Validation of a commercial receptor kit Sulfasensor Honey for the screening of sulfonamides in honey according to Commission Decision 2002/657/EC

The Sulfasensor Honey kit is a receptor test dedicated to the screening of sulphonamide residues respectively in different matrices. The aim of this project was to evaluate and validate this kit according to the Community Reference Laboratory (CRL) guideline for the validation of screening methods to achieve the French control plan for honey. The test is robust, quick (90 min for 40 samples), easy to perform and easy to read. The false-positive rate was estimated to be 12.5%. The detection capabilities CCβ of the kit were lower than or equal to 25 μg kg(-1) for sulfamethazine, sulfamerazine, sulfathiazole and sulfapyridine, and between 25 and 50 μg kg(-1) for sulfadiazine and sulfadimethoxine, 150 μg kg(-1) for sulfaquinoxaline, and 1000 μg kg(-1) for sulfamethoxazole and sulfamethizole. Sulfanilamide was not detected by the kit. The kit was applicable to a wide variety of honeys (different floral and geographical origins, liquid or solid). This kit was used to implement the French control plan for the detection of antibiotic residues in honey in 2010 in parallel with an HPLC method. However, in 2011 the kit was replaced by an LC-MS/MS method for the screening and confirmation of sulfonamide residues in honey, which detects all the sulfonamides of interest.     

Analysis of 27 antibiotic residues in raw cow’s milk and milk-based products – validation of Delvotest® T

Delvotest® T was evaluated for its capability at detecting residues of 27 antibiotics in raw cow’s milk and in some dairy ingredients (skimmed and full-cream milk powders). The kit was used as a screening tool for the qualitative determination of antibiotics from different families in a single test. Results delivered by such a method are expressed as ‘positive’ or ‘negative’, referring to the claimed screening target concentration (STC). Validation was conducted according to the European Community Reference Laboratories’ (CRLs) residues guidelines of 20 January 2010 and performed by two laboratories, one located in Europe and the other in Asia. Five criteria were evaluated including detection capability at STC, false-positive (FP) rate, false-negative (FN) rate, robustness and cross-reactivity using visual reading and Delvoscan®. STCs were set at or below the corresponding maximum residue limit (MRL), as fixed by European Regulation EC No. 37/2010. Four antibiotics (nafcillin, oxytetracycline, tetracycline and rifaximin) out of 27 had a false-negative rate ranging from 1.7% to 4.9%; however, it was still compliant with the CRLs’ requirements. Globally, Delvotest T can be recommended for the analysis of the surveyed antibiotics in raw cow’s milk, skimmed and full-cream milk powders. Additional compounds were tested such as sulfamethazine, spiramycin and erythromycin; however, detection at the corresponding MRL was not achievable and these compounds were removed from the validation. Other drugs from the sulfonamide, aminoglycoside or macrolide families not detected by the test at the MRL were not evaluated in this study. Regarding the reliability of this rapid test to milk-based preparations, additional experiments should be performed on a larger range of compounds and samples to validate the Delvotest T in such matrices.     

Validation of a commercial receptor kit Sulfasensor® Honey for the screening of sulfonamides in honey according to Commission Decision 2002/657/EC

The Sulfasensor® Honey kit is a receptor test dedicated to the screening of sulphonamide residues respectively in different matrices. The aim of this project was to evaluate and validate this kit according to the Community Reference Laboratory (CRL) guideline for the validation of screening methods to achieve the French control plan for honey. The test is robust, quick (90?min for 40 samples), easy to perform and easy to read. The false-positive rate was estimated to be 12.5%. The detection capabilities CCβ of the kit were lower than or equal to 25?µg?kg^?1 for sulfamethazine, sulfamerazine, sulfathiazole and sulfapyridine, and between 25 and 50?µg?kg^?1 for sulfadiazine and sulfadimethoxine, 150?µg?kg^?1 for sulfaquinoxaline, and 1000?µg?kg^?1 for sulfamethoxazole and sulfamethizole. Sulfanilamide was not detected by the kit. The kit was applicable to a wide variety of honeys (different floral and geographical origins, liquid or solid). This kit was used to implement the French control plan for the detection of antibiotic residues in honey in 2010 in parallel with an HPLC method. However, in 2011 the kit was replaced by an LC-MS/MS method for the screening and confirmation of sulfonamide residues in honey, which detects all the sulfonamides of interest.     

