Stability of Tylosin in Honey Impact on Residue Analysis Don Noot, Tom Thompson

Transcription

1 Stability of Tylosin in Honey Impact on Residue Analysis Don Noot, Tom Thompson

2 Background Information collaboration with Agriculture and Agri-Food Canada project leader: Dr. Steve Pernal (Beaverlodge, AB) American foulbrood (AFB) disease caused by spore forming bacteria (paenibacillus larvae subsp. larvae) AFB highly lethal to bees and easily spread between colonies previously controlled by prophylactic use of oxytetracycline (OTC) OTC-resistant strains of AFB recently encountered

3 Background Information (cont d) AAFC examining use of alternative antimicrobials for control of OTC-rAFB carrying out field trials with lincomycin and tylosin 1. efficacy of antimicrobial treatment 2. bee mortality rates 3. persistence of antimicrobial residues in honey AFLB involved in residue analysis work since 2002 Developed LC-MS and then LC-MS/MS method for determination of lincomycin and tylosin residues in honey

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5 Persistence of Antimicrobial Residues in Honey numerous studies performed by various researchers regarding stability of tylosin in honey degradation profiles examined by either: antimicrobial activity (presumed to be due to active parent compound, tylosin A) HPLC analysis specifically for tylosin A various groups in Canada, USA and Europe have developed methods for tylosin residues in harvested honey methods focused on detection of residues of tylosin A

6 Degradation of Tylosin A However: Hamill et al. (Antibiotica et Chemotherapia, 1961): identified desmycosin as antimicrobially active degradation product of tylosin A Paesen et al. (Journal of Pharmaceutical and Biomedical Analysis, 1995): tylosin A degrades almost exclusively to tylosin B (desmycosin) in acidic aqueous solutions

7 Significance of Tylosin A Degradation in Honey ph of honey ranges from 3.4 to 6.1 (ave = 3.9) Kochansky (Journal of Apicultural Research, 2004): reported presence of desmycosin in honey t 1/2 for desmycosin > t 1/2 for tylosin A total antimicrobial activity = tylosin A + desmycosin

8 Determination of Tylosin A and Desmycosin in Honey by LC-ESI-MS/MS Advantages of analytical methodology: very simple sample preparation LC-MS/MS both highly specific and sensitive screening and confirmation performed simultaneously rapid turnaround time possible little variation in matrix effect due to similarity of honey samples produced in Alberta

9 Determination of Tylosin A and Desmycosin in Honey - sample preparation Liquefy honey (60 C) 1. centrifuge 2. remove bulk debris 2 g honey to 10 ml with 30% ACN in water Analyze by LC-ESI-MS/MS

10 Gradient Elution Program (total run time = 28 min) Time (min) 0.04% HFBA in Water ACN EtAc Flow Rate (ml/min)

11 Determination of Tylosin A and Desmycosin in Honey by LC-ESI-MS/MS to MS/MS from LC to MS/MS from LC analytical column analytical column separation of sugars to waste LC effluent to MS/MS ( min)

12 Determination of Tylosin A and Desmycosin in Honey by LC-ESI-MS/MS Compound Quantitation transition Cone voltage (V) Collision energy (ev) Confirmation transition Cone voltage (V) Collision energy (ev) Tylosin > > Desmycosin > > Roxithromycin >

13 Determination of Tylosin A and Desmycosin in Honey by LC-ESI- MS/MS (50 ppb in honey) tylosin A roxithromycin (ISTD) desmycosin

14 Analytical Method Validation spike levels: 10, 60, 180 µg kg -1 six replicates at each level three replicate experiments on different days average recoveries ranged from % over all levels typical %RSD <5% LOQs: tylosin A = 2 µg kg -1 desmycosin = 4 µg kg -1

15 Investigation of Tylosin A Degradation in Honey include desmycosin in analysis for antimicrobials in honey honey samples from AAFC field trials honey samples from Surveillance & Extension project in collaboration with AB Beekeepers in-laboratory study of stability of tylosin A in honey study desmycosin formation

