SIMULTANEOUS SCREENING OF ANTIBIOTICS IN HONEY WITH BIOCHIP ARRAYS

Randox Laboratories Limited, 55 Diamond Road, Crumlin, Co Antrim, BT29 4QY, UK.

Porter, J.; McConnell, R.I.; McGarrity, M.C., Bell, B.; O’Loan, N.; Tohill, A.; Fitzgerald, S.P.

Introduction

Conclusion

ResultsMethodology

Randox Laboratories Limited, 55 Diamond Road, Crumlin, County Antrim, BT29 4QY, United KingdomT +44 (0) 28 9442 2413 F +44 (0) 28 9445 2912 E marketing@randox.com I www.randox.com

The use of antibiotic drugs in apiculture is globally restricted. There are currently no European Union antibiotic Maximum Residue Limits (MRLs) set, as antibiotics are not approved for treatment of honey bees in Europe, however this is not always the case outside Europe. High levels of honey imports and the emergence of antibiotic resistance have led to growing concerns for public health.

Biochip Array Technology enables the simultaneous determination of multiple analytes from a single sample, which consolidate testing and increases efficiency. Simultaneous determination of several antibiotics when screening honey samples reduces the quantity of samples to be assessed by confirmatory analysis.

Here we report the application of Biochip Array Technology to the multi-analyte screening of antimicrobials in honey. This represents a valuable analytical tool, which provides consolidation of testing.

The simultaneous immunoassays for the multi-analyte determination of drug residues in honey were performed on the semi-automated bench top analyser Evidence Investigator (EV3602).

Biochip (9mm x 9mm)Semi-automated analyser Evidence Investigator

Biochip Carrier(3x3 biochips)

• Competitive chemiluminescent immunoassays are employed for the simultaneous determination of antibiotics in the biochip arrays.

• The capture molecules are immobilised and stabilised on the biochip surface defining microarrays.

• The biochip is also the vessel where the immunoreactions take place.

• Simultaneous detection of chemiluminescent immunoreactions using digital imaging technology.

• The system incorporates dedicated software to process and archive the multiple data generated.

Biochips are supplied in a carrier (3x3 biochips per carrier), which are placed in the handling tray supplied, which has the capacity to accommodate six carriers (54 biochips).

Anti Microbial Array IICeftiofurQuinolonesStreptomycinTetracyclinesThiamphenicolTylosinAnti Microbial Array II (EV3524)Anti Microbial Array II Control (AMC5035)

Anti Microbial Array IIIAHDAMOZAOZSEMChloramphenicolAnti Microbial Array III (EV3695)Anti Microbial Array III Control (AMC5036)

Test Menu

Antimicrobial array kits (include multianalyte calibrators), controls and Evidence Investigator analyser were manufactured by Randox Laboratories Ltd. (Crumlin, UK). Assays were performed in accordance with manufacturer’s instructions.

Summary of sample preparationFor antimicrobial arrays I Plus and II a simple sample procedure is necessary to prepare the honey sample for application to both arrays. One gram of sample is dissolved and diluted in wash buffer provided with the kit and then applied to the biochip.

For antimicrobial array III 1g of honey is derivatised, followed by solvent extraction, dried down and resuspended in sample diluent before being applied to the biochip.

Antimicrobial Array 1 Plus Limits of detection (LOD)

Assay LOD (ppb)

Calibration range** (ppb)

Sulphadimethoxine 5.0* 0-20Sulphadiazine 5.0 0-20Sulphadoxine 5.0 0-20

Sulphamethizole 5.0 0-20Sulphachlorpyridazine 5.0 0-20Sulphamethoxazole 1.6 0-31

Sulphamethoxypyridazine 5.0 0-20Sulphamonomethoxine 20.0 0-120

Sulphamerazine 5.0 0-20Sulphisoxazole 5.0 0-20Sulphathiazole 5.0 0-20

Sulphamethazine 5.0 0-20Sulphaquinoxaline 5.0 0-20

Sulphapyridine 8.0 0-20Trimethoprim 9.0 0-10

*A higher LOD of 10 ppb is recommended for the screening of unprocessed raw honey samples.** The calibration range may vary slightly with the batch of calibrators

Specificity• Fifteen simultaneous immunoassays

were specific for the targets.• The sulphamethoxypyridazine assay

presented 56% cross-reactivity with sulphaethoxypyridazine.

• The sulphamethoxazole assay presented 92% cross-reactivity with sulphamethizole.