Proficiency testing for the evaluation of the ability of European Union-National Reference laboratories to determine aflatoxin M1 in milk at levels corresponding to the new European Union legislation

In 1992, the European Union set up a network of National Reference Laboratories and charged the Community Reference Laboratory with the responsibility to design a proficiency testing scheme for assessing the analytical ability of laboratories involved in the official control of aflatoxin M1 in milk. Since 1996, two exercises of proficiency testing have been performed on samples of milk powder and liquid milk at various levels of aflatoxin M1 contents. The trials were conducted according to ISO Guide 43, in particular for the homogeneity testing of sample batches and for the calculation of laboratory z-scores. The National Reference Laboratories officially designated by their governments participated in this programme. Samples were naturally-contaminated milk obtained by feeding cows with aflatoxin B1-contaminated feed. The levels of aflatoxin M1 in the samples ranged from 0.2 to 0.7 microg/kg in milk powder and from 0.05 to 0.07 microg/l in liquid milk. These levels were chosen as being close to the European Union-regulated limit of 0.05 microg of aflatoxin M1 per litre. The results produced by laboratories were compiled and statistically analysed to detect any outlying results and to calculate the individual z-scores. Except for one laboratory in each exercise, all laboratories exhibited acceptable or questionable z-scores. The interlaboratory relative standard deviation for reproducibility (RSDR) obtained for both 1996 and 1998 exercises were in the range 15.7-30.3%. Compared with other published studies, this indicates a very good precision for the performance of this laboratory network in the analysis of traces of aflatoxin M1 in milk.     

Proficiency study for the determination of nitrofuran metabolites in shrimps

A proficiency test for the determination of nitrofuran metabolites in shrimp tissue was organized in the first half of 2003. This test was intended to allow the participants to use their routine method and to assess their competence on this specific analysis. The participation in this proficiency test was offered to all the National Reference Laboratories (NRLs) of the European Union (EU) in charge of the analysis of nitrofurans, to Official Laboratories of the then 10 Candidate Countries for entry in EU and to some countries exporting food to the EU. The participants (20) analysed nitrofuran metabolites in eight randomly coded frozen samples including three blank samples. All participants performed a confirmatory method using liquid chromatography/mass spectrometry to detect total nitrofuran metabolite residues. Both qualitative and quantitative analyses of the results were investigated. Qualitatively, 16 laboratories out of 20 gave the correct interpretation of the results in term of compliant/non-compliant sample. Quantitatively, laboratory performance was evaluated by calculating the z-scores.     

In-house validation of PremiTest, a microbiological screening test with solvent extraction, for the detection of antimicrobial residues in poultry muscles

PremiTest, a microbial inhibition test for the screening of antimicrobial residues, was validated according to the criteria established by Decision 2002/657/EC. Sensitivity, detection capability (CCβ), specificity, selectivity, robustness and applicability were evaluated. The methodology involves the technique of solvent extraction, which increases the detection capability of the test for a wider range of antibiotics. The following CCβ values in poultry muscle were found: penicillin G ≤ 12.5 μg kg(-1), total sulfonamides ≤ 75 μg kg(-1), erythromycin 75 μg kg(-1) and lincomycin 50 μg kg(-1). The detection capability of chlortetracycline was equal to its maximum residue limit (100 μg kg(-1)) and the method did not detect gentamicin (1000 μg kg(-1)), for which no MRL is established in poultry muscle. Specificity evaluated in relation to different analytes and matrices did not detect any interferences in the tests results; whilst the robustness showed that the pH neutralisation point of the extract affects the analytical results and the kits' performance. Only the screening of tetracyclines requires the analysis of extracts without pH neutralisation. The results of the validation process showed that this method is acceptable for screening β-lactam, sulfonamide and macrolide antimicrobial groups in the National Residues and Contaminants Control Programme (PNCRC), and that for this it is fit for purpose.     

