Submitted to: Rapid Communications in Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/21/2003
Publication Date: 2/21/2003
Citation: FAGERQUIST, C.K., LIGHTFIELD, A.R. ANALYSIS FOR B-LACTAM ANTIBIOTICS IN KIDNEY TISSUE BY LIQUID CHROMATOGRAPHY WITH ELECTROSPRAY IONIZATION AND SELECTIVE REACTION MONITORING/TANDEM ION TRAP MASS SPECTROMETRY. RAPID COMMUNICATIONS IN MASS SPECTROMETRY. 2003.
Interpretive Summary: Beta-lactams are an important class of antibiotics used extensively in both humans and food-producing animals (e.g. cattle, swine, sheep, goats, turkeys and chickens) to treat infections. There is concern that the overuse of antibiotics in livestock may lead to the emergence of antibiotic-resistant bacterial strains that may cross from livestock to humans. Another health concern is that antibiotic residues in edible tissues may lead to an allergic response in hypersensitive individuals. In consequence, there is a real need among regulatory and monitoring agencies (e.g. FSIS) to have antibiotic detection techniques that are simple, sensitive, rapid, inexpensive, rugged and quantitative. The current FSIS technique for detection of beta-lactam antibiotics in edible tissue is a bioassay that is non-specific, semi-quantitative, time-consuming and labor intensive. We have developed a simple, rapid, sensitive and specific method for the detection of beta-lactam antibiotics in edible tissue using a simple extraction and clean-up with liquid chromatography/mass spectrometry detection. Eleven beta-lactams antibiotics were analyzed in fortified and incurred beef kidney tissue. For almost all the beta-lactams, we obtained confirmation of fortified samples at the established tolerances (or target levels). A side-by-side comparison of analysis of incurred tissue with our method and the FSIS bioassay showed good agreement. Based on this investigation, a simple, sensitive, rapid, and specific screening technique for beta-lactams in kidney tissue is now available for use by regulatory and monitoring agencies.
Technical Abstract: Eleven B-lactams antibiotics were analyzed in fortified and incurred beef kidney tissue using high-performance liquid chromatography/selective reaction monitoring/tandem ion trap mass spectrometry. The analytes included: deacetylcephapirin, amoxicillin, cephapirin, desfuroylceftiofur cysteine disulfide (DCCD, a biomarker of ceftiofur), ampicillin, cefazolin, Pen G, oxacillin, cloxacillin, naficillin and dicloxicillin. Pen V was used as an internal standard. The mass spectrometer used for this analysis is a quadrupole ion trap mass spectrometer with electrospray ionization (ESI). Analytes were extracted with acetonitrile and water. Clean-up was performed by solid-phase extraction. Limits of confirmation (LOC) in matrix-matched calibration curve samples were: deacetylcephapirin: 5 ppb; amoxacillin: 10 ppb; cephapirin: 5 ppb; DCCD: 25 ppb; ampicillin: 5 ppb; cefazolin: 5 ppb; Pen G: 5 ppb; oxacillin: 5 ppb; cloxacillin: 5 ppb; naficillin: 5 ppb; dicloxacillin: 50 ppb. We obtained adequate recoveries at the established tolerances (or target levels) of the analytes. We tested our method with incurred kidney tissue which had previously been tested by a microbial assay. A comparison between our LC/MS(n) method and the bioassay show, in general, good correspondence between the results of the two techniques. In several cases, our method was more sensitive than the bioassay. We found that selected-reaction monitoring tandem mass spectrometry was superior in the detection of analytes in incurred tissue than the previously used full spectrum tandem mass spectrometry. We also obtained a tandem mass spectra of DCCD which is significantly at variance with previously published spectra.