In-house validation of an ELISA method for screening of semicarbazide in eggs

An enzyme-linked immunosorbent assay (ELISA) method is described for the semi-quantitative determination of semicarbazide (SEM), the marker residue for the banned nitrofuran drug, nitrofurazone, in chicken eggs. The sample homogenate is subjected to acid hydrolysis and derivatisation with o-nitrobenzaldehyde, followed by ethyl acetate/hexane extraction and detection by ELISA. The ELISA procedure has been validated using 0.3, 1.0 and 3 microg kg(-1) of SEM in fortified samples. Detection capability (CC(ss)) was based on the acceptance of 5% false compliant results for a given concentration level according to Commission Decision 2002/657/EC and was determined to be 0.3 microg kg(-1) with a respective limit of detection of 0.13 microg kg(-1). A validated LC-MS/MS method was used for the analysis of incurred egg samples and the results compared with ELISA. A good correlation between the results obtained from ELISA and LC-MS/MS within the concentration range 0.12-20.3 microg kg(-1) was observed in samples collected from chickens fed with a medicated ration of nitrofurazone (r = 0.992, n = 14). Validated ELISA enabled reliable monitoring of SEM levels in eggs collected from incurred chickens over a 90-day period.     

Kinetics of semicarbazide and nitrofurazone in chicken eggs and egg powders

The accumulation, depletion and partitioning of semicarbazide (SEM) and its parent compound nitrofurazone (NFZ) in eggs were studied using hens fed NFZ at therapeutic and sub-therapeutic levels. Dietary NFZ correlated strongly with NFZ and total SEM in eggs, while 28% of observed SEM was present in the form of parent NFZ. Depletion half-life in eggs was 2.4 days for SEM and 1.1 days for NFZ. NFZ accumulated preferentially in yolk (57–63%) as opposed to albumen, while 71–80% of SEM was found in yolk. In whole egg, 29% of SEM was present as tissue-bound residues compared with 80% in breast muscle. Whilst NFZ and SEM were partly degraded by pasteurization and spray drying, sufficient NFZ remained to suggest it might be detectable in egg powders when SEM is observed at low µg kg^?1 concentrations. NFZ was detectable in whole eggs during ingestion of only 0.1% of the therapeutic NFZ dose, making detection of intact NFZ in eggs a feasible means to prove conclusively the administration of this banned compound.     

Comparative study of three screening tests,two microbiological tube tests,and a multi-sulphonamide ELISA kit for the detection of antimicrobial and sulphonamide residues in eggs

The screening of antimicrobial residues in eggs is an especially important subject. Three different commercial kits for the screening of sulphonamides and other antimicrobials in eggs were validated in accordance with Decision 2002/657/EC: one enzyme-linked immunoabsorbant assay (ELISA) kit multi-sulphonamides (from RAISIO Diagnostics) and two microbiological tests (a Premi® test from DSM and an Explorer® kit from Zeu-Inmunotec). The false-positive rates were lower than 2% for all kits. The detection capabilities (CCβ) have to be as low as possible for banned substances and lower than the maximum residue limit (MRL) when MRLs have been set. The sensitivity of the Premi® test was better than that of the Explorer® test, probably because of the dilution of the eggs before the Explorer® test was used. The CCβ values towards most of the tested sulphonamides were satisfactory with the Premi® test (≤ 100 µg kg^?1). Performance in a proficiency test for the detection of sulphonamides in eggs with the Premi® test confirmed these results. The detection capabilities of tetracycline and doxycycline were at the level of the MRL or twice the MRL maximum. The detection capabilities for chlortetracycline and oxytetracycline were higher (four to six times the MRL). The detection capabilities for amoxicillin, neomycin, tylosin and erythromycin were lower than their respective MRLs. Detection capabilities for sulphonamides were much lower for the ELISA kit than for microbiological tests. The ELISA kit could be recommended for the targeted screening of sulphonamides in eggs. On the other hand, the Explorer® and Premi® tests could be used as wide screening tests allowing the detection of most of the antimicrobial families.     