Determination of 105 antibiotic,anti-inflammatory,antiparasitic agents and tranquilizers by LC-MS/MS based on an acidic QuEChERS-like extraction

A procedure for screening 105 veterinary drugs in foods by liquid chromatography tandem mass-spectrometry (LC-MS/MS) is presented. Its scope encompasses raw materials of animal origin (milk, meat, fish, egg and fat) but also related processed ingredients and finished products commonly used and manufactured by food business operators. Due to the complexity of the matrices considered and to efficiently deal with losses during extraction and matrix effects during MS source ionisation, each sample was analysed twice, that is ‘unspiked’ and ‘spiked at the screening target concentration’ using a QuEChERS-like extraction. The entire procedure was validated according to the European Community Reference Laboratories Residues Guidelines. False-negative and false-positive rates were below 5% for all veterinary drugs whatever the food matrix. Effectiveness of the procedure was further demonstrated through participation to five proficiency tests and its ruggedness demonstrated in quality control operations by a second laboratory.     

Validation of a Five Plate Test,the STAR protocol,for the screening of antibiotic residues in muscle from different animal species according to European Decision 2002/657/EC

The STAR protocol is a Five Plate Test (FPT) developed several years ago at the Community Reference Laboratory (CRL) for the screening of antimicrobial residues in milk and muscle. This paper presents the validation of this method according to European Decision 2002/657/EC and to an internal guideline for validation. A validation protocol based on ‘simulated tissues’ and on a list of 16 representative antimicrobials to be validated was implemented in our laboratory during several months for the STAR protocol. The performance characteristics of the method were determined (specificity, detection capabilities CCβ, applicability, ruggedness). In conclusion, the STAR protocol is applicable to the broad-spectrum detection of antibiotic residues in muscles of different animal species (pig, cattle, sheep, poultry). The method has good specificity (false-positive rate = 4%). The detection capabilities were determined for 16 antibiotics from different families in relation to their respective maximum residue limit (MRL): beta-lactams (penicillins and cephalosporins ≤ MRL), tetracyclines (≤MRL and ≤2.5 MRL), macrolides (2 MRL), quinolones (≤2 MRL), some sulphonamides (≤3 MRL), and trimethoprim (2 MRL). However, the sensitivity of the STAR protocol towards aminoglycosides (>8 MRL) and florfenicol (≤10 MRL) was unsatisfactory (>>MRL). The two objectives of this study were met: firstly, to validate the STAR protocol according to European Decision 2002/657/EC, then to demonstrate that the validation guideline developed to implement this decision is applicable to microbiological plate tests even for muscle. The use of simulated tissue appeared a good compromise between spiked discs with antibiotic solutions and incurred tissues. In addition, the choice of a list of representative antibiotics allowed the reduction of the scope of the validation, which was already costly in time and effort.     

基于免疫原理的7种磺胺类兽药残留快速检测试剂结果准确性评估

目的 评估市场上5种基于免疫原理的快速检测试剂盒检测7种磺胺类兽药残留的结果准确性.方法 分别用5种快速检测试剂盒与GB/T 21316-2007《动物源性食品中磺胺类药物残留量的测定液相色谱-质谱/质谱法》对样品中的7种磺胺类兽药残留进行测定,验证快速检测试剂盒的检测准确性,考察5种快速检测试剂盒在选择不同的样品基质...     

牛奶中氯霉素残留量测定能力验证结果分析

目的对牛奶中氯霉素残留量测定的能力验证结果进行分析。方法按照中国合格评定国家认可委员会规定的方法进行均匀性检验和稳定性检验。以Z比分数作为统计量对参加实验室的检测结果进行评价,选择全部参加实验室上报结果的中位值作为指定值、标准化四分位距作为能力评定标准差。结果牛奶中氯霉素残留检测项目能力验证实验室满意率为84.8%,可疑结果率为0,不满意率为15.2%。国家级和省级检验机构的满意率相对较高。结论在本次能力验证中,大多数实验室均能准确检测牛奶中氯霉素的残留。对不达标机构,通过本次能力验证查找存在的问题,可以帮助实验室整改,进一步提升检验能力。     

