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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
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