Validation of a liquid chromatography-tandem mass spectrometry screening method to monitor 58 antibiotics in milk: a qualitative approach

A multi-residue method was developed for monitoring antibiotic residues in milk using liquid chromatography coupled to a tandem quadrupole mass spectrometer (LC/MS-MS). Two very short extractions followed by two LC/MS-MS acquisitions allowed the screening of 58 antibiotics belonging to eight different families (penicillins, cephalosporins, sulfonamides, macrolides, lincosamides, aminoglycosides, tetracyclines, and quinolones). This method is currently implemented in the laboratory in a qualitative way, i.e. monitoring the presence or absence of residue in a sample and identification of the analyte before the confirmation step. In order to assess the performance of this method, a validation strategy described in an internal guideline for the validation of screening methods was applied. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest (maximum residue limit, MRL) at least. According to European Commission Decision 2002/657/EC, the suitable sensitivity of a screening method can be demonstrated when the CCβ is below or equal to the MRL and so the false-compliant rate below or equal to 5% at the MRL level. The validation scheme was established in order to take into account various variability factors: the apparatus response, the interday repeatability, the matrix effect, etc. The results of the validation clearly demonstrate the suitability of this method for the detection and identification of more than 50 antibiotics and they are in agreement with the results obtained in routine analysis.     

A novel approach to simultaneous screening and confirmation of regulated pharmaceutical compounds in dietary supplements by LC/MS/MS with an information-dependent acquisition method

The commercial success of synthetic phosphodiesterase-5 (PDE-5) inhibitors (viz. sildenafil, vardenafil and tadalafil) for erectile dysfunction (ED) has led to their widespread use as adulterants in dietary supplements (DSs). Reports on adulteration by ED drugs or their analogues in DSs suggest they may cause a serious threat to human health. The problem is becoming more complex as hidden and structurally modified analogues are continuously being reported. To analyse known drugs and their analogues, three commonly used PDE-5 inhibitors, naturally existing icariin and yohimbin, and their 19 analogues were analyzed in this study. They were identified using ion-spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM). This MRM procedure gave a limit of detection of less than 0.02?ng?ml^?1 for the 24 compounds, selectivity of fragmentation using MRM for 2.5?–?8.5?min in a single run and peak height repeatability of coefficient of variation of 3.9?–?31.8%. An IDA method using the MRM scans to detect the presence of known analytes was set up and added to a built-in library for screening for PDE-5 inhibitors. These MRM experiments were used to trigger product ion scans using a hybrid quadrupole-linear ion trap instrument. The product ion scan was compared and confirmed by a library search of MS/MS spectra acquired from a reference standard. To search for new analogues of PDE-5 inhibitors, a precursor ion scan of an expected ion m/z 283, which was one of the mass fragments from the analogues of sildenafil or vardenafil, was performed and fragmentation of the precursor ion, by combining a precursor ion scan with automatic confirmation using EPI spectra, was acquired. Of the 37 DSs tested, two were eventually found to be adulterated with yohimbin and vardenafil, respectively. The approach proposed in this study would be valuable in characterizing chemical constituents of drug residues and their analogues with identical chemical substructures from complex natural and synthetic sources in DSs using an information-dependent acquisition-enhanced product ion (IDA–EPI) scan.     

Why semicarbazide (SEM) is not an appropriate marker for the usage of nitrofurazone on agricultural animals

A comprehensive global database on semicarbazide (SEM) in foodstuffs and food ingredients is presented, with over 4000 data collected in foods such as seafood (crustaceans, fish powders), meat (beef, chicken powders), dairy products (e.g. raw milk, milk powders, whey, sweet buttermilk powder, caseinate, yoghurt, cheese), honey and other ingredients. The results provide evidence that the presence of SEM in certain dairy ingredients (whey, milk protein concentrates) is a by-product of chemical reactions taking place during the manufacturing process. Of the dairy ingredients tested (c. 2000 samples), 5.3% showed traces of SEM > 0.5 µg/kg. The highest incidence of SEM-positive samples in the dairy category were whey (powders, liquid) and milk protein concentrates (35% positive), with up to 13 µg/kg measured in a whey powder. Sweet buttermilk powder and caseinate followed, with 27% and 9.3% positives, respectively. SEM was not detected in raw milk, or in yoghurt or cheese. Of the crustacean products (shrimp and prawn powders) tested, 44% were positive for SEM, the highest value measured at 284 µg/kg. Fish powders revealed an unexpectedly high incidence of positive samples (25%); in this case, fraudulent addition of shellfish shells or carry-over during processing cannot be excluded. Overall, the data provide new insights into the occurrence of SEM (for dairy products and fish powders), substantially strengthening the arguments that SEM in certain food categories is not a conclusive marker of the use of the illegal antibiotic nitrofurazone.     

