Alpha 1-acid glycoprotein affinity columns for the clean-up of adrenergic drugs.
alpha 1-Acid glycoproteins (AAGs) have a structure resembling beta-adrenergic receptors and bind several basic drugs in plasma. Chromatographic columns were prepared by linking epsilon-NH2 groups of AAG lysines to a Sepharose 4B support, in order to purify by affinity chromatography adrenergic drugs of possible use in animal production. Loading capacities, binding efficiency, memory effects and matrix interferences from urine samples were studied. The method developed involves sample application in buffered media (pH 7.4), washing with 5 ml 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 AAG were able 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%, respectively. 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 recovery of 97.8%; no matrix interferences were observed.
Brambilla
G
G
Istituto Superiore di Sanità, Laboratorio di Medicina Veterinaria, Rome, Italy.
Fiori
M
M
Curiel
I
I
Serpe
L
L
Gallo
P
P
D000318:Adrenergic beta-Agonists / Q000032:analysis* / Q000652:urine; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002976:Clenbuterol / Q000032:analysis* / Q000652:urine; D004350:Drug Residues / Q000032:analysis*; D006133:Growth Substances / Q000032:analysis* / Q000652:urine; D009961:Orosomucoid; D019155:Veterinary Drugs / Q000032:analysis / Q000652:urine
D000318:Adrenergic beta-Agonists; D006133:Growth Substances; D009961:Orosomucoid; D019155:Veterinary Drugs; D002976:Clenbuterol