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Simultaneous Screening of Antibiotics in Honey with Biochip Arrays

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Simultaneous Screening of Antibiotics in Honey with Biochip Arrays SIMULTANEOUS SCREENING OF ANTIBIOTICS IN HONEY WITH BIOCHIP ARRAYS Porter, J.; McConnell, R.I.; McGarrity, M.C., Bell, B.; O’Loan, N.; Tohill, A.; Fitzgerald, S.P. Randox Laboratories Limited, 55 Diamond Road, Crumlin, Co Antrim, BT29 4QY, UK. Introduction The use of antibiotic drugs in apiculture is globally restricted. There Biochip Array Technology enables the simultaneous determination Here we report the application of Biochip Array Technology to the are currently no European Union antibiotic Maximum Residue of multiple analytes from a single sample, which consolidate multi-analyte screening of antimicrobials in honey. This represents a Limits (MRLs) set, as antibiotics are not approved for treatment of testing and increases efficiency. Simultaneous determination valuable analytical tool, which provides consolidation of testing. honey bees in Europe, however this is not always the case outside of several antibiotics when screening honey samples reduces Europe. High levels of honey imports and the emergence of antibiotic the quantity of samples to be assessed by confirmatory analysis. resistance have led to growing concerns for public health. Methodology Results The simultaneous immunoassays for the multi-analyte determination Antimicrobial Array 1 Plus of drug residues in honey were performed on the semi-automated Limits of detection (LOD) bench top analyser Evidence Investigator (EV3602). Assay LOD Calibration range** Specificity (ppb) (ppb) • Fifteen simultaneous immunoassays were specific for the targets. Sulphadimethoxine 5.0* 0-20 • The sulphamethoxypyridazine assay Sulphadiazine 5.0 0-20 presented 56% cross-reactivity with Sulphadoxine 5.0 0-20 sulphaethoxypyridazine. Sulphamethizole 5.0 0-20 • The sulphamethoxazole assay Sulphachlorpyridazine 5.0 0-20 Sulphamethoxazole 1.6 0-31 presented 92% cross-reactivity with Semi-automated analyser Biochip (9mm x 9mm) Biochip Carrier Sulphamethoxypyridazine 5.0 0-20 sulphamethizole. Evidence Investigator (3x3 biochips) Sulphamonomethoxine 20.0 0-120 Sulphamerazine 5.0 0-20 Precision: Sulphisoxazole 5.0 0-20 • Competitive chemiluminescent immunoassays are employed Typical intra-assay precision (n=20) Sulphathiazole 5.0 0-20 <14% for different concentration levels. for the simultaneous determination of antibiotics in the Sulphamethazine 5.0 0-20 biochip arrays. Sulphaquinoxaline 5.0 0-20 10/611,624/SF,11/655/5F • The capture molecules are immobilised and stabilised on the Sulphapyridine 8.0 0-20 biochip surface defining microarrays. Trimethoprim 9.0 0-10 10/623/5F • The biochip is also the vessel where the immunoreactions *A higher LOD of 10 ppb is recommended for the screening of unprocessed raw honey samples. take place. ** The calibration range may vary slightly with the batch of calibrators • Simultaneous detection of chemiluminescent immunoreactions using digital imaging technology. • The system incorporates dedicated software to process and archive the multiple data generated. Antimicrobial Array II Limits of Detection (LOD) Specificity Biochips are supplied in a carrier (3x3 biochips per carrier), Assay LOD Calibration Assay Compound %Cross- Assay Compound %Cross- which are placed in the handling tray supplied, which has the (ppb) range* (ppb) reactivity reactivity capacity to accommodate six carriers (54 biochips). Quinolones Norfloxacin 100 Ceftiofur Ceftiofur 100 Ceftiofur 2.31 0-7.0 Pefloxacin 84 Thiamphenicol Florphenicol 100 Quinolones 3.44 0-11.5 Enrofloxacin 76 Thiamphenicol 53 Streptomycin 7.56 0-75.0 Ciprofloxacin 59 Streptomycin Streptomycin 100 Test Menu Tetracyclines 8.65 0-2.5 Ofloxacin 57 