E. Daeseleire, L. Mortier, Ph. Delahaut and G. Huyghebaert

Determination of concentration levels of anticoccidials in eggs due to the presence of low levels of those compounds in feed for laying hens caused by carryover at the

feeding mill

E. Daeseleire, L. Mortier, Ph. Delahaut and G. Huyghebaert

Brussels, 03/05/2005

Anticoccidials

compounds that are widely used as feed additives to prevent and treat avian coccidiosis

avian coccidiosis:• contagious amoebic disease affecting livestock• carried by unicellular organisms belonging to the genus Eimeria• causes poor weight gain, reduced egg production, intestinal lesions resulting in diarrhoea and related health problems, can cause death• most important parasitic disease in poultry


Legislation

• Regulation (EC) N° 1831/2003 on additives for use in animal nutrition

• although pure medicines, regarded as additives : not subject to MRL-legislation (MRL = maximum residue limit)

• phasing out by 31/12/2012; Commission shall submit a report on the use of these substances as feed additives and available alternatives before 1/1/2008

• No MRLs available “zero tolerance” principle : no residues allowed


N

N

CH3

CH3O2N

dimetridazoleN

N

NCl

Cl

Cl

CN

O O

H

diclazuril

N

Cl

N

NH

H

NN

H

Cl

robenidine

main metabolite

N

N

CH3

CH2OHO2N

dimetridazole-OH

Selected compounds

• dimetridazole

• halofuginone

• robenidine

diclazuril

nicarbazin

N

H

N

H

O

4,4'- dinitrocarbanilide (DNC)

N

NH3C

CH3

OH

2-hydroxy-4,6-dimethylpyrimidine (HDP)

an equimolar mixture of

halofuginone

N

H

HOO

O

Cl

Br N

N


LC-MS/MS-method

• HPLC (high performance liquid chromatography)

– instrument : 2695 Alliance LC system (Waters)

– conditions :

columnWaters C18, 5 µm, 150

mm x 2.1 mm i.d.

pre-columnAlltima C18, 5 µm, 7.5 x

2.1 mm i.d.

eluent AH2O/ACN : 95/5 + 0.1%

HCOOH

eluent B ACN

flow rate 0.25ml/min

column temperature

35°C

injection volume

10 µl

gradiëntgradient elution

Isocratic for narasin

• MS (mass spectrometry)

– instrument : Quattro Ultima Pt

(Waters Micromass)

– MS/MS : principle of MRM : fragmentation of precursor ion into product ions


Mass spectrometric conditions

compoundionisation

modePrecursor ion

(m/z)cone (V)

Product ions (m/z) *: most abundant

collision energy (eV)

dimetridazole ES + 142.1 5096.1 *81.2

1220

2-hydroxy dimetridazole

ES + 158.1 40140.1*55.2

914

halofuginone ES + 416.0 50100.2*120.1

2018

robenidine ES + 334.1 50155.1*138.1

1824

nicarbazin ES - 301.1 35137.1*107.1

830

diclazuril ES -404.9406.9

5050

334.1*336.1

1616

narasin ES + 787.4 80431.2*531.2

5043


• Animal experiment :

– location : Department of animal nutrition and husbandry, Merelbeke

– Laying hens : ISA-brown, selected on egg production and body weight, in week 30 of laying cycle at start of the experiment

– Period of adjustment : 4 weeks before the experiment hens were placed in the cages

– feed : mash of 11.7 MJ MEn/kg, containing 16 % crude protein

– housing : 4 hens/cage, conventional conditions of ventilation, temperature (18-22°C) and light (16h light/day)


• GROUPS : 5 compounds

diclazuril, halofuginone, robenidine, nicarbazin, dimetridazole

x 2 concentrations/compound100% (= max. conc. in feed), 5% (carry-over)

+ blank group

compound additive/premix conc. 100%

diclazuril Clinacox 1 mg/kg

halofuginone Stenerol 3 mg/kg

robenidine Cycostat 36 mg/kg

nicarbazin Maxiban 40 mg/kg nica+40 mg/kg narasin

dimetridazole standaard Sigma 200 mg/kg

dimetridazole :

only 1 group (100%) as this is a forbidden

compound (Annex 4) TOTAL : 10 groups x 12 hens/group


• PREPARATION OF FEED :at DVV

• per group 40 kg feed was prepared

• first 5 %, than 100 % concentration

• between 5 % and 100 %, 3 rinses were scheduled

• between 2 different compounds, 5 rinses were done

ANALYSIS FEED SAMPLES :

• feed samples were analysed with a validated LC-MS/MS method

• the rinses in between were negative : no carry-over took place


compound group theoretical conc. measured conc.

