Solid-phase extraction followed by liquid chromatography-mass spectrometryfor trace determination of beta-lactam antibiotics in bovine milk

A confirmatory assay able to unambiguously identify and quantify10 approved -for-use beta -lactam antibiotics in milk below stipulated U.S. and EU tole rance levels is presented. beta -Lactams are extracted from 10 mt of intact milk by a Carbograph 4 cartridge. After solvent removal, residue reconstit ution, and filtration, a completely transparent and uncolored extract is in jected into a liquid chromatography -mass spectrometry (LC-MS) instrument e quipped with an electrospray (ES) ion source and a single quadrupole. Durin g the chromatographic run, the ES/MS system is operated first in the positi ve-ion mode (PI) and then in the negative-ion (NI) mode. This is done to ci rcumvent matrix interferences resulting in remarkable signal weakening of t he last-eluted analytes, when detecting them as [M+H](+) adductions. MS dat a acquisition is performed by a time-scheduled three-ion selected ion monit oring program. At the 5 ng/mL level, recoveries of the beta -lactams are be tween 70 (nafcillin) and 108% (cephalin), with relative standard deviations ranging between 5 (oxacillin) and 11% (amoxicillin and ceftiofur). The res ponse of the ES/MS detector is linearly related to injected amounts up to 5 00 ng, irrespective of the chemical characteristics of the beta -lactams an d the acquisition mode selected (PI or NI modes). Limits of quantification, based on a minimal value of the signal-to-noise ratio of 10, were estimate d to be within 0.4 (cephalin) and 3 ng/mL (dicloxacillin). Analyses of milk samples taken after intramammary application of amoxicillin showed that 1. 2 ng/mL of this penicillin was still present 6 days after treatment. At thi s concentration level, the identification power of the method is not weaken ed, as signals of the three product ions of amoxicillin are still well dist inguishable from the background noise.