Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory tests.

Residues of the tetracycline group of antibiotics were quantified in pork and chicken muscle tissue that had previously been screened with a microbiological inhibition test and an immunological method. Pieces of frozen pork and chicken meat were screened on a pH 6 culture medium seeded with Bacillus subtilis. An aqueous extract of the inhibitor-positive samples was then screened with a group-specific commercial ELISA kit, able to detect levels of oxytetracycline, chlortetracycline, tetracycline and doxycycline corresponding with the European MRL or lower. The cut-off value of the ELISA was set at a B/B0 value of 75%. Finally, confirmation and quantification were performed using a validated HPLC method with fluorescence detection. The fluorescence was induced by complexation of the tetracyclines with the zirconium cation which is added post-column to the HPLC eluate. This fluorescence makes it possible to quantitate residues below one-half of the MRL. To gain additional qualitative information some samples were also analysed with LC-MS-MS. ELISA analysis demonstrated the presence of residues of tetracyclines in 12 out of 19 inhibitor-positive pork samples and in 19 out of 21 inhibitor-positive chicken samples. Doxycycline was detected with HPLC in 10 of these 12 pork samples and in 18 out of 19 chicken samples. The two other ELISA positive pork samples contained oxytetracycline, while no tetracyclines were found in one ELISA positive chicken meat sample. The correlation between the ELISA B/B0 values and the actual levels determined with the HPLC method was poor, whereas a better correlation was observed between the inhibition zones and the doxycycline levels. Our results indicate that an inhibition test with a medium at pH 6 and B. subtilis as test organism is well suited to screen pork and chicken muscle tissue for residues of tetracycline antibiotics. Since many positive samples contained doxycycline levels below the MRL, a confirmatory method is necessary to quantify the residues.     

Identification of myosin heavy chain isoforms in porcine longissimus dorsi muscle by electrophoresis and mass spectrometry

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS‐PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA‐F8 (anti‐MHC slow/I), SC‐71 (anti‐MHC 2a and 2x), 10F5 (anti‐MHC 2b), and BF‐35 (anti‐MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS‐PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS‐PAGEtechnique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.     

Development of a polymerase chain reaction and capillary gel electrophoresis method for the detection of chicken or turkey meat in heat-treated pork meat mixtures

A polymerase chain reaction and capillary gel electrophoresis (PCR-CGE) method with ultraviolet (UV) or laser induced fluorescence detection (LIF) was established for the detection of chicken or turkey in heat-treated pork meat mixtures. Mitochondrial DNA samples extracted from heat treated meat were amplified with their corresponding specific primers yielding PCR products between 200 and 300 bp. LIF detection was superior than UV detection in terms of precision and sensitivity for the study of DNA fragments. The CGE-LIF method was highly reproducible and accurate for determining DNA fragment size. The PCR-CGE-LIF was sensitive since a significant fluorescent signal was obtained at the minimum admixture level employed of 1% in meat mixtures. Thus, the PCR-CGE-LIF method established was useful for the detection of chicken or turkey in heat treated meat mixtures and may prove to be useful for the detection of poultry meat in pork processed products.     

Determination of rendering plant sterilization conditions using a commercially available ELISA test kit developed for detection of cooked beef.

A commercially available enzyme-linked immunosorbent assay (ELISA) test kit developed for detection of cooked beef in meat samples was used to determine appropriate heat treatment of rendered materials. An improved extraction procedure increased the absolute difference in R-values between 2 rendered materials treated under different conditions (average temperature 129 and 134 degrees C, respectively). To evaluate the influence of the main sterilization parameters on ELISA results, a factorial design approach was used. The parameters investigated were temperature, time, particle size, and meat composition. Lean meat samples containing beef and pork were sterilized under strictly controlled conditions in a laboratory autoclave. The experiments demonstrated that the R-values obtained with the ELISA test kit for beef are strongly influenced by temperature and time, whereas particle size has a minor influence. The proportion of bovine material did not have any impact on R-values. Autoclave-processed lean meat samples were analyzed by using an ELISA test kit for pork, which was validated in a collaborative trial. The ELISA test kit for pork proved to be more sensitive for the investigated parameters, thus verifying and extending previous investigations.     

Western blot detection of brain material in heated meat products using myelin basic protein and neuron-specific enolase as biomarkers

An assay based on Western blotting and detection of central nervous system (CNS)-specific antigens was developed to detect brain tissue in processed (heated) meat products. Bands of antigen-bound primary antibodies were visualised through secondary anti-antibodies labelled with peroxidase, which generated chemiluminescence documented by a photographic film. Ponceau-S staining before antibody incubation and molecular mass information on detected antigens after immunoreactions added information supporting correct identification of brain tissue in the meat products. In this approach B50/growth-associated protein (B50), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), neurofilament (NF), neuron-specific enolase (NSE) and synaptophysin (Syn) proteins were detected in raw luncheon meat and a liver product enriched with brain tissue at a level of 5% (m/m). Only MBP and NSE were considered suitable biomarkers for detection of 1% (m/m) brain tissue in meat products pasteurised at 70 °C or sterilised at 115 °C. The use of an anti-monkey MBP instead of anti-human MBP enabled speciation of the CNS material whether from bovine and ovine brains or from porcine brain tissue. This immunoblot assay potentiates the analysis of approximately 70 samples within 8 h, including sample preparation and the simultaneous probing of NSE and MBP target antigens.     

Determination of Pyrethroid Residues in Pork Muscle by Immunoaffinity Cleanup and GC-ECD

A method was developed to determine six pyrethroids (tau-fluvalinate, fenpropathrin, λ-cyhalothrin, cyfluthrin, α-cypermethrin and deltamethrin) in pork muscle by immunoaffinity column cleanup and gas chromatography-electron capture detection. Spiked pork muscle samples at 5, 20, 50 μg kg^?1 were extracted with petroleum spirit-diethyl ether (1:1, v/v). Fat was eliminated by liquid–liquid partition between acetonitrile and petroleum spirit. An immunoaffinity column (IAC) was used for further cleanup. The IAC column was prepared by coupling the polyclonal antibodies to the protein A sepharose gel and the resulting affinity gel columns were sufficiently stable for multiple reuse. Target compounds were adsorbed at pH 7.4 and after extensive washing, eluted with 3 mL methanol. Recoveries of the six pyrethroids were typically >70%. The detection limit was 2 μg kg^?1 for λ-cyhalothrin and α-cypermethrin and 5 μg kg^?1 for cyfluthrin, deltamethrin, fenpropathrin and tau-fluvalinate. Repeat analyses of pork muscle samples showed good repeatability. The method was applied to detect residues in meat samples.     

Meat species identification by linear discriminant analysis of capillary electrophoresis protein profiles

The objective of this study was to utilize linear discriminant analysis (LDA) in the interpretation of capillary electrophoresis-sodium dodecyl sulfate polymer-filled capillary gel electrophoresis (CE-SDS) meat protein profiles for the identification of meat species. The specific objectives were 1) to collect quantitative data on water-soluble and saline-soluble proteins of different meat species obtained by CE-SDS and 2) to apply LDA on collected CE-SDS protein data for the development of a pattern recognition statistical model useful in the differentiation of meat species. Samples were raw beef top and eye round, boneless fresh pork ham and loin, turkey leg and breast meat, and mechanically deboned turkey meat collected on six different occasions, making a total of 42 samples. Additionally, 14 samples were used as test samples to determine the classification ability of the procedure. Quantitative protein data obtained by CE-SDS was used to generate separate LDA models for either water- or saline-soluble protein extracts. Although a saline solution was a more efficient meat protein-extracting agent, as shown by a higher total protein concentration and a larger number of peaks, water-soluble CE-SDS protein profiles gave more distinctive discrimination among meat species. The correct classification given by LDA on water-soluble protein data was 100% for all meat species, except pork (94%). Conversely, the correct classification on saline-soluble protein data was 88% for beef and mechanically deboned turkey meat, and 94% and 100% for turkey and pork meat, respectively. LDA proved to be a useful pattern recognition procedure in the interpretation of CE-SDS protein profiles for the identification of meat species.     

A Novel and Reusable Electrochemical Genosensor for Detection of Beef Adulteration

This work describes the construction of a genosensor based on a graphite electrode modified with an reduced graphene oxide/poly(3-hidroxybenzoic acid) nanocomposite with an specific DNA oligonucleotide for detection of cattle mitochondrial DNA, in order to certify beef purity. Electrochemical and morphological analyses indicate that the genosensor allows duplex formation with the DNA of pure sample of beef lysate. The genosensor was selective, identifying up to 1 % (w/w) of pork in beef samples, showing good reproducibility and stability within six weeks of storage, and can be reused four times, being a great tool for the evaluation of beef purity, with application in the meat production and marketing chain.     

Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food matrices

The penicillin-binding protein PBP 2x* from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the detection and determination of penicillins and cephalosporins with intact beta-lactam structure in milk, bovine and porcine muscle juice, honey and egg. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and digoxigenin as functional groups (DIG-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses anti-digoxigenin F(ab) fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more antibody fragments are bound via the digoxigenin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present. A fractional factorial design was applied to detect chemometrically effects and interactions of the assay parameters. For optimisation of the significant parameters a Box-Behnken design was used. The assay has been developed for various food matrices as screening test with the option for a quantitative assay, when the identity of the residual beta-lactam is known (e.g. elimination studies). Cefoperazon, cefquinome, cefazolin, cloxacillin, ampicillin and benzylpenicillin could be detected at levels corresponding to 1/2 EU maximum residue limit (MRL) in milk, meat juice from muscle tissue of different species, egg and honey (where applicable) without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.     

基于增强型脂质去除固相小柱净化结合液相色谱-串联质谱法测定猪肉和猪肝中的双甲脒农药及其代谢物

建立了采用增强型脂质去除(EMR-Lipid)固相小柱净化结合液相色谱-串联质谱(LC-MS/MS)测定猪肉和猪肝中双甲脒 (AMZ) 及其3种代谢物2,4-二甲基苯胺(DMA)、单甲脒(DMPF)和2,4-二甲基苯基甲酰胺(DMF)残留量的方法。 样品经乙腈蛋白沉淀及盐析提取,通过Captiva EMR-Lipid 过滤小柱净化,滤膜过滤后,采用Agilent ZORBAX Eclipse Plus C₁₈柱(50 mm×2.1 mm,1.8 μm)分离,以0.1%甲酸水溶液与0.1%甲酸乙腈为流动相进行梯度洗脱,采用电喷雾离子源正离子方式扫描,多反应监测模式进行检测。 结果表明,在猪肝与猪肉中AMZ、DMA线性范围为1～200 μg/kg,DMPF和DMF的线性范围为0.1～200 μg/kg,相关系数(R²)均大于0.991;AMZ、DMA、DMPF和DMF的方法定量限(S/N=10)分别为0.6、0.6、0.05、0.05 μg/kg;对空白猪肉和猪肝进行0.1、1、5、50 μg/kg 4个浓度水平的加标实验,回收率在60.2%～127.4%之间,相对标准偏差均低于12%。 该方法简便、快捷,适用于猪肉和猪肝中双甲脒及其代谢物残留量的同时测定。     

ELISA for semicarbazide and its application for screening in food contamination

The development of a direct competitive enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies specific for semicarbazide (SEM) is described. Molecular modelling of the hapten mimics and other key components of the assay system was conducted to explain antibody properties in relation to hapten design. The small aliphatic molecule SEM was coupled to 3-carboxybenzaldehyde to produce carboxyphenyl-SEM (CPSEM), for the generation of specific antibodies. Five rabbits produced antibodies against NPSEM (used in direct and indirect ELISA formats) exhibiting a 50% binding inhibition level (IC₅₀ values) of 0.06-2.28 μg L⁻¹ in assay buffer for SEM. The most sensitive indirect assay based on the antibody MVK39 showed a high dynamic range providing a linear readout in the range of 0.01-0.2 μg L⁻¹. Antibody MVK31 (IgG) allowed specific SEM detection at an IC₅₀ = 0.14 μg L⁻¹ in direct ELISA and was evaluated using solvent extracted SEM-spiked porcine and baby food samples. Recovery levels determined from fortified samples (0.5, 1.0, 1.5, 5, 10 and 20 μg kg⁻¹) of porcine and baby food ranged from 82.9 to 105.3%, respectively, with a coefficient of variation less than 15.5%. Respective detection capability and threshold of the assay for porcine muscle, set on the basis of acceptance of no false negative results, was 0.3 and 0.11 μg kg⁻¹.     

Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan

An ELISA was developed for routine examination for extensive monitoring and screening programs for the residues of salbutamol in swine serum, animal feed, meat, and meat-related products destined for human consumption in Taiwan. Objectives of the study were to investigate the use of a new immunoassay for the detection of salbutamol residues in swine meat and animal feed samples, and to compare with a commercial kit in field test screens. A fast, simple and reliable sample preparation method for the determination of salbutamol was established. Field trials with 222 swine meat and 120 animal feed samples that were taken from local meat markets, auction markets and feed mills. The application and the results of two ELISA kits (a homemade and a commercial kit) for the screening of salbutamol were presented. Adopting 2 μg kg⁻¹ salbutamol as a cut-off value for swine meat, the commercial β-agonist ELISA had a sensitivity of 85.3% and a specificity of 95.2% versus GC-MS at a cut-off of 2 μg kg⁻¹. The homemade salbutamol ELISA had a sensitivity of 100% and a specificity of 90.9% and gave no false-negative rate results. Furthermore, adopting 20 μg kg⁻¹ salbutamol as a cut-off value for animal feed, both the commercial and homemade ELISA showed 100% sensitivity and 100% specificity of the assays. In conclusion, a sensitive, specific salbutamol polyclonal antibody-based ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of β-agonists.     

Monitoring of streptomycin and dihydrostreptomycin residual levels in porcine meat press juice and muscle via solid-phase fluorescence immunoassay and confirmatory analysis by liquid chromatography after post-column derivatization

A solid-phase fluorescence immunoassay (SPFIA) that was primarily developed for detection of antibiotic residues in milk was qualitatively applied for the pre-screening of the residues of aminoglycoside antibiotics, streptomycin and dihydrostreptomycin, in meat press juice. The confirmation of both analytes was performed using a validated method of highperformance liquid chromatography with post-column derivatization. The analytical performance was demonstrated by the analysis of pork meat samples spiked at three concentration levels, ranging from 0.25 to 2.5 ppm for each analyte. In general, the recoveries ranged from 80.4 to 81.5% and from 79.6 to 84.4% for streptomycin and dihydrostreptomycin, respectively, with relative standard deviations lower than 6%. The limits of detection were 0.1 and 0.15 ppm for streptomycin and dihydrostreptomycin, respectively, and the limits of quantification of 0.35 and 0.5 ppm are below the maximum residue limits of Codex, the European Union, and the Korean Food and Drug Administration (ranging from 0.5 to 0.6 ppm). Eight real samples collected from the Seoul area were first monitored using SPFIA, and none of them were found positive. These findings are in good accordance with those observed by HPLC analysis. To the best of our knowledge, this is the first report to monitor the aminoglycoside residues in pork meat press juice using SPFIA.     

Development of an optical surface plasmon resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry meat

The aim of this study was to develop an optical biosensor inhibition immunoassay, based on the surface plasmon resonance (SPR) principle, for use as a screening test for 13 (fluoro)quinolones, including flumequine, used as veterinary drugs in food-producing animals. For this, we immobilised various quinolone derivatives on the sensor chip and tested binding of a range of different antibodies (polyclonal and one engineered antibody) in the presence and absence of free (fluoro)quinolones. The main challenge was to detect flumequine in an assay giving good results for the other compounds. One antigen–antibody combination proved satisfactory: polyclonal antibodies raised against a dual immunogen and, on the sensor chip, a fluoroquinolone derivative. It was the first time that this concept of the bi-active antibody was described in the literature.The assay, optimised for detection in three matrices (poultry muscle, fish, and egg), was tested on incurred samples prepared by liquid extraction followed by two washing steps. This rapid, simple method proved adequate for detecting at least 13 (fluoro)quinolones at concentrations below established maximum residue levels (MRLs). The reference molecule norfloxacin could be detected in the range of 0.1–10 μg kg⁻¹ in extracts of egg and poultry meat and in the range of 0.1–100 μg kg⁻¹ in extracts of fish. The determined midpoints of these calibration curves were about 1, 1.5 and 3 μg kg⁻¹ in poultry meat, egg and fish, respectively.     

Pre-column derivatization of sulfa drugs with fluorescamine and high-performance liquid chromatographic determination at their residual levels in meat and meat products.

A rapid, sensitive and selective high-performance liquid chromatographic method is described for simultaneous determination of eight sulfa drugs in meat and meat products using pre-column derivatization with fluorescamine. The drugs are sulfisomidine, sulfadiazine, sulfamerazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline. The method includes blender extraction of 3-g samples with chloroform, partition with 3 M hydrochloric acid, derivatization with fluorescamine at pH 3.0 and subsequent high-performance liquid chromatographic analysis on a C18 column with fluorescence detection at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. The drugs were separated with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C. The average recovery from samples fortified at 0.1 ng/g was 92.6% with a coefficient of variation of 6.2%. The detection limit was 0.01 ng/g for sulfaquinoxaline and 0.005 ng/g for the other seven drugs. The method was field-tested in a survey of 37 samples including beef (five), pork (seven), chicken (seven), ham (five), sausage (eight), bacon (two) and roast beef (three). Sulfadimidine was detected in one pork sample at the level of 0.295 ng/g and in ham at 0.178 ng/g.     

基于液相色谱-串联质谱法的肉类特征肽段鉴别及掺假测定

建立了液相色谱-串联质谱技术鉴别肉类特征肽段及定量检测羊肉中常见外源肉掺假的方法。样品经蛋白质提取、胰蛋白酶水解和固相萃取小柱净化后,利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）和Proteinpilot软件,实现蛋白质和多肽的鉴定;再通过基本局部比对搜索工具（BLAST）与Uniprot数据库对比分析,筛选出羊肉、鸭肉、猪肉和鸡肉的20个物种特征性多肽标志物;最后利用高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）系统对羊肉、鸭肉、猪肉和鸡肉的特征性多肽进行了验证和多反应监测（MRM）定量研究。将鸭肉、猪肉和鸡肉分别按照质量分数为1%、5%、10%、20%、50%的比例掺加到羊肉中,得到鸭肉最低掺假检出限为0.25%、猪肉最低掺假检出限为0.17%、鸡肉最低掺假检出限为0.10%。     

Multiresidue chromatographic method for the determination of macrolide residues in muscle by high-performance liquid chromatography with UV detection.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of tilmicosin, tylosin, spiramycin, and its major metabolite neospiramycin was developed that is suitable for porcine, bovine, and poultry muscles. Macrolide residues were extracted from muscle with acetonitrile, fat was removed by liquid-liquid extraction with isooctane, and the extract was then cleaned on Bond Elut C18 cartridges. The HPLC separation was performed on an Inertsil ODS3 C18 column (150 x 4 mm) with 0.05% trifluoroacetic acid-acetonitrile in a gradient mode. Two different chromatographic gradients were used for tilmicosin-tylosin and spiramycin-neospiramycin, and the detection wavelengths were 287 and 232 nm, respectively. The method was validated from 1/2 the maximum residue limit (MRL) to 4 times the MRL with pork muscle samples. Mean recoveries were 60, 63.5, 51, and 42% for tilmicosin, tylosin, spiramycin, and neospiramycin, respectively. The detection limits are 15 micrograms/kg for tilmicosin and tylosin, 30 micrograms/kg for spiramycin, and 25 micrograms/kg for neospiramycin. Linearity, precision, and accuracy of the method were also tested.     

Application of ion-exchange cartridge clean-up in food analysis. II. Determination of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in meat using liquid chromatography with ultraviolet detection.

A multiresidue analytical method was developed for the simultaneous determination of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in meat. The method involves the use of an ion-exchange cartridge for sample clean-up followed by ion-pair high-performance liquid chromatography with ultraviolet detection. The recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from pork muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg were in the range of 77-90, 73-95 and 80-93% with coefficients of variation of 0.5-1.7, 1.6-4.4 and 3.2-6.6%, respectively. For beef muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg, the recoveries of these compounds were 83-92, 71-86 and 77-90% with coefficients of variation of 1.7-4.4, 2.6-7.0 and 3.9-6.4%, respectively. The detection limits for each penicillin were 0.02 mg/kg in meat.     

Mechanistic aspects of electrochemical hydride generation for cadmium

A reversed-phase chromatographic method has been developed and optimised in order to detect and quantitate soybean proteins in commercial heat-processed meat products. The optimised conditions consisted of a linear binary gradient tetrahydrofurane-water-0.05% trifluoroacetic acid at a flow rate of 1 mL/min. Meat products were defatted with acetone and soybean proteins were extracted with a buffered solution at pH 9.60. The injection of this extract into the chromatographic system enabled the detection of soybean proteins in heat-processed meat products in about 12 min. The method enabled the detection and quantitation of additions of 0.38% (w/w) and 0.63% (w/w), respectively, of soybean proteins (related to 10 g of initial product). The method has been proven to be precise with relative standard deviations (R.S.D.) for repeatability, intermediate precision, and internal reproducibility lower to 7.0%. Recoveries obtained for spiked meat products were close to 100% and no matrix interferences were observed. The application of the method to commercial heat-processed meat products in whose formulation soybean proteins were present yielded soybean protein contents ranging from 0.90% to 1.54%, below the maximum levels established by regulations.