Comparison of a fluoroquinolone surface plasmon resonance biosensor screening assay with established methods

The performance of a previously developed immunochemical biosensor screening method for fluoroquinolone (FQ) antibiotics in poultry muscle, fish and egg was compared with established methods. Blank sample material of the target matrices was individually spiked with the FQs at half maximum residue levels. Homogeneity of the test materials was confirmed by liquid chromatography–mass spectrometry (LC–MS/MS). Identical sets of spiked samples as well as incurred samples from a previous feeding experiment were sent to three independent laboratories and analysed by LC–MS/MS, a microbiological assay and the new biosensor assay. The new method correctly identified all contaminated samples and demonstrated advantages in sensitivity and analysis time compared to the microbiological screening assay.     

Development and validation of an LC-MS/MS method for the simultaneous analysis of 28 specific narcotic adulterants used in dietary supplements

The purpose of this study was to develop a method to analyse the concentration of multiple illegal narcotics present in dietary supplements. To this end, we established and optimised a procedure using LC-MS/MS simultaneously to analyse 28 narcotic compounds in various forms of dietary supplements, including powders, tablets, liquids and capsules. In addition, candy and cookies that have also had detected cases of adulteration were also analysed. The specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), stability and recovery for these methods were validated accordingly. The LOD and LOQ of the LC-MS/MS ranged from 0.01–50.0 to 0.03–100 ng g^?1, respectively. The linearity of these results was good (r² > 0.99), with intra- and inter-day precision values of 0.2–5.2% and 0.2–4.8%, respectively. Further, the intra- and inter-day accuracies of this method were 97.0–103.4% and 94.6–103.1%, respectively. The stability RSD was less than 7.8%. The mean recovery for this LC-MS/MS procedure was 81.1–117.4%, with an RSD less than 9.8%. Following the validation of our method, we analysed 47 commercially available dietary supplements obtained in Korea. Whilst none of these samples had detectable amounts of the 28 specified narcotic adulterants, our novel LC-MS/MS procedure can be utilised comprehensively and continually to monitor illegal drug adulteration in various forms of dietary supplements.     

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg^-1) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.     

Formation of semicarbazide (SEM) in food by hypochlorite treatment: is SEM a specific marker for nitrofurazone abuse?

Semicarbazide (SEM) is considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone. It is therefore used as a marker for nitrofurazone abuse. Recently, there has been concern about other sources of SEM in tissue samples, which are not linked to the illegal use of nitrofurazone. The present studies have shown that SEM can occur naturally, e.g. in algae, shrimps and eggs, and is formed from natural substances, e.g. arginine and creatine. A significant formation of SEM was observed in samples treated with hypochlorite commonly used in food processing for disinfection or bleaching. SEM was formed in different kinds of nitrogen compound-containing samples (0.3-20 μg kg^-1) after treatment with 1% active chlorine. It was detected in the mg kg^-1 range after hypochlorite treatment (0.015% active chlorine) of creatine. Lower levels were also formed from creatinine, arginine and urea. SEM present in hypochlorite-treated carrageenan proved mostly to occur in the tissue-bound form. Therefore, differentiation between SEM from nitrofurazone abuse and SEM originating from natural constituents (due to hypochlorite treatment) seems not to be unambiguously possible.     

Development and validation of an LC-MS/MS method for the quantification of tiamulin,trimethoprim, tylosin,sulfadiazine and sulfamethazine in medicated feed

A new multi-compound method for the analysis of veterinary drugs, namely tiamulin, trimethoprim, tylosin, sulfadiazine and sulfamethazine was developed and validated in medicated feeds. After extraction, the samples were centrifuged, diluted in Milli-Q water, filtered and analysed by high performance liquid chromatography coupled to tandem mass spectrometry. The separation of the analytes was performed on a biphenyl column with a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in Milli-Q water. Quantitative validation was done in accordance with the guidelines laid down in European Commission Decision 2002/657/EC. Method performances were evaluated by the following parameters: linearity (R² < 0.99), precision (repeatability <14% and within-laboratory reproducibility <24%), recovery (73.58–115.21%), sensitivity, limit of detection (LOD), limit of quantification (LOQ), selectivity and expanded measurement uncertainty (k. = 2). The validated method was successfully applied to the 2 medicated feeds obtained from the interlaboratory studies and feed manufactures from Spain in August 2017. In these samples, tiamulin, tylosin and sulfamethazine were detected at the concentration levels declared by the manufacturers. The developed method can therefore be successfully used to routinely control the content and homogeneity of these antibacterial substances in medicated feed.