鸡肉糜样中金刚烷胺检测能力验证研究

目的设计鸡肉糜样中金刚烷胺残留检测能力验证研究(proficiency testing,PT)项目,评价实验室对鸡肉中金刚烷胺残留量的检测能力。方法采用单因素方差分析和t检验对样品均匀性和稳定性进行分析。对能力验证结果进行稳健统计分析,通过Z比分数评价实验室检测能力。结果在P为0.05显著水平,制备100包样品均匀性符合要求且在整个能力验证计划周期内保持稳定,满足能力验证计划要求;17个省、市的42家实验室参加了本次能力验证,其中35家结果为满意,满意率为83.3%。结论参加能力验证的绝大多数实验室可以准确检测金刚烷胺,表明鸡肉中金刚烷胺残留量检测水平总体良好。     

Development and Comparison of Sample Preparation Techniques for Chromatographic Analysis of Sulfonamide Residues in Bovine Milk

Most studies to determine sulfonamide residues in milk samples have used solid-phase extraction as the sample preparation technique. However, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, introduced in 2003, has been used in the extraction of various compounds in food matrices. This study aimed to evaluate two sample preparation techniques: solid-phase extraction and QuEChERS, for chromatographic analysis of sulfonamides (sulfathiazole, sulfamethazine, and sulfadimethoxine) in bovine milk. The chromatographic parameters and the QuEChERS extraction procedure were developed by using different experimental designs, obtaining good peak resolution, recovery, precision, accuracy, linearity, selectivity, and limits of detection and quantification. In contrast, using solid-phase extraction, acceptable recoveries and selectivity were not achieved, despite the number of articles published that have applied this sample preparation technique for sulfonamide analysis. As a result of the experiments performed, probably sulfonamides are retained together with other components of the matrix in the sample pretreatment step (prior to its addition in the cartridge containing solid phase), which is an important part of solid-phase extraction with raw whole milk. Therefore, QuEChERS is a better method than solid-phase extraction for the analysis of sulfonamide residues in milk. Validation tests demonstrated that the method is appropriate, within the maximum residue limit (0.1 mg kg^?1). Moreover, it was possible to use a lower amount of solvent compared with previously published articles (6 mL against 10 or 15 mL).     

Use of proficiency samples to assess diagnostic laboratories in France performing a Trichinella digestion assay

Routine diagnosis of animal trichinellosis for food safety and trade relies on a method of artificial digestion to free Trichinella muscle larvae from meat for subsequent identification by microscopy. As part of a quality control system, the French National Reference Laboratory (NRL) initiated ring trials to determine the sensitivity of the test performed in the 72 routine diagnostic laboratories in France. A method was devised to obtain calibrated meat samples containing known numbers of capsules with Trichinella spiralis muscle larvae. This method was based on an incomplete artificial digestion of Trichinella-infected mice carcasses to allow the collection of intact Trichinella capsules. Capsules were placed into a meatball of 100 +/- 2 g of pork and horsemeat to produce proficiency samples. Three categories of samples were prepared: small (3 to 5 capsules), medium (7 to 10), and large (12 to 15). The sensitivity was expressed as the percentage of muscle larvae recovered from each proficiency sample. Reproducibility was tested with ring trials organized between two NRLs (France and Canada), and a reference sensitivity of 84.9% was established. National ring trials were then organized in France, with the 72 routine diagnostic laboratories each receiving four proficiency samples per session. After five sessions, an improvement in the digest test sensitivity was observed. Results at the fifth session indicated sensitivities of 78.60% +/- 23.70%, 81.19% +/- 19.59%, and 80.52% +/- 14.71% muscle larvae for small, medium, and large samples, respectively. This study supports the use of proficiency samples to accurately evaluate the performance of routine diagnostic laboratories that conduct digestion tests for animal trichinellosis diagnosis.     

Microbiological and chemical identification of antimicrobial drugs in kidney and muscle samples of bovine cattle and pigs

