stability of tylosin in honey impact on residue analysis noot - 2006 wctow.pdf · presented at the...
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Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Stability of Tylosin in HoneyImpact on Residue AnalysisDon Noot, Tom Thompson
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Background Information
• collaboration with Agriculture and Agri-Food Canadaproject 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
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Background Information (cont’d)
• AAFC examining use of alternative antimicrobials for control of OTC-rAFB
• carrying out field trials with lincomycin and tylosin1. efficacy of antimicrobial treatment2. bee mortality rates3. persistence of antimicrobial residues in honey
• AFLB involved in residue analysis work since 2002Developed LC-MS and then LC-MS/MS method for determination of lincomycin and tylosin residues in honey
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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 honeyt1/2 for desmycosin > t1/2 for tylosin A
• total antimicrobial activity = tylosin A + desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Determination of Tylosin A and Desmycosin in Honey - sample preparation
Analyze by LC-ESI-MS/MS
2 g honey to 10 mL with 30% ACN in water
Liquefy honey (60 °C)1. centrifuge
2. remove bulk debris
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Gradient Elution Program(total run time = 28 min)
Time(min)
0.04% HFBA in Water
ACN EtAc
10 -
-
-
100
100
-
-
-
100
100
-
-
100
19 - 100 0.5
10
Flow Rate(mL/min)
0 90 0.2
0.2
0.2
0.5
0.5
0.5
0.2
9 -
18 -
23 90
10 -
12 -
17 -
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Determination of Tylosin A and Desmycosin in Honey by LC-ESI-MS/MS
separation of sugars LC effluent to MS/MS(6.5 – 9.6 min)
to MS/MS
towaste
fromLC
to MS/MS fromLC
analytical column
analytical column
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Determination of Tylosin A and Desmycosin in Honey by LC-ESI-MS/MS
CompoundQuantitation
transition
Conevoltage
(V)
Collision energy
(eV)
Confirmation transition
Conevoltage
(V)
Collision energy
(eV)
Tylosin 916.5 > 174.0
45 40 916.5 > 772.5
45 30
Desmycosin 772.5 > 174.0
45 29 772.5 > 156.0
45 40
Roxithro-mycin
837.4 > 158.0
45 40 - - -
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Determination of Tylosin A and Desmycosin in Honey by LC-ESI-MS/MS
(50 ppb in honey)
tylosin A
roxithromycin(ISTD)
desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Analytical Method Validation
• spike levels: 10, 60, 180 µg kg-1
six replicates at each levelthree replicate experiments on different days
• average recoveries ranged from 96 – 107% over all levels
• typical %RSD <5%
• LOQs: tylosin A = 2 µg kg-1
desmycosin = 4 µg kg-1
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Investigation of Tylosin A Degradation in Honey
• include desmycosin in analysis for antimicrobials in honey
honey samples from AAFC field trialshoney samples from Surveillance & Extension project in collaboration with AB Beekeepers
• in-laboratory study of stability of tylosin A in honeystudy desmycosin formation
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Stability Study Experimental Design
• weigh out 2.00 + 0.05 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
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Stability of Antimicrobials in Honey Stored at 34°C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 5 10 15 20 25Weeks Stored
Con
cen
trati
on
(u
g/g
)
Tylosin A Desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Stability of Antimicrobials in Honey Stored at 34°C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 5 10 15 20 25Weeks Stored
Con
cen
trati
on
(n
mole
s/g
)
Tylosin A Desmycosin Tylosin A + Desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Stability of Antimicrobials in Honey Stored at 20°C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 5 10 15 20 25Weeks Stored
Con
cen
trati
on
(u
g/g
)
Tylosin A Desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Stability of Antimicrobials in Honey Stored at 20°C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 5 10 15 20 25Weeks Stored
Con
cen
trati
on
(n
mole
s/g
)
Tylosin A Desmycosin Tylosin A + Desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Stability of Antimicrobials in Honey Stored at -18°C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 5 10 15 20 25Weeks Stored
Con
cen
trati
on
(u
g/g
)
Tylosin A Desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Preliminary Conclusions from Stability Study
• tylosin A degrades relatively rapidly at 34°C (hive temperature)
desmycosin concentrations initially rise and then level offmass balance decreases with storage time at 34°C
• tylosin A begins to degrade slowly in honey when stored at 20°C
desmycosin simultaneously slowly increasesmass balance remains constant for storage up to 20 weeks at 20°C
• tylosin A is extremely stable in honey when stored at -18°C
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Results from Controlled Field Studies
Tylosin A and Desmycosin Concentrations in Harvested Honey from Controlled Field Trials
0
20
40
60
80
100
120
140
160
180
2001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Con
cent
ratio
n (u
g/kg
)
Tylosin A
Desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Results from Controlled Field Studies
Tylosin A and Desmycosin Concentrations in Harvested Honey from Controlled Field Trials
0
50
100
150
200
2501 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Con
cent
ratio
n (n
mol
/kg)
Tylosin A
Desmycosin
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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 ABextreme 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 Limitfor tylosin at 60 µg kg-1
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Future Considerations – Impact of Desmycosin Residues
(results in ppb)
Initial Tylosin A (2004)
Repeat Tylosin A (2005)*
Desmycosin(2005)
Tylosin A + Desmycosin
honey #1 30 31 33 64
53
114
85
74
honey #2 25 28 25
honey #3 51 49 65
honey #4 39 38 47
honey #5 32 33 41
* samples stored frozen
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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: ??
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
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
Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.
Detection of Desmycosin Residuespractical application
(results in ppb)Tylosin A
LevelDesmycosin
Level
honey a 5 7
honey b 62 44
honey c 27 6
honey d 53 30
Several honey samples from “controlled” application in Manitoba -odd ratio prompted questions that revealed inconsistencies.
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