Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/2/2004
Publication Date: 1/26/2005
Citation: Fagerquist, C.K., Lightfield, A.R., Lehotay, S.J. 2005. Confirmatory and quantitative analysis of b-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry. Analytical Chemistry 5(77).1473-1482. Interpretive Summary: Antibiotics are widely used to treat infections, as well as to increase bulk, in food-producing animals. Regulatory agencies have established tolerance levels for antibiotic residues in edible tissues. The monitoring of antibiotic residues in edible tissues is important because of the hypersensitivity of some individuals to antibiotics as well as the emergence of antibiotic-resistant strains of bacteria. In consequence, developing simple, rapid, sensitive and quantitative methods for the detection of chemical residues in foods is crucial both for public health as well as facilitating the removal of trade barriers to American food exports caused by dissonant international standards used in the monitoring chemical residues in foods. A simple, rapid, rugged, sensitive, specific and quantitative method has been developed for the detection of 10 beta-lactam antibiotics in beef kidney tissue. The method involves simple solvent extraction, dispersive solid-phase extraction clean-up and liquid chromatography tandem mass spectrometry detection (LC/MS/MS). The method is confirmatory and quantitative in fortified samples below tolerance levels for the five beta-lactam antibiotics that have established tolerances. For the other five beta-lactam antibiotics the method quantitates to 5 ng/g. Recoveries in fortified samples were better than 70 % for all antibiotics except DCCD which had a recovery of 58%. The method was also tested on 30 incurred samples that had previously been tested by FSIS using their approved microbial assay. The LC/MS/MS analysis generally agreed with the microbial assay for 23 samples although LC/MS/MS is able to specifically identify which antibiotic is present whereas the microbial assay cannot. In addition, in six of the 23 samples, the LC/MS/MS method detects a second antibiotic as being present that is undetected by the microbial assay. For samples which do not fall into the "general agreement" category, the most serious discrepancy involves two samples where the LC/MS/MS method detects a violative level of a cephalosporin beta-lactam in the first sample (and a possibly violative level in the second) whereas the microbial assay identifies these samples as having only violative levels of a penicillin beta-lactam. The microbial assay was designed to distinguish between cephalosporin and penicillin antibiotics but fails to correctly identify the type of antibiotic present in these two samples.
Technical Abstract: A simple, rapid, sensitive, specific and rugged method for the confirmation and quantitation of beta-lactam antibiotics in fortified and incurred bovine kidney tissue has been developed. The method uses a simple solvent extraction/deproteinization, dispersive solid-phase extraction (dispersive-SPE) clean-up and liquid chromatography tandem mass spectrometry (LC/MS/MS) for confirmation and quantitation. LC/MS/MS analysis was performed using time-scheduled events and multiple-reaction monitoring (MRM) on a triple-quadrupole mass spectrometer with an electrospray ionization source. Control tissue was fortified with 10 beta-lactams: deacetylcephapirin (an antimicrobial metabolite of cephapirin), amoxicillin, desfuroylceftiofur cysteine disulfide (DCCD, an antimicrobial metabolite of ceftiofur), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, naficillin, and dicloxacillin. Penicillin V was used as external standard. Average recoveries of 70% or better were obtained for all beta-lactams except DCCD which had an average recovery of 58%. The LC/MS/MS method was able to demonstrate quantitative recoveries at established tolerance levels and provide confirmatory data for unambiguous analyte identification. Dispersive-SPE clean-up greatly simplifies and accelerates sample preparation and improves overall recoveries compared with conventional SPE clean-up. The method was also tested on 30 incurred bovine kidney samples obtained from the USDA Food Safety and Inspection Service which had previously tested the samples using the approved semi-quantitative microbial assay. The results from the quantitative LC/MS/MS analysis were in general agreement with the microbial assay for 23 samples although the LC/MS/MS method is superior in that it can specifically identify which antibiotic is present and quantitate its concentration, whereas the microbial assay can only identify the type of beta-lactam present and report a concentration with respect to penicillin G. In addition, for six of the 23 samples, LC/MS/MS analysis detected a penicillin AND a cephalosporin beta-lactam as being present, whereas the microbial assay detected only a penicillin beta-lactam. For samples which do not fall into the "general agreement" category, the most serious discrepancy involves two samples that were penicillin (+) violative by the microbial assay, whereas LC/MS/MS analysis detected only violative levels of either deacetylcephapirin (in the first) and possibly violative levels of desfurorylceftiofur (in the second). Deacetylcephapirin and DCCD are cephalosporin beta-lactams not penicillins.