16 Stability Study Experimental Design weigh out g portions of blank honey spike all honey samples with 2 µg of tylosin A Store tubes in the dark at 3 temperatures: 34 C (average hive temperature) 20 C (room temperature) -18 C (freezer) at pre-determined intervals randomly pull 3 tubes from each of the storage temperatures and analyze for tylosin A and desmycosin

17 Stability of Antimicrobials in Honey Stored at 34 C Tylosin A Desmycosin Concentration (ug/g) Weeks Stored

18 Stability of Antimicrobials in Honey Stored at 34 C Tylosin A Desmycosin Tylosin A + Desmycosin Concentration (nmoles/g) Weeks Stored

19 Stability of Antimicrobials in Honey Stored at 20 C Tylosin A Desmycosin Concentration (ug/g) Weeks Stored

20 Stability of Antimicrobials in Honey Stored at 20 C Tylosin A Desmycosin Tylosin A + Desmycosin Concentration (nmoles/g) Weeks Stored

21 Stability of Antimicrobials in Honey Stored at -18 C Tylosin A Desmycosin Concentration (ug/g) Weeks Stored

22 Preliminary Conclusions from Stability Study tylosin A degrades relatively rapidly at 34 C (hive temperature) desmycosin concentrations initially rise and then level off mass balance decreases with storage time at 34 C tylosin A begins to degrade slowly in honey when stored at 20 C desmycosin simultaneously slowly increases mass balance remains constant for storage up to 20 weeks at 20 C tylosin A is extremely stable in honey when stored at -18 C

23 Results from Controlled Field Studies recommended practice is to apply tylosin in fall based on field studies with fall application and summer harvest: desmycosin ranges from 61 to 117% of tylosin A concentrations in terms of µg kg -1 on average, desmycosin = 87% of tylosin A desmycosin ranges from 72 to 138% of tylosin A concentrations in terms of nmol kg -1 on average, desmycosin = 103% of tylosin A

24 Results from Controlled Field Studies Tylosin A and Desmycosin Concentrations in Harvested Honey from Controlled Field Trials Concentration (ug/kg) Tylosin A Desmycosin

25 Results from Controlled Field Studies Tylosin A and Desmycosin Concentrations in Harvested Honey from Controlled Field Trials Concentration (nmol/kg) Tylosin A Desmycosin

26 Use of Tylosin to Control AFB AAFC study report to Health Canada may assist with lincomycin and tylosin registration for apiculture tylosin currently available for use in AB extreme cases only (proof of r-afb) must obtain veterinarian s prescription Health Canada MRL for OTC in honey: 300 µg kg -1 Health Canada has proposed Working Residue Limit for tylosin at 60 µg kg -1

27 Future Considerations Impact of Desmycosin Residues (results in ppb) Initial Tylosin A (2004) Repeat Tylosin A (2005)* Desmycosin (2005) Tylosin A + Desmycosin honey # honey # honey # honey # honey # * samples stored frozen

28 Future Considerations Impact of Desmycosin Residues (cont d) major EU accredited lab reports total tylosin: total tylosin = tylosin A + tylosin B (desmycosin) if tylosin A is undetected, but desmycosin detected: honey considered to contain tylosin residues interpretation of data versus establishment of MRL for tylosin residues in honey:??

29 General Considerations for Storage of Honey with Incurred Residues storing honey at ambient temperatures may decrease tylosin A concentration but total antimicrobial residues (tylosin A + desmycosin) will not decrease as rapidly tylosin A is extremely stable when stored at freezer temperatures honey is generally not stored at this type of temperature, however: if producers store bulk honey (e.g., in drums) in unheated sheds over winter, residues may persist for extended periods of time

30 Detection of Desmycosin Residues practical application Several honey samples from controlled application in Manitoba - odd ratio prompted questions that revealed inconsistencies. (results in ppb) Tylosin A Level Desmycosin Level honey a 5 7 honey b honey c 27 6 honey d 53 30

31 Questions?