TURBOFLOW TECHNOLOGY AND LIQUID CHROMATOGRAPHY-TANDEM MASS
SPECTROMETRY FOR MULTI CLASS DETERMINATION OF ANTIBIOTICS IN MILK
C.P.B.Martins1, J. DiBussolo2, R. Zuiderent3, A. Stolker4, R.J. Peters 4
1Thermo Fisher Scientific, Barcelona, Spain, [email protected]
2 Thermo Fisher Scientific, Franklin, USA
3 Thermo Fisher Scientific, Breda, The Netherlands
4 RIKILT, Institute of Food Safety, The Netherlands
Abstract: A novel method for the determination and quantification of antibiotics residues in milk samples is presented. It employs the use of TurboFlow on-line extraction coupled to Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS). The advantage of this method is the drastic reduction of sample preparation time when comparing with the normal offline sample preparation methods. The automated on-line extraction was performed using TurboFlow technology which exploits the difference between large and small molecules and column chemistry to retain compounds of interest while matrix molecules flow to waste. The analysis was performed by LC-MS/MS in the selective reaction monitoring mode (SRM). Introduction: The Global Food Market has become more competitive and equally cost responsive, hence the need for analytical procedures that permit high sample throughput as well as higher sensitivity in combination with good reproducibility. LC-MS/MS is a powerful tool in food analysis, especially when combined with an automated on-line extraction that reduces matrix interferences. The use of less sample handling steps improves also the performance characteristics of the method like recovery, repeatability and reproducibility. However, most analytical techniques developed for quantitative analysis of antibiotic residues in food have been based on methods involving SPE followed by LC-MS. Only recently, methods employing on-line sample preparation have been reported, but usually for a specific class of compounds, rather than a multi-class method. Methodology: Liquid milk products were mixed with a diluent of 50% acetonitrile and 50% water containing 50 mM ammonium acetate and 7.5 mM disodium-EDTA. Triplicate samples were spiked with nine antibiotics at various concentrations (5 to 500 ng/mL). After centrifugation, the supernatants were placed in plastic vials. Neat standards of antibiotics were prepared in diluent. Using an Aria TLX-4 (four-channel, two-dimensional LC) system, standards and prepared samples were injected into Cyclone and Cyclone-P TurboFlow columns connected in series. From each injection, the extracted antibiotics were automatically transferred to a Hypersil C8 column where they were separated by a methanol gradient. The analytes were eluted into a heated electrospray source of a Quantum Ultra tandem mass spectrometer.
salinomycin, spiramycin, sulfamethazine and tetracycline were detected and quantified using positive-ion SRM. Peak areas of spiked milk samples were compared to those of neat standards to assess recovery and matrix interferences. Results: Even without internal standards, quantitative results proved to be fairly linear in the concentration range of 5 to 500 µg/L as well as reproducible and precise (RSD 0.4-14 %). The limits of detection were between 0.1 and 5.2 µg/L.
Also, the results obtained show good reproducibility even when testing different milk brands. Higher fat content seem to influence the precision of the method only at the highest level of the range studied (500 ppb). At the 100 ppb level, the matrix effect is minimal for salinomycine Na, spiromycin, tetracycline, oxytetracycline and sulphametazine. However, values between 70 and 85% were found for phenylbutazone and albendazole, difloxacin. Further work will include the analysis of real samples and raw milk. Conclusions: The automated TurboFlow-LC-MS/MS method developed permits simple sample preparation while minimizing matrix interferences for the detection of various antibiotics in milk products down to ug/L levels. Accurate quantitation of those compounds subject to residual matrix interferences could be accomplished by using a suitable internal standard.
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