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

Abstract

A previously reported multiresidue method for the analysis of 11 important β-lactams (amoxicillin, ampicillin, cefazolin, cephalexin, cloxacillin, desfuroylceftiofur cysteine disulfide (DCCD), deacetylcephapirin, dicloxacillin, nafcillin, oxacillin, and penicillin G) 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 C 18 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 [ 13C 6]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 β-lactams, except for DCCD, 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–MS/MS (electrospray positive ionization) performance was achieved by using 0.1% formic acid as an additive in both parts of the mobile phase, in water and in acetonitrile. 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.