Evaluation of liquid chromatographic behavior of cephalosporin antibiotics using restricted access medium columns for on-line sample cleanup of bovine milk

Microsample deproteinization of bovine milk was carried out on-line using a series of restricted access medium (RAM) bovine serum albumin (BSA) columns: C-8, C-18, phenyl, and cyano. The four different columns prepared showed a high percentage of protein exclusion using water as the mobile phase and provided an appropriate retention profile for a series of five cephalosporin antibiotics (cefoperazone, cephacetril, cephalexin, cephapirin, and ceftiofur). Chromatographic conditions such as washing time, buffer pH, and type and percentage of organic modifier were fully evaluated with respect to the protein elution profile and retention of the antibiotic by RAM column. One of these columns was chosen to develop and validate a method for the determination of cefoperazone in bovine milk. The system used in this work was composed of a RAM-BSA phenyl column coupled to a C18 analytical column. The standard curve was linear over the range 0.100-2.50 mu g/mL. The limits of quantification and detection were 0.100 and 0.050 mu g/mL, respectively. The developed method showed high intermediate precision (CV of 2.37-2.63%) and accuracy (90.7-94.3%) with adequate sensitivity for drug monitoring in bovine milk samples.