MULTI-ANALYTE SCREENING OF TWELVE COCCIDIOSTAT RESIDUES IN ANIMAL FEED USING BIOCHIP ARRAY TECHNOLOGY

Tohill, A., McBride, A., Porter, J., McConnell, R.I., FitzGerald, S.P.

Randox Food Diagnostics, 55 Diamond Road, Crumlin, CoAntrim BT29 4QY, UK

INTRODUCTION

Coccidiostats are antiprotozoal agents that act upon Coccidia parasites in livestock. These drugs are often administered through the use of medicated animal feeds with often more than one drug being used at once. Prophylactic use of medicated coccidiostat feeds increases the risk of drug residue carry over into meat for human consumption. Maximum Residue Limits (MRLs) have been put in place for a number of coccidiostat residues.

With the aim to increase the screening capacity, this study reports the application of biochip array technology to the simultaneous determination of coccidiostat residues from a single animal feed sample with minimal sample preparation.

CONCLUSION

This study indicates applicability of biochip array technology to the multiplex determination of coccidiostat residues in feed. The methodology requires a simple sample preparation and can be applied to the detection

of coccidiostat residues in feed at different concentration levels. The use of this technology increases the screening capacity and facilitates the surveillance of feed samples.

METHODOLOGY

Simultaneous chemiluminescent immunoassays, defining discrete test sites on the biochip surface, were applied to the semi-automated Evidence Investigator analyser (EV3602, Randox Food Diagnostics, Crumlin, UK).

Biochip (9mm x 9mm)

Semi-automated analyser Evidence Investigator

Biochip carrier (3 x 3 biochips)

Clopidol (CPL)Decoquinate (DEQ)Diclazuril (DZL)Halofuginone (HAL)

Imidocarb (IMO)Lasalocid (LAS)Maduramicin (MAD)Monensin (MON)

Nicarbazin (NZN)Robenidine (ROB)Salinomycin/narasin (SAL)Toltrazuril (TZL)

Test Menu

After a simple liquid: liquid extraction followed by centrifugation, the feed samples are applied to the biochip. The final extract dilution can be chosen to suit specific needs.

Feed Sample Preparation

RESULTS

Precision

Intra-assay precision (n=20), expressed as CV(%), was <15% for all the assays across different concentration levels.13.287.313RDS

13.217. 237, 238, 251, 253, 266.313RDS

Specificity/Cross-reactivity (CR)

Analyte Compound CR (%)

ClopidolClopidol

Nequinate

100

135

Decoquinate Decoquinate 100

DiclazurilDiclazuril

Clazuril

100

12

Halofuginone Halofuginone 100

Imidocarb Imidocarb dipropionate 100

Lasalocid Lasalocid A 100

MaduramicinMaduramicin

ammonium100

MonensinMonensin

Monensin A

100

89

NicarbazinDinitrocarbanilide

Nicarbazin

100

98

Robenidine Robenidine 100

Salinomycin/

Narasin

Salinomycin

Narasin

100

130

Toltrazuril

Toltrazuril sulphone

Toltrazuril sulphoxide

Toltrazuril

100

145

7

15.358, 359, 313RDS

Sensitivity

Analyte

Sensitive Detection Elevated Detection

LOD (mg/kg) LOD (mg/kg)

Clopidol 400.0 3000.0

Decoquinate 125.0 937.5

Diclazuril 17.5 131.3

Halofuginone 2.5 18.8

Imidocarb 1.0 7.6

Lasalocid 25.0 188.0

Maduramicin 3.8 28.1

Monensin 3.8 28.1

Nicarbazin 0.5 3.8

Robenidine 20.0 150.0

Salinomycin/

Narasin6.3 46.9

Toltrazuril 6.3 46.9

Examples of typical calibration curves

12.197, 198, 242.313RDS

0

3500

0 42 6 8 10 12 14 16

3000

Maduramicin Ammonium (ng/ml)

Rela

tive

Ligh

t Uni

ts (R

LU) 2500

2000

1500

1000

500

0

3500

0 42 6 8 10 12 14 16 18 20 22 24 26 28

3000

Salinomycin HCL (ng/ml)

Rela

tive

Ligh

t Uni

ts (R

LU) 2500

2000

1500

1000

500

0

3500

0

42 6

5 10 15 20 25 30 35 40 45 50 55 60 65 70 8075 85

12 14 16

3000

Robenidine HCL (ng/ml)

Rela

tive

Ligh

t Uni

ts (R

LU)

2500

5000

4500

4000

2000

1500

1000

500

0

3500

4000

0 42 6 8 10 12 14 16 18 20 22 24 26 28

3000

Toltrazuril Sulphone

Rela

tive

Ligh

t Uni

ts (R

LU)

2500

2000

1500

1000

500

15.350.313RDS

0LAS NZN IMO TZL MAD SAL CPL MON ROB DEQ HAL DZL

Coccidiostat tested

Drug concentration (mg/kg)

Biochip Result

Assigned Value

Assigned Value Range

Biochip array performance testing for multiple Coccidiostat residues for QC material (FAPAS feed sample)

20

40

60

80

112.0 mg/kg

80.0 mg/kg

60.9 mg/kg

48.0 mg/kg

100

120

140

160

106.9 mg/kg

83.6 mg/kg

76.3 mg/kg

45.0 mg/kg

139.2 mg/kg

122.6 mg/kg

99.4 mg/kg

59.6 mg/kg

Sample Analysis