Streamlining methodology for the multiresidue analysis of beta-lactam antibiotics in bovine kidney using liquid chromatography-tandem mass spectrometry

Authors: Mastovska, Katerina; Lightfield, Alan

Submitted to: Journal of Chromatography A
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/8/2008
Publication Date: 7/11/2008
Citation: Mastovska, K., Lightfield, A.R. 2008. Streamlining methodology for the multiresidue analysis of beta-lactam antibiotics in bovine kidney using liquid chromatography-tandem mass spectrometry. Journal of Chromatography A. 1202:118-123.

Interpretive Summary: Beta-lactam antibiotics (penicillins and cephalosporins) are one of the most widely applied type of antimicrobial drugs in current veterinary practice. Monitoring of their residues in edible tissues is important because of the hypersensitivity (allergic reactions) of some individuals to beta-lactams, the emergence of antibiotic-resistant strains of bacteria, and international trade issues. Thus, regulatory agencies and food producers are faced with an increasing demand for more sensitive, rapid, and reliable analytical methods for antibiotic analysis. A previously reported multiresidue method for the analysis of 11 important beta-lactams in bovine kidney has been improved and further streamlined by using a portion rather than the entire extract and by employing syringeless filter vials for final extract filtration and storage. Based on the results of the stability study and liquid chromatography (LC) mobile phase tests, previously used methanol has been eliminated from the entire method, including the LC-tandem mass spectrometry analysis. The streamlined method and information about beta-lactam stability in different solvents will benefit regulatory and other laboratories involved in residue analysis of antibiotics.

Technical Abstract: A previously reported multiresidue method for the analysis of 11 important beta-lactams in bovine kidney has been further streamlined. The method is based on a simple extraction using acetonitrile-water (4:1, v/v), followed by dispersive solid-phase extraction clean-up with C18 sorbent, concentration of an extract aliquot, and filtration of the final extracts using syringeless filter vials, which are used for the sample introduction in the liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The recoveries have been improved by adding the internal standard 13C6-sulfamethazine to the homogenized sample before the extraction step, which enabled a proper control of the volume changes during the sample preparation. Average recoveries of fortified samples were 87-103% for all beta-lactams, except for desfuroylceftiofur cysteine disulfide, which had an average recovery of 60%. Based on the results of the stability study and LC mobile phase tests, methanol has been eliminated from the entire method, including the LC-MS/MS analysis. The best overall LC-ESI(+)-MS/MS performance was achieved by using 0.1% formic acid as an additive in both parts of the mobile phase, in water and in acetonitrile. However due to the stability issues, formic acid should not be added to the final extracts prior to the LC-MS/MS analysis to match the mobile phase composition. To prevent carry-over in the LC-MS/MS analysis, the LC method was divided into two parts: one serving as an analytical method for injection of the sample and elution of the analytes and the other one, starting at a highly organic mobile phase composition, being dedicated for injection of a solvent, washing of the system, and equilibration of the column to the initial conditions of the analytical method. In this way, a blank solvent is injected after each sample, but these in-between injections contribute minimally to the overall sample throughput.