Microbiological and chemical identification of antimicrobial drug residues was attempted in 95 kidney and 76 muscle samples from 58 cattle, 36 pigs and one horse which had revealed kidneys positive to an inhibitor test. Information on pre-slaughter medication with one antimicrobial drug was available for 63% of the carcasses. Microbiological identification was performedbyagar diffusionusing 17 or 18combinationsof eight test bacteria, varying medium pH and three substances blocking the action of certain antimicrobials. Sampleactivity patterns compiled from inhibition zone diameters on test plates were compared with those obtained with standard antimicrobial solutions both visually and by locating the minimal sum of absolute pairwise differences over the tests. Chemical identification of residues was based on liquid chromatography. In kidney samples containing one microbiologicallyidentified antimicrobial the two methods gave fully consistent results with tetracyclines (15/15) and fluoroquinolenes (8/8). Preparation and storage of the kidney samples before chemical analyses appeared to influencethechemicalidentification of penicillin G. The results were consistent in 37 of the 41 samples stored without homogenization at - 70oC. The residue was identified by chemical means only in six and neither microbiologicallynorchemicallyinfour kidneysamples with information on pre-slaughter medication. The same residue as in the kidney samples was identifiable microbiologically in 41% of the muscle samples of the same carcasses. The results show that the microbiolo gical method is well suited for identification of antibiotic residues. They indicate further that an enhanced resolution with a reduced combination of plates is attainable.     

DETECTION OF ANTIMICROBIAL DRUG RESIDUES IN KENYAN MILK

The improved Dutch tube diffusion test was used to study the occurrence of inhibitory substances in raw bulk milk samples within the Nakuru District in Kenya. Initially the detection limits of the method were verified using milk standards spiked with selected antibiotics. Addition of penicillinase to inhibitor-positive samples was used for preliminary identification of penicillin G-type antibiotics and residue levels were estimated against a standard curve constructed by means of a B. stearothermophilus disc assay. The two-tube test was used to screen 1109 field samples of which 229 (21%) were suspect positive. The identification procedure confirmed 165 samples (14.9%) to contain penicillin G-type residues of which 118 contained levels exceeding the established EU MRL for penicillin G (4 μg/kg). This study indicates that antibiotic residues are prevalent in milk within the Nakuru district of Kenya. It suggests that the improved tube diffusion test in combination with a multiplate system could be useful for qualitative and quantitative identification of antimicrobial drug residues in milk.     

Residue analysis of tetracyclines in poultry muscle: shortcomings revealed by a proficiency test.

A proficiency test for tetracycline drug residues in poultry muscle was organized according to the guidelines of International Laboratory Accreditation Cooperation (ILAC) ILAC-G13:2000 (2000). For the proficiency test, three test materials were prepared. The homogeneity and stability of the materials during the study were demonstrated. Sixteen laboratories accepted the invitation to participate in the proficiency test; 11 laboratories reported results within the time frame of the study. Most notably, only four of the participating laboratories complied with the definition of the maximum residue limit (MRL) concerning the inclusion of 4-epimers as stated in European Commission Regulation 281/96 (1996). Most participants reported values for the decision limit (CCalpha) and detection capability (CCbeta) and hence were already in compliance with European Commission 2002/657/EC (2002) for this aspect of method validation. However, some CCalpha and CCbeta values were not in agreement with the actual within-laboratory reproducibility calculated from the results reported in this proficiency test. Although most laboratories obtained satisfactory results, it is clear that an effort is needed to include 4-epiOTC, 4-epiTC and 4-epiCTC in the analytical methods. Moreover, reconsideration of values determined for CCalpha and CCbeta with respect to their accuracy may be necessary in some cases.     

PREVALENCE OF ANTIMICROBIAL RESIDUES IN PREPROCESSED AND PROCESSED COWS' MILK IN TRINIDAD

The prevalences of antimicrobial residues were determined in cows' bulk raw milk from 16 collection centers, in pasteurized milk obtained from sale outlets, and in raw, pasteurized and sterilized milk in the processing line of a plant in Trinidad. The Delvotest SP 5 Pack test kits were used to detect the antimicrobial residues in milk. Thirty-one (10.3%) of 300 bulk milk samples contained antimicrobial residues with penicillin responsible for 90.3% of all the positive samples which originated from 8 (50.0%) of the 16 collection centers. The prevalence of residues in pasteurized milk was 21.3% compared to 8.3% detected in sterilized milk. The difference was not statistically significant (P ± 0.05; X²). Pasteurized milk obtained from sale outlets but originating from two other processing plants had antimicrobial residue prevalence of 0% and 8.3%, respectively. It is concluded that the presence of antimicrobial residues in raw and processed milk in Trinidad could be of public health and economic significance .     