Abbreviations

AAFCO – Association of American Feed Control Officials; TYL – tylosin; TIAM – tiamulin fumarate; TRIM – trimethoprim; SDZ – sulfadiazine; SMZ – sulfamethazine; UV – ultraviolet detector; FLD – fluorescence detector; HPLC – high performance liquid chromatography; MS/MS – tandem mass spectrometry; LOD – limit of detection; LOQ – limit of quantification; CV – coefficient of variation; SD – standard deviation; U – uncertainty     

Strategies for the screening of antibiotic residues in eggs: comparison of the validation of the classical microbiological method with an immunobiosensor method

ABSTRACT

Efficient screening methods are needed to control antibiotic residues in eggs. A microbiological kit (Explorer® 2.0 test (Zeu Inmunotech, Spain)) and an immunobiosensor kit (Microarray II (AM® II) on Evidence Investigator? system (Randox, UK)) have been evaluated and validated for screening of antibiotic residues in eggs, according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods. The e-reader? system, a new automatic incubator/reading system, was coupled to the Explorer 2.0 test. The AM II kit can detect residues of six different families of antibiotics in different matrices including eggs. For both tests, a different liquid/liquid extraction of eggs had to be developed. Specificities of the Explorer 2.0 and AM II kit were equal to 8% and 0% respectively. The detection capabilities were determined for 19 antibiotics, with representatives from different families, for Explorer 2.0 and 12 antibiotics for the AM II kit. For the nine antibiotics having a maximum residue limit (MRL) in eggs, the detection capabilities CCβ of Explorer 2.0 were below the MRL for four antibiotics, equal to the MRL for two antibiotics and between 1 and 1.5 MRLs for the three remaining antibiotics (tetracyclines). For the antibiotics from other families, the detection capabilities were low for beta-lactams and sulfonamides and satisfactory for dihydrostreptomycin (DHS) and fluoroquinolones, which are usually difficult to detect with microbiological tests. The CCβ values of the AM II kit were much lower than the respective MRLs for three detected antibiotics (tetracycline, oxytetracycline, tylosin). Concerning the nine other antibiotics, the detection capabilities determined were low. The highest CCβ was obtained for streptomycin (100 µg kg^–1).     

Improved microbial screening assay for the detection of quinolone residues in poultry and eggs

An improved microbiological screening assay is reported for the detection of quinolone residues in poultry muscle and eggs. The method was validated using fortified tissue samples and is the first microbial assay to effectively detect enrofloxacin, difloxacin, danofloxacin, as well as flumequine and oxolinic acid, at or below their EU maximum residue limits (MRL). The accuracy of the assay was shown by analysing incurred tissue samples containing residue levels around the MRL. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of the quinolone concentration in these samples showed that the test plate can be used semi-quantitatively, allowing the definition of an “action level” as an inhibition zone above which a sample can be considered “suspect”. The presented assay is a useful improvement or addition to existing screening systems.     

Immunoassay based on monoclonal antibodies versus LC-MS: deoxynivalenol in wheat and flour in Southern Brazil

An indirect competitive enzyme-linked immunosorbent assay (ELISA) method using a monoclonal antibody for deoxynivalenol (DON) detection in wheat and flour was standardised and validated (detection limit?=?177.1?µg?kg^?1) and its performance was compared with LC-MS, quantification limit?=140?µg?kg^?1). DON recovery ranged from 88.7% to 122.6% for wheat grain and from 70.6% to 139.3% for flour. Among the 38 wheat samples evaluated, DON was detected in 29 samples (76.3%) by ic-ELISA (281.6–12?291.4?µg?kg^?1) and in 22 samples (57.9%) by LC-MS (155.3–9906.9?µg?kg^?1). The 0.93 correlation coefficient between ic-ELISA and LC-MS data in 19 positive DON wheat samples demonstrated the reliability and efficiency of ic-ELISA. Results indicated that standardised ic-ELISA was suitable for DON screening in wheat samples and the need for continuous monitoring of mycotoxin levels in foodstuffs.     

