alpha(1)-Acid glycoprotein affinity columns for the clean-up of adrenergicdrugs

alpha(1)-Acid glycoproteins (AAGs) have a structure resembling beta-adrener gic receptors and bind several basic drugs in plasma. Chromatographic colum ns were prepared by linking epsilon-NH2 groups of AAG lysines to a Sepharos e 4B support, in order to purify by affinity chromatography adrenergic drug s of possible use in animal production. Loading capacities, binding efficie ncy, memory effects and matrix interferences from urine samples were studie d. The method developed involves sample application in buffered media (pH 7 .4), washing with 5 mi of PBS, and elution with 4 ml of 1% v/v acetic acid. Under these conditions no memory effect was observed. Loading capacity is correlated with the physiological plasma binding rate (PB) of the drug. For clenbuterol (PB 50%) and anilino-like related drugs, 5 mg of FLAG were abl e to bind about 15 x 10(-6) g of drug, with a 100% recovery from the column . Repeatability and reproducibility, expressed as RSD, were 4.2 and 5.4%, r espectively. The calculated AAG:drug molar ratio was 4.5:1, indicating 22% of the AAG bound to the column retained drug affinity. Among phenolic-like agonists, salbutamol (PB 5%), fenoterol and isoxsuprine hardly interacted, whereas nylidrin, ritodrine and bamethan showed more effective binding. We also checked binding of other drugs of possible use in veterinary medicine. Application of the AAG column to spiked bovine urine revealed a mean recov ery of 97.8%; no matrix interferences were observed.