Stability of Tylosin in Honey Impact on Residue Analysis Noot - 2006 WCTOW.pdf · Presented at the...

Presented at the 41st Annual Western Canada Trace Organic Workshop, Vancouver, B.C.

Stability of Tylosin in HoneyImpact on Residue AnalysisDon Noot, Tom Thompson


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


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


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


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


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


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


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


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 -



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



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 - - -



(50 ppb in honey)

tylosin A

roxithromycin(ISTD)

desmycosin


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


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


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


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



0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Con

cen

trati

on

(n

mole

s/g

)

Tylosin A Desmycosin Tylosin A + Desmycosin



0.0

0.2

0.4

0.6

0.8

1.0

1.2


Con

cen

trati

on

(u

g/g

)




0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4


Con

cen

trati

on

(n

mole

s/g

)

Tylosin A Desmycosin Tylosin A + Desmycosin


Stability of Antimicrobials in Honey Stored at -18°C

0.0

0.2

0.4

0.6

0.8

1.0

1.2


Con

cen

trati

on

(u

g/g

)



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


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



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



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


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


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


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: ??


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


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.


Questions?

Stability of Tylosin in Honey Impact on Residue Analysis Noot - 2006 WCTOW.pdf · Presented at the...

Documents

Transcript of Stability of Tylosin in Honey Impact on Residue Analysis Noot - 2006 WCTOW.pdf · Presented at the...