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
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