Waters Corporation, 34 Maple St. Milford, MA …

1 IntroductionApiculture relies on antibiotics to prevent disease propagating through the densely populated bee overuse of antibiotics in honey bee colonies can cause high levels of residues in honey products, whichbecomes a public health issue. Additionally, bacteria that have developed resistance to the applied antibioticscan pose an increased threat to both human and animal health. Consequently, antibiotics become less effectiveagainst bacteria and there will be fewer alternatives available for the successful treatment of producers search for these alternative antibiotics such as nitrofurans to treat disease. Nitrofuran antibiotics metabolize rapidly with an in vivo half-life in the order of hours, making parent drugdetection ineffective.

2 An LC/MS/MS method is described for the quantification of nitrofuran metabolite residues (AOZ, AMOZ, 1-Aminohydantion(AH), Semicarbizide (SC)) in commercially available honey. The metaboliteresidues were extracted from the honey samples by first dissolving the honey in HCl. The samples were cleaned,derivatized and then enriched using Oasis HLB solid phase extraction devices. The metabolite residues wereresolved chromatographically using a XTerra MS C18analytical column. Positive ion electrospray massspectrometry was used to quantify and confirm the parent ion [M+H]+and fragments for each target of Honey SamplesThe honey samples were prepared for analysis using a two step SPE process (Figure 1).

3 The first step providesa simple pass through clean-up to fractionate the analytes from the bulk of the matrix. This dramatically improvesthe subsequent derivitazation procedure. The second SPE protocol provides additional clean-up as well asproviding a sample enrichment factor of 10 to1 (2g Honey into 200 L). A 2 g sample of honey was diluted with 5 mL of M HCl and prepared using the procedure outlined inFigure 1. The eluent was quantitatively collected and 300 L of 50 mM 2-nitrobenzaldehyde in DMSO is addedfor derivitization. The sample was hydrolyzed and derivatized for 18 hours at 37 C. The sample was cooledto room temperature and adjusted to pH 7 by addition of 6 mL of M K2 HPO4prior to SPE Step DETERMINATION OF NITROFURAN METABOLITE residues IN HONEYK evin M.

4 Jenkins, Michael S. YoungWaters corporation , 34 Maple St. Milford, MA*The pass through fraction and washsteps are quantitatively combined intoa single 15 mL capped sample mL methanol, 2 mL waterPass-Through*2 g prepared honey in 5 mL of N HClWash 1*2 mL waterCollect Quantitatively*Figure 1: SPE Protocol SummarySPE Step 1 removes polyphenolic constituents, waxes, and organic contaminants. The sugars and the underivatizednitrofuran metabolites pass through the cartridge. This cartridge is discarded. The sugars are removed postderivatization in SPE Step 2 before the final SPE elution of the derivatized nitrofuran Step 1 SPE Step 2 NOONH2NH2 NNH2 OHNONH2 ONONH2 NNHOONO2 OHCNOONNO2NO2 NNNH2 OHNO2 NNNHOONO2 NONONOH+,H2O, 37oC, 18 hrNitrofuran Derivative StructuresAOZS emicarbizide (SC)1-Aminohydantion (AH)AMOZO asis HLB, 3 cc, 60 mgPart No.

5 WAT094226 Oasis HLB, 3 cc, 60 mgPart No. WAT094226 Condition/Equilibrate1 mL methanol, 1 mL waterLoadprepared honey in sample pH 7 Wash 12mL waterWash 22 mL 30% methanol in waterElute3 mL 2% FA in 90:10 MTBE/methanolEvaporate and Reconstitutein 200 L Mobile PhaseDry20 minutes ApplicationNOTEHPLC ConditionsInstrument: Alliance 2695 Separations ModuleColumn: XTerra MSC18, m, x 100 mmPart Number: 186000404 Flow Rate: mL/minMobile Phase: Isocratic 70 % 20 mM ammonium formate pH ,30 % : LTemperature: 30 CMS ConditionsInstrument: Waters Micromass Quattro Micro Interface.

