Development of a Monoclonal Antibody-Based Immunochromatographic Assay Detecting Ractopamine Residues in Swine Urine

A lateral-flow immunochromatographic assay (LFIA) using monoclonal antibody was developed for the rapid detection of ractopamine residues in swine urine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC₅₀) of the lateral-flow immunochromatographic test strip under an optical density scanner were calculated to be 0.1?±?0.013 and 0.71?±?0.056 ng/mL, respectively. The cut-off level was observed with the naked eye of 1 ng/mL for detection of ractopamine residue in swine urine. Parallel analysis of swine urine samples with ractopamine showed comparable results obtained from the LFIA and LC-MS/MS. Therefore, the described lateral-flow test strip can be used as a reliable, rapid and cost-effective on-site screening technique for the determination of ractopamine residues in swine urine.     

Simultaneous separate and group determination of tylosin and tilmicosin in foodstuffs using single antibody-based immunoassay

An approach to modify the specificity of competitive indirect ELISA for determination of veterinary macrolides tylosin (TYL) and tilmicosin (TMN) using different structural design of coating antigen is described. The homologous and heterologous assay formats using rabbit antiserum against BSA-TYL conjugate were developed. The first format allowed selective determination of TYL (cross-reactivity for TMN was negligible). Heterologous hapten desmycosin (DMN) based conjugate being immobilised on the plates changed the specificity to group recognition (cross-reactivity for TMN was 103.4%). Using two coating antigens but single antibody and the same TYL standard solutions the described tandem test was capable for simultaneous determination of the respective analytes with their possible differentiation. The detection limit of assay that was far below the current MRLs and achieved 0.07 ng mL⁻¹ for TYL and 0.14 ng mL⁻¹ for TMN allowed to minimise matrix effect by sample dilution. The ELISAs of antibiotics in milk, eggs, honey and chicken muscle were optimised and showed acceptable recovery rate. A range of foodstuff samples was analysed using the developed tandem test.     

Determination of sulphonamide residues in cattle meats by the Charm-II system and validation with high performance liquid chromatography with fluorescence detection

The purpose of this study was determination of sulphonamide residues (sulphanilamide, sulphadiazine, sulphathiazole, sulphamerazine, sulphamethazine, sulphamethoxazole, and sulphadimethoxine) in cattle meat by the Charm II technique and the validation of sulphonamide levels by high performance liquid chromatography with fluorescence detector (HPLC-FLD). Of 157 meat samples, 9 samples (5.73%) were found positive by the Charm II method. To make quantitative confirmation of sulphonamide content of positive samples, HPLC-FLD was used and four samples were confirmed as positive. In HPLC analysis, the limit of detection (LOD) was in the range 8–15 μg/kg and the limit of quantification (LOQ) was 13–25 μg/kg. Average recoveries of sulphonamides ranged from 44.6% to 81% with relative standard deviations below 6% (n = 6). In conclusion, we consider that the results obtained in field screening by only using the Charm II system, as is common practice in Turkey and worldwide, are inadequate and thus the results should be confirmed by sensitive systems like HPLC.     

Detection of adulterants such as sweeteners materials in honey using near-infrared spectroscopy and chemometrics

Near-infrared (NIR) spectroscopy combined with chemometrics methods has been used to detect adulteration of honey samples. The sample set contained 135 spectra of authentic (n = 68) and adulterated (n = 67) honey samples. Spectral data were compressed using wavelet transformation (WT) and principal component analysis (PCA), respectively. In this paper, five classification modeling methods including least square support vector machine (LS-SVM), support vector machine (SVM), back propagation artificial neural network (BP-ANN), linear discriminant analysis (LDA), and K-nearest neighbors (KNN) were adopted to correctly classify pure and adulterated honey samples. WT proved more effective than PCA, as a means for variables selection. Best classification models were achieved with LS-SVM. A total accuracy of 95.1% and the area under the receiver operating characteristic curves (AUC) of 0.952 for test set were obtained by LS-SVM. The results showed that WT-LS-SVM can be as a rapid screening technique for detection of this type of honey adulteration with good accuracy and better generalization.     

Evaluation of antioxidant activity and total phenolics of selected Czech honeys

Honey serves as a good source of natural antioxidants, which are effective in reducing the risk occurrence of heart disease, cancer, cataracts, different inflammatory processes and immune-system decline. In the fresh selected Czech honey samples originated mainly from the region North Moravia antioxidant activity and total polyphenol content were determined. A total of 40 honey samples (multifloral, lime, rape, raspberry, mixture and honeydew honeys) native to different stations gained in the period from May by August year 2006 were analysed. Total phenolics (TP) content was determined by the modified Folin-Ciocalteau method [TP was expressed as mg of gallic acid equivