3.5, and 2.0 ng/g with CVs of 8.9, 11.6, and 7.2%, respectively.