Multi-residue method for the confirmation of four avermectin residues in food products of animal origin by ultra-performance liquid chromatography-tandem mass spectrometry

A confirmatory method was developed for the rapid determination of abamectin, ivermectin, doramectin and eprinomectin residues in various food products of animal origin, such as pork muscle, pork liver, fish and milk. Samples were homogenized, extracted and de-proteinized by acetonitrile, cleaned via two-step cleaning procedure using Bond Elut C(18) SPE columns and then alumina-N cartridges. All the four avermectin residues in different animal-food products were simultaneously separated and determined by ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) within 3.5 min. Data acquisition under positive ESI-MS/MS was performed by applying multiple reaction monitoring (MRM) for both identification and quantification, and mass spectrometric conditions were optimized to increase selectivity and sensitivity. The matrix-matched calibration curves for different matrices, such as pork muscle, pork liver, fish and milk, were constructed and the interference effect of different sample matrices on the ionization was effectively eliminated. The UPLC-MS/MS method was validated with satisfactory linearity, recovery, precision and stability. Matrix-matched calibration curves of abamectin, ivermectin, doramectin and eprinomectin in four different matrices were linear (r(2)( )≥ 0.990, goodness-of-fit coefficients ≤12.8%) in the range 2.5-200 μg kg(-1). The limits of detection and quantification for the four avermectins were in the range 0.05-0.68 and 0.17-2.27 μg kg(-1), respectively. Recoveries were 62.4-104.5% with good intra- and inter-day precision. The method was rapid, sensitive and reliable, and can be applied to the quantitative analysis of avermectin residues in different animal-food products.     

Effects of feeding pasteurized waste milk to dairy calves on phenotypes and genotypes of antimicrobial resistance in fecal Escherichia coli isolates before and after weaning

The aim of this study was to evaluate the effects of feeding pasteurized waste milk (pWM) to calves on antimicrobial resistance of fecal Escherichia coli at both phenotypic and genotypic levels. Fifty-two Holstein female calves (3 ± 1.3 d of age) were fed 1 of the 2 different types of milk: milk replacer (MR) without antimicrobials or pWM with β-lactam residues until weaning at 49 d of age. Fecal swabs of all calves were obtained on d 0, 35, and 56 of the study and 3 E. coli isolates per sample were studied. Phenotypic resistance was tested by the disk diffusion method against a panel of 12 antimicrobials. A total of 13 resistance genes consisting of β-lactam, sulfonamide, tetracycline, and aminoglycoside families were examined by PCR. Feeding pWM to calves increased the presence of phenotypic resistance to ampicillin, cephalotin, ceftiofur, and florfenicol in fecal E. coli compared with MR-fed calves. However, the presence of resistance to sulfonamides, tetracyclines, and aminoglycosides was common in dairy calves independent of their milk-feeding source, suggesting other factors apart from the feeding source are involved in the emergence of antimicrobial resistance.     

Accuracy of BRT and Delvotest microbial inhibition tests as affected by composition of ewe's milk

The presence of drug residues in ewe's milk samples can be determined by microbial assays. The main limitation of these tests is the large number of false-positive results associated with them. False-positive results can be explained by the interaction of certain substances naturally existing in ewe's milk with the growth of the microorganism used in the test. In this study, milk chemical composition (fat, protein, lactose, total solids), somatic cell counts (SCCs), free fatty acid concentrations, and lactoperoxidase system components were determined in order to investigate their influence on the rate of false-positive results for the BRT and Delvotest microbiological inhibitor tests. Milk samples were obtained after morning milking of Manchega ewes at 15, 30, 45, 60, 75, 90, 105, 120, and 135 days after parturition. The animals did not receive any kind of treatment or medicated feed throughout the experiment. The false-positive rates for BRT and Delvotest were 3.75 and 2.4%, respectively. When the logistic regression model was applied, the percentages of total solids for positive samples were significantly different from those for negative samples (16.90 versus 18.42% for BRT, 16.05 versus 18.45% for Delvotest), while the SCC logarithmic transformation was significantly higher for the positive samples than for the negative samples (5.38 versus 5.11 log units for BRT, 5.32 versus 5.11 log units for Delvotest). Moreover, Delvotest-positive samples exhibited thiocyanate concentrations higher than those of Delvotest-negative samples (8.18 mg/liter versus 6.85 mg/liter). Further analyses are needed to confirm the possible presence of antimicrobial residues in this particular type of milk sample.