HPLC determination of cefotaxime and cephalexine residues in milk and cephalexine in veterinary formulation

An HPLC method was developed and validated for the determination of the cephalosporins cefotaxime and cephalexine in skimmed bovine milk. The analytical column, Kromasil C-18 (250 mm x 4.0 mm, 5 mu m) was operated at ambient temperature. Mobile phase consisted of CH3OH-acetate buffer (pH = 4.0) and it was delivered isocratically at a flow rate of 1.0 mL center dot min(-1). Total analysis time was less than 5 min. Caffeine was used as internal standard (5 ng center dot mu L-1). UV detection was performed at 265 nm. Method validation was performed by means of intra-day (n = 5) and inter-day accuracy and precision (n = 8), sensitivity and linearity. Limits of detection (LOD) and limits of quantification (LOQ) were 0.1 and 0.3 ng center dot mu L-1, respectively. The method was applied to the analysis of a veterinary drug (CEPOREX) containing cephalexine. The results were quite accurate with the relative error varying from -8.0 to -3.5%. Solid-phase extraction was applied to remove all matrix interference from milk samples. High extraction recoveries (average 84-121%) were achieved by using Abselut NEXUS cartridges with acetonitrile as eluent and a rinsing step with water and n-butanol. A pre-concentration step was necessary in a 1/10 level to reach the EU MRL concentration level (100 mu g center dot kg(-1)). RSD values were less than 7% for both cephalosporins.