Dihydrostreptomycin 182 Thiamphenicol 0.66 0-5 Enoxacin 54 Tylosin 1.01 0-5 Pipemidic acid 36 Tylosin Tylosin 100 Anti Microbial Array I Plus Anti Microbial Array II 09/1026/346 Fleroxacin 32 Tilmicosin 37 Sulphadimethoxine Ceftiofur * The calibration range may vary slightly with the batch of calibrators Levofloxacin 32 Tetracyclines Tetracycline 100 Sulphadiazine Quinolones Nadifloxacin 27 4-epitetracycline 87 Sulphadoxine Streptomycin Orbifloxacin 23 Rolitetracycline 67 Precision Danofloxacin 20 4-epioxytetracycline 52 Sulphamethizole Tetracyclines Typical intra-assay precision (n=20) <8% for Sulphamethoxazole Thiamphenicol Marbofloxacin 16 Oxytetracycline 52 different concentration levels. Oxolonic acid 12 Chlortetracycline 51 Sulphamethoxypyridazine Tylosin 08/324,352/295 Difloxacin 8 Demeclocycline 41 Sulphachlorpyridazine Anti Microbial Array II (EV3524) Anti Microbial Array II Control (AMC5035) Pazufloxacin 7 Doxycycline 23 Sulphamerazine Sarafloxacin 6 4-epichlortetracycline 20 Sulphamonomethoxine Methacycline 11 Sulphisoxazole Anti Microbial Array III 08/367, 377, 382, 387/295 Sulphathiazole AHD Sulphamethazine AMOZ Antimicrobial Array III Sulphaquinoxaline AOZ Limits of Detection (LOD) Specificity Sulphapyridine SEM Assay LOD Calibration Assay Compound %Cross Reactivity Trimethoprim Chloramphenicol (ppb) range* (ppb) AHD 4-NP-AHD 100 Anti Microbial Array I Plus (EV3775) Anti Microbial Array III (EV3695) Antimicrobial PLUS I Multianalyte Control (AMC5084) Anti Microbial Array III Control (AMC5036) AHD 0.2 0-10 Nitrofurantoin 41.9 AMOZ 0.2 0-10 AMOZ 4-NP-AMOZ 100 AOZ 0.3 0-10 Furaltadone 40.7 SEM 1.0 0-20 AOZ 4-NP-AOZ 100 Chloramphenicol 0.2 0-5 Furazolidone 8.3 Antimicrobial array kits (include multianalyte calibrators), controls 08/083/296, 09/108/296 SEM 4-NP-SEM 100 and Evidence Investigator analyser were manufactured by Randox *The calibration range may vary slightly with the batch of calibrators 5-Nitro-2-furaldehyde semicarbazone 13.6 Laboratories Ltd. (Crumlin, UK). Assays were performed in Chloramphenicol Chloramphenicol 100 Chloramphenicol glucuronide 75.1 accordance with manufacturer’s instructions. Abbreviations 08/102/296, 09/121/296 AHD: 1-aminohydantoin hydrochloride Summary of sample preparation AMOZ: 5-methylmorpholino-3-amino-2-oxazolidinone Precision For antimicrobial arrays I Plus and II a simple sample procedure AOZ: 3-amino-2-oxazolidinone SEM: semicarbazide Typical intra-assay precision (n=20) <10% for different concentration levels. is necessary to prepare the honey sample for application to both 08/084/296 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 Conclusion before being applied to the biochip. • The results show the applicability of biochip array technology for the simultaneous screening of antimicrobials per sample of honey Once prepared honey samples are applied to the biochip arrays 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 Number of test results for 45 samples nitrofuran metabolites and chloramphenicol are simultaneously determined on a third array. • Broad detection of quinolones and tetracyclines. 675 in less than 270 in less than 225 in less than • With the Evidence Investigator analyser 54 biochips can be handled at one time and the system incorporates dedicated software to 2.5 hours 2.5 hours 2.5 hours process and archive the multiple data generated. Antimicrobial Antimicrobial Antimicrobial • These biochip arrays represent an excellent multi-analytical tool for the screening of different antibiotics in honey samples on one Array 1 Plus Array II Array III platform and they are also applicable to other matrices. Randox Laboratories Limited, 55 Diamond Road, Crumlin, County Antrim, BT29 4QY, United Kingdom T +44 (0) 28 9442 2413 F +44 (0) 28 9445 2912 E [email protected] I www.randox.com.
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