% (compared to theoretical value)

diclazuril100% 1000 µg/kg 926 µg/kg 93

5% 50 µg/kg 47 µg/kg 94

halofuginone100% 3000 µg/kg 1475 µg/kg 49

5% 150 µg/kg 162 µg/kg 108

robenidine100% 36 mg/kg 39 mg/kg 108

5% 1800 µg/kg 1597 µg/kg 89

nicarbazin+narasin

100%nicarbazine : 40 mg/kg 41 mg/kg 103

narasine : 40 mg/kg 41 mg/kg 103

5%nicarbazine : 2000 µg/kg 2114 µg/kg 106

narasine : 2000 µg/kg 2144 µg/kg 107

dimetridazole 100% 200 mg/kg 101 mg/kg 51

results analysis feed containing anticoccidials :


a. halofuginone

first pos. sample plateau concentration concentration below CCα

5% day 3 (18 µg/kg) 30 µg/kg 8 days after withdrawal feed


0

100

200

300

400

500

600

-2 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33

day

µg

/kg

hfg

- 5

0% g

rou

p

0

5

10

15

20

25

30

35

40

µg

/kg

hfg

- 5

% g

rou

phalofuginone-50%

halofuginone- 5%


b. dimetridazole


DMZ day 2 (676 µg/kg) 650 µg/kg 9 days after withdrawal feed

DMZ-OH day 2 (1513 µg/kg) 1700 µg/kg 13 days after withdrawal feed

0

500

1000

1500

2000

2500

-2 2 5 8 11 14 17 20 23 26

day

µg

/kg

dimetridazole

dimetridazole-OH


c. robenidine




0

200

400

600

800

1000

1200

1400

1600

1800

-2 2 5 8 11 14 17 20 23 26 29 32 35 38

day

µg

/kg

ro

b -

100

% g

rou

p

0

10

20

30

40

50

60

70

80

µg

/kg

ro

b -

5%

gro

up

robenidine - 100%

robenidine - 5%


d. narasin




0

20

40

60

80

100

120

-2 2 5 8 11 14 17 20 23 26 29

day

µg

/kg

nar

- 1

00%

gro

up

0

12

34

5

67

8

µg

/kg

nar

- 5

% g

rou

p

narasine - 100%

narasine - 5%


e. nicarbazin (dinitrocarbanilide)



100% day 3 (200 µg/kg) 7000 µg/kg > 23 days after withdrawal feed

0

10002000

3000

40005000

6000

70008000

9000

-2 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

day

ppb

dnc

in 1

00%

gro

up

0

50100

150

200250

300

350400

450

ppb

dnc

in 5

% g

roup

dnc-100%

dnc-5%


f. diclazuril



100% day 3 (18 µg/kg) 110 µg/kg > 22 days after withdrawal feed

0

20

40

60

80

100

120

140

-2 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35

days

ppb

DIC

LA -

100%

gro

up

0

1

2

3

4

5

6

7

ppb

DIC

LA -

5% g

roup

diclazuril-100%

diclazuril-5%


Advice 24-2004 of the scientific committee of the Belgian Federal Food Agency:

action limit of 10 µg/kg for diclazuril, narasin, nicarbazin and robenidine

compound group max. concentration # days after withdrawal < 10 µg/kg

diclazuril100 % 100 µg/kg 11

5 % 5 µg/kg -

halofuginone50% 501 µg/kg 10

5% 36 µg 4

robenidine100% 1535 µg/kg 15

5% 70 µg/kg 8

narasin100% 97 µg/kg 8

5% 7 µg/kg -

nicarbazin 100 % 6500 µg/kg 19

5 % 300 µg/kg 11

dimetridazole 50%DMZ 711 µg/kg (dag 7) 5

DMZ-OH 1915 µg/kg (dag 3) 6

Documents

E. Daeseleire, L. Mortier, Ph. Delahaut and G. Huyghebaert