Precision:Typical intra-assay precision (n=20) <14% for different concentration levels.

Assay LOD (ppb)

Calibration range* (ppb)

Ceftiofur 2.31 0-7.0Quinolones 3.44 0-11.5

Streptomycin 7.56 0-75.0Tetracyclines 8.65 0-2.5

Thiamphenicol 0.66 0-5Tylosin 1.01 0-5

Assay Compound %Cross-reactivity

Assay Compound %Cross- reactivity

Quinolones Norfloxacin 100 Ceftiofur Ceftiofur 100Pefloxacin 84 Thiamphenicol Florphenicol 100

Enrofloxacin 76 Thiamphenicol 53Ciprofloxacin 59 Streptomycin Streptomycin 100

Ofloxacin 57 Dihydrostreptomycin 182Enoxacin 54

Pipemidic acid 36 Tylosin Tylosin 100Fleroxacin 32 Tilmicosin 37

Levofloxacin 32 Tetracyclines Tetracycline 100Nadifloxacin 27 4-epitetracycline 87Orbifloxacin 23 Rolitetracycline 67Danofloxacin 20 4-epioxytetracycline 52Marbofloxacin 16 Oxytetracycline 52Oxolonic acid 12 Chlortetracycline 51

Difloxacin 8 Demeclocycline 41Pazufloxacin 7 Doxycycline 23Sarafloxacin 6 4-epichlortetracycline 20

Methacycline 11

Antimicrobial Array II Limits of Detection (LOD) Specificity

08/367, 377, 382, 387/295

PrecisionTypical intra-assay precision (n=20) <8% for different concentration levels.08/324,352/295

Antimicrobial Array III Limits of Detection (LOD)

Assay LOD (ppb)

Calibration range* (ppb)

AHD 0.2 0-10AMOZ 0.2 0-10AOZ 0.3 0-10SEM 1.0 0-20

Chloramphenicol 0.2 0-508/083/296, 09/108/296*The calibration range may vary slightly with the batch of calibrators

Assay Compound %Cross Reactivity

AHD 4-NP-AHD 100Nitrofurantoin 41.9

AMOZ 4-NP-AMOZ 100Furaltadone 40.7

AOZ 4-NP-AOZ 100Furazolidone 8.3

SEM 4-NP-SEM 1005-Nitro-2-furaldehyde semicarbazone 13.6

Chloramphenicol Chloramphenicol 100Chloramphenicol glucuronide 75.1

08/102/296, 09/121/296

09/1026/346* The calibration range may vary slightly with the batch of calibrators

PrecisionTypical intra-assay precision (n=20) <10% for different concentration levels.

• The results show the applicability of biochip array technology for the simultaneous screening of antimicrobials per sample of honey on the Evidence Investigator analyser.

• Simple sample dilution is only required for measurement of 15 sulphonamides, trimethoprim, quinolones, streptomycin, tylosin (tilmicosin), tetracyclines, thiamphenicol (florphenicol) on two biochip arrays. Following derivatisation and solvent extraction, four nitrofuran metabolites and chloramphenicol are simultaneously determined on a third array.

• Broad detection of quinolones and tetracyclines.• With the Evidence Investigator analyser 54 biochips can be handled at one time and the system incorporates dedicated software to

process and archive the multiple data generated.• These biochip arrays represent an excellent multi-analytical tool for the screening of different antibiotics in honey samples on one

platform and they are also applicable to other matrices.

10/623/5F

10/611,624/SF,11/655/5F

Anti Microbial Array I PlusSulphadimethoxineSulphadiazineSulphadoxineSulphamethizoleSulphamethoxazoleSulphamethoxypyridazineSulphachlorpyridazineSulphamerazineSulphamonomethoxineSulphisoxazoleSulphathiazoleSulphamethazineSulphaquinoxalineSulphapyridineTrimethoprimAnti Microbial Array I Plus (EV3775)Antimicrobial PLUS I Multianalyte Control (AMC5084)

AbbreviationsAHD: 1-aminohydantoin hydrochlorideAMOZ: 5-methylmorpholino-3-amino-2-oxazolidinoneAOZ: 3-amino-2-oxazolidinoneSEM: semicarbazide

08/084/296

Specificity

Once prepared honey samples are applied to the biochip arrays

Number of test results for 45 samples

675 in less than 2.5 hours

Antimicrobial Array 1 Plus

270 in less than 2.5 hours

Antimicrobial Array II

225 in less than 2.5 hours

Antimicrobial Array III