6 Positive Electrospray (ESI+) Optics:Capillary - kVExtractor - 4 VRF Lens - VSource Block Temperature - 150 CDesolvation Temperature - 350 CMRM Parameters: e AOZ AMOZ SCAHS emicarbizide External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)Analyte AreaAOZ External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)Analyte AreaAMOZ External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)Analyte AreaAH External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)Analyte AreaFigure 2: Representative calibrationcurves spiked into blank honey.

7 Note:Suitable deuterated internal standardswere not available at the time ofthis study. The resulting non-linearcalibration curves for SC and AHwere fit to a quadratic function. Figure 4: Representative chromatograms of a 400 ng/Kgspiked honey sampleFigure 3. Left Side Column (SPE Step 1): The columnon the left shows the retained matrix resulting fromthe initial sample pass-through. This column is discardedand the passed-through sample is then Side Column (SPE Step 2): The column on theright shows the sample clean-up after the finalanalyte (V)Collision (V)AOZ236 1342812 AMOZ335 2912812SC209 1922510AH249 1782815 e AOZ AMOZ SCAHS emicarbizide External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)Analyte AreaAOZ External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)Analyte AreaAMOZ External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)Analyte AreaAH External 1500 2000 2500 3000 3500 4000 Concentration (ng/kg)

8 Analyte AreaResultsSPE Step 1 SPE Step 2 Waters , Micromass, Alliance, Oasis and XTerra are trademarks of Waters corporation . All trademarks are property of their respective owners. 2004 Waters corporation . Printed in the Offices:Austria and European Export(Central South Eastern Europe, CIS and Middle East)(43) 1 8771807 Australia(61) 2 9933 1777 Belgium(32) 2 7261000 Brazil(55) 11 5543 7788 Canada800 252 4752 China (8610) 84518918 CIS/Russia (7) 095 336 7000 Czech Republic (42) 02 6171 1384 Denmark(45) 46 598080 Finland (358) 9 506 4140 France(33) 1 30 48 72 00 Germany(49) 6196 40 06 00 Hong Kong(852) 2964 1800 Hungary(36) 1 350 5086 India and India Subcontinent(91) 80 28371900 Ireland(353) 1 448 1500 Italy(39) 02 (81) 3 3471 7191 Korea(82) 2 820 2700 Mexico(5255) 5524 76 36 The Netherlands31 (0)76-50 87 200 Norway(47) 63 84 60 50 Poland(48)

9 22 833 4400 Puerto Rico(787) 747 8445 Singapore(65) 6278 7997 Spain(34) 93 600 93 00 Sweden(46) 8 555 11500 Switzerland(41) 62 889 2030 Taiwan(886) 2 2543 1898 United Kingdom(44) 208 238 6100 All other countries: Waters corporation 478 2000/800 252 Sample 1 AnalyteRSD (%)* Sample 2 AnalyteRSD (%)* (ng/kg)200200300200 Table 1: Relative standard deviation obtained from two different lots of honey spiked at 500 ng/Kg (ppt). Sample 1 is raw wildflower honey and Sample 2 is buckwheat honey, both commercially available. Metabolite recovery was greater than 85% post-derivatization for each analyte. The blanks used for spiking tested negative before the and ConclusionsThe SPE protocol described in this paper provides sampleenrichment and cleanup acceptable for the routine determinationof nitrofuran metabolites in honey.

10 Results obtained fromfortified honey samples indicate that the limit of quantification(LOQ) was below 300 derivatization is necessary for chromatographicretention for the small, polar nitrofuran metabolites. Unfortunately,derivatization in the presence of unwanted matrix reducesthe reaction efficiency and may increase matrix side-reactionsthat can interfere with the LC analysis. The two-step SPEprocedure described in this method was optimized to minimizethe matrixeffects prior to derivatization. This procedurereduced the undesirable matrix interferences while minimizingthe amount of derivatization reagent required to achieve asuccessful reaction.