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Microsoft powerpoint - euroresidue_2012

Multi residue method for the detection of antimicrobial agents in Dried Distillers Grains by liquid chromatography-high resolution mass spectrometry George Kaklamanos and Ursula Vincent
European Commission – DG Joint Research Centre, IRMM, Retieseweg 111, B-2440 Geel, Belgium IntroductionDried Distillers Grains (DDGs) are produced as a co-product from bio-ethanol production from grain. It is a non-animal based, high protein livestock feed supplement, produced from the distillation and dehydration process during ethanol production. Ethanol fermentation routinely becomes contaminated with bacteria, as a result yeast converts starch to ethanol, but bacteria convert those same sugars to lactic or acetic acid. Hence, if the bacteria get out of control, ethanol production yields can drop significantly. To overcome this disability and control the population of bacteria, producers may use different kind and large amounts of antimicrobials, possessing a threat to human and animal health. To address such threat, the presence of antibiotics in DDGs must be controlled. To enable the detection of antimicrobial agents in Dried Distillers Grains (DDGs), a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed including a wide range of classes of antibiotics (authorized coccidiostats, banned coccidiostats, macrolides, tetracyclines, quinolones and sulphonamides). The compounds were extracted with a mixture of organic solvents, filtered, centrifuged and directly injected to the LC-HRMS. The ionization mode was heated- Electrospray ionization (H-ESI) in positive and negative full scan mode. Analysis was performed with a high resolving power of 50.000 to achieve acceptable mass accuracy. The method proved to have acceptable accuracy and is capable to provide highly selective qualitative and quantitative analysis.
Sample preparation: 3 g of feed
Liquid-solid extraction with a mixture of ACN/MeOH/H O containing 1% formic acid. Sonication,
centrifugation, filtration and injection to the LC-HRMS.
HPLC: Gradient RP-HPLC, mixture of Internal standards
Altima HP C18, 150x3.2 mm, 5 μm column, column temperature: 35°C, mobile phase: MeOH (0.5% formic acid) and H O (0.5% formic acid), flow rate 350 ml.min-1, injection volume 10 μl.  Atmospheric Pressure ionization: H-ESI Ionization
Spray voltage: 4KV, sheath gas: 60 arb, aux gas: 10 arb, capillary temperature: 275 οC, heated m/z= 734.46118-734.47586 F: FTMS {1,1} + p ESI Full ms  Exactive Orbitrap-HRMS:
Scan range: 140-940 m/z, resolution 50.000, polarity: positive and negative switching, AGC: 3x106, maximum inj. time: 20 ms.
m/z= 458.18760-458.19676 F: FTMS {1,1} + p ESI Full ms [140.00-940.00] MS  10 points were evaluated for the matrix-matched calibration curves, covering a dynamic m/z= 142.05968-142.06252 F: FTMS {1,1} + p ESI Full ms range from 25-1000 ng g-1 (Internal and External calibration).
Validation experiments were carried out by performing 3 replicates per sample and 3 repetitions for each concentration level over 3 days.
 4 concentration levels were tested 100, 200, 250 and 500 ng g-1.
m/z= 334.05874-334.06542 F: FTMS {1,1} + p ESI Full ms [140.00-940.00] MS A/A Compound
Concentration
Recovery
A/A Compound
Concentration
Recovery
Halofuginone hydrobromide
Amprolium
Nicarbazin
Clopidol
Diclazuril
Ethopabate
Semduramicin sodium
Dimetridazole
Decoquinate
Ronidazole
Monensin sodium
Furazolidone
Lasalocid sodium
Marbofloxacin
Chromatogram of a spiked DDG sample containing 48 antimicrobial agents; representative compounds from each class, Maduramicin ammonium
Norfloxacin
namely sulfamethoxazole, erythromycin, minocycline, dimetridazole, robenidine and enrofloxacin are displayed in the Salinomycin sodium
Ciprofloxacin
figure. The spiked concentration is of 100 ng g-1 (mass accuracy at 5 ppm). Danofloxacin
Robenidine
Enrofloxacin
Lincomycin
Sarafloxacin
Spiramycin
Difloxacin
Tilmicosin
Cinoxacin
 A mass resolving power of 50.000 was required to achieve mass accuracy lower than 10ppm.
 Data obtained showed satisfactory precision and accuracy.
Tylosin tartrate
Oxolinic acid
 All compounds were identified and quantified over a wide dynamic range with R2 ranging from Rosamicin
Sulfathiazole
Erythromycin
Sulfamerazine
 In order to test the method as a building up point more compounds were added (covering 11 Clarithromycin
Sulfamethazine
different chemical groups) and were successfully identified, showing the potential of the Minocycline
Sulfamethoxypyridazine
Tetracycline
Sulfadimethoxine
 The utilization of liquid chromatography coupled to the high resolution Orbitrap proved to be a powerful tool for routine analysis of antimicrobial agents in DDG control.
Oxytetracycline
Sulfachloropyridazine
Chlortetracycline
Sulfamethoxazole
Methacycline
Sulfamonomethoxine
Doxyxycline
Sulfaquinoxaline
Dr Ursula Vincent
European Commission • Joint Research Centre
Institute for Reference Materials and MeasurementsFood safety and Quality UnitTel. +32 (0)14 57 1207 • Email: ursula.vincent@ec.europa.eu

Source: http://www.qsaffe.eu/new%20pdfs/WP1_Euroresidue_2012.pdf

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