Validation by collaborative trial of an isotope dilution liquid chromatographic tandem mass spectrometric method to determine the content of acrylamide in roasted coffee

A method for the determination of acrylamide in roasted coffee was subjected to method validation by collaborative trial. The aim was to extend the scope of a method already standardized for the determination of acrylamide in bakery and potato products to include roasted coffee. Modifications of the already standardized method were therefore kept to a minimum. The method was based on aqueous extraction of the roasted coffee matrix and solid-phase extraction (SPE) clean-up followed by isotope dilution high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The test portion of the sample was spiked with stable isotope-labelled acrylamide and extracted on a mechanical shaker with n-hexane and water for 1 h. The sample extract was centrifuged, the organic phase discarded, and a portion of the aqueous extract further cleaned-up by SPE on an Isolute Multimode cartridge followed by a second clean-up step on an Isolute ENV + cartridge. The volume of the acrylamide-containing fraction eluted from the second SPE column was reduced by evaporation and analysed by LC-MS/MS. Three coffee samples and one aqueous acrylamide standard solution were sent to eleven laboratories from eight European Union Member States. All samples were sent as blind duplicates. Based on the reported results, the relative standard deviations for reproducibility (RSD_R) were 11.5% at a level of 160 µg kg^?1, 10.1% at a level of 263 µg kg^?1, and 9.6% at a level of 585 µg kg^?1. The values for repeatability (RSD_r) in those materials ranged from 1.0% to 3.5%. The method performance parameters satisfied internationally accepted criteria. Hence, the method would be suitable for the enforcement of regulatory limits.     

Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies

Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 μg/L and a LOQ of 56.4 μg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices.     

ELISA快速测定小麦中硫丹及硫丹硫酸酯

研究应用酶联免疫方法，快速检测小麦中硫丹及硫丹硫酸酯。小麦经80％甲醇提取后，用PBS／T缓冲液稀释5倍后，酶联免疫方法直接测定。方法平均回收率为73．3％～88．8％，线性范围1．5μg／L～25μg／L，最低检测限0．4μg／L，变异系数〈10．03％。经气相色谱／质谱联用方法验证，灵敏度、重现线和准确度均符合快速检测要求。该法具有快速、简便、低耗、易操作等特点。     

Validation of two ELISA kits for the screening of tylosin and streptomycin in honey according to the European decision 2002/657/EC

Antibiotics are mixed with the food of bees to fight against diseases. No maximum residue limits have been set for honey. Recommended concentrations (RCs) have been published by European Union Reference Laboratories for tylosin and streptomycin. The objective of this project was to select and validate enzyme-linked immunosorbent assay (ELISA) kits for the screening of tylosin and streptomycin/dihydrostreptomycin residues to be implemented in the French honey control plan. Four ELISA kits for tylosin and five ELISA kits for streptomycin were evaluated. At the end, one kit each was selected and validated for tylosin (TECNA AB620) and streptomycin (Europroxima). Both ELISA kits for tylosin and streptomycin are specific, robust, fast and easy-to-use tests. The detection capability CCβ of tylosin A was less than or equal to 10?µg?kg^?1 (half the RC). The CCβ of desmycosin (the hydrolysed product of tylosin A in acidic conditions) is approximately 200?µg?kg^?1, which is five times the RC for tylosin (20?µg?kg^?1). Thus, this kit is fit for the screening of tylosin A but is unsuitable to detect desmycosin. The detection capability CCβ of streptomycin was less than or equal to 10?µg?kg^?1 (one fourth the RC). The cross-reactivity with dihydrostreptomycin was equal to 136%. Both ELISA kits were applicable to a wide variety of honey (single flower and multiflower, different floral origins, different geographic origins, different consistencies [liquid or solid] and different colours).     

Evidence of non-extractable florfenicol residues: development and validation of a confirmatory method for total florfenicol content in kidney by UPLC-MS/MS

The parent compound florfenicol (FF) is a broad-spectrum antibacterial compound licensed in the UK for use in cattle, pigs and the aquaculture industry. The analysis of porcine tissues in this study demonstrates that significant amounts of solvent non-extractable FF-related residues are present in incurred tissues (kidney and muscle) from treated animals. The results indicate that methods based on solvent extraction alone may carry a significant risk of reporting false-negative results. The use of a strong acid hydrolysis step prior to solvent extraction of tissue samples is necessary for an accurate estimate of the total tissue FF content. A robust and sensitive method for the determination of total FF residue content in kidney samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. This method covers the synthetic amphenicol drug FF and its metabolites, measured as the marker residue florfenicol amine (FFA) as per Commission Regulation (EU) No. 37/2010. Non-extractable and intermediate metabolites are converted to the hydrolysis product FFA, and then partitioned into ethyl acetate. Extracts are solvent exchanged prior to a dispersive solid-phase extraction step, then analysed using an alkaline reverse-phase gradient separation by UPLC-MS/MS. The method was validated around the maximum residue levels (MRLs) set out in Regulation (EU) No. 37/2010 for bovine kidney in accordance with Commission Decision No. 2002/657/EC. The following method performance characteristics were assessed during a single laboratory validation study: selectivity, specificity, sensitivity, linearity, matrix effects, accuracy and precision (decision limit (CCα) and detection capability (CCβ) were determined).     

Evaluation and validation of two microbiological tests for screening antibiotic residues in honey according to the European guideline for the validation of screening methods

Two microbiological kits based on Bacillus stearothermophilus (Eclipse 50® and Premi®Test) have been evaluated and validated according to the European guideline for the validation of screening methods (January 2010) and in relation to the concentrations recommended by the EU-RL in 2007. Both tests are robust, a fast method and easy to implement. Both tests are applicable to a very large variety of honeys from different floral and geographical origins (rosemary, lavender, scrub, heath, alder, forest, lemon, acacia, chestnut, raspberry, mountain and flowers) as well as honey of different colours (from blank honey to brown honey, including yellow and orange honey). A satisfactory false-positive rate of 5% was obtained for the Eclipse 50® test. The observed detection capabilities CCβ of the Eclipse 50® kit were: chlortetracycline (>75?µg?kg^?1), oxytetracycline (≤200?µg?kg^?1), tetracycline (>100?µg?kg^?1), cloxacillin (≤40?µg?kg^?1), tylosin (≤200?µg?kg^?1), desmycosin (>400?µg?kg^?1), sulfadiazine (≤300?µg?kg^?1), sulfadimethoxine (≤250?µg?kg^?1), sulfamerazine (>300?µg?kg^?1), sulfamethazine (>1000?µg?kg^?1), sulfamethizole (>75?µg?kg^?1), sulfamethoxazole (≤25?µg?kg^?1), sulfanilamide (?1000?µg?kg^?1), sulfaquinoxaline (>75?µg?kg^?1), sulfathiazole (≤250?µg?kg^?1) and lincomycin (>1500?µg?kg^?1). These levels were all higher than the recommended concentrations where they exist. Due to its lack of sensitivity, it cannot be recommended for reliable routine use. The observed CCβ of the Premi®Test kit were: chlortetracycline (10?µg?kg^?1), oxytetracycline (>10?µg?kg^?1), tetracycline (≤10?µg?kg^?1), cloxacillin (≤5?µg?kg^?1), tylosin (≤10?µg?kg^?1), desmycosin (≤15?µg?kg^?1), sulfadiazine (≤25?µg?kg^?1), sulfadimethoxine (≤25?µg?kg^?1), sulfamerazine (≤25?µg?kg^?1), sulfamethazine (≤25?µg?kg^?1), sulfamethizole (≤25?µg?kg^?1), sulfamethoxazole (≤10?µg?kg^?1), sulfanilamide (≤25?µg?kg^?1), sulfaquinoxaline (≤10?µg?kg^?1), sulfathiazole (25?µg?kg^?1) and lincomycin (≤25?µg?kg^?1). The Premi®Test kit could be recommended for reliable use in routine control due to its low detection capabilities (except for aminoglycosides), but the disadvantage is a high false-positive rate of 14%.