Poster_martinsclaudia_alimentos

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.

Source: http://www.4rseem.uji.es/abstracts/M48_POSTER_MartinsClaudia_alimentos.pdf

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