Determination of antibacterials in animal feed by pressurized liquid extraction followed by online purification and liquid chromatography-electrospray tandem mass spectrometry

Abstract

A fully automated method has been developed for analysis of eighteen antibacterial compounds, including penicillins, cephalosporins and sulfonamides, in animal feed with limits of quantification in the range 0.25-5.79 microg kg(-1). The method is based on pressurized liquid extraction of 3 g homogenized feed with water and online clean-up of 500 microL of the extract with C(18)HD cartridges. The purified sample was directly analysed by liquid chromatography-electrospray tandem mass spectrometry (SPE-LC-ESI-MS-MS). Chromatographic separation was achieved within 10 min by use of a C(12) Phenomenex Hydro-RP reversed-phase analytical column and a mobile phase gradient (water + 0.1% formic acid-methanol + 0.1% formic acid). The method was validated, revealing capability for detection of concentrations as low as 0.09 microg kg(-1), decision limits (CCalpha) and detection capabilities (CCbeta) in the range 10-174 microg kg(-1) and 22-182 microg kg(-1), respectively, and inter-day precision ranging from 0.7 to 8.3%. Recovery, with internal standard correction, was in the range 93-134% for all analytes. The method was then applied to analysis of fifteen feed samples, nine of which contained at least one antimicrobial at concentrations between 0.006 and 1.526 mg kg(-1). The performance data and results from the method were compared with those from a previous method developed by our group, using offline SPE, by analyzing the same set of samples by both methods. The online SPE approach resulted in slightly improved sensitivity, with LODs of 0.09-2.12 microg kg(-1) compared with 0.12-3.94 microg kg(-1) by the offline approach. In general, better recovery was achieved by use of online purification (for 72% of the analytes) and the correlation between the two methods was good. The main advantages of the new online method are rapid and automated sample pre-treatment, and reduction of sample manipulation, enabling high-throughput analysis and highly accurate results. Because of all these characteristics, the proposed method is applicable and could be deemed necessary within the field of food control and safety.