Development and validation of a quantitative confirmatory method for 30 β-lactam antibiotics in bovine muscle using liquid chromatography coupled to tandem mass spectrometry

- Carbapenems and other β-lactams can be analysed by LC-MS/MS without derivatisation.
- More accurate results can be achieved by controlling temperature during analysis.
- Methanol cannot be used in β-lactam analysis because it degrades analytes.
- The use of isotope standards negates matrix effects and improves method precision.

A method was developed for the confirmatory and quantitative analysis of 30 β-lactam antibiotic residues in bovine muscle. The method includes 12 penicillins (amoxicillin, ampicillin, cloxacillin, dicloxacillin, mecillinam, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin), 12 cephalosporins (cefacetrile, cefadroxil, cephalexin, cefalonium, cefazolin, cefoperazone, cefotaxime, cefquinome, cefuroxime, desacetyl cephapirin, desfuroylceftiofur cysteine disulfide, desfuroylceftiofur dimer), five carbapenems (biapenem, doripenem, ertapenem, imipenem, meropenem) and faropenem. Samples were extracted using a simple solvent extraction with acetonitrile:water (80:20, v/v) and C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) detection. Chromatography was performed on a reversed phase CSH C18 column, using a binary gradient separation comprising of 0.01% formic acid and 0.2 mM ammonium acetate in water (mobile phase A) and 0.01% formic acid in acetonitrile (mobile phase B). The mass spectrometer was operated in the positive electrospray ionisation mode (ESI(+)). Validation was performed following the 2002/657/EC guidelines. Trueness ranged between 69% and 143% and precision ranged between 2.0% and 29.9% under within-laboratory reproducibility conditions. The developed method uses minimal sample preparation and 30 test samples can be analysed by a single analyst in a single day. To the best of our knowledge, this is the first method for carbapenems in foodstuff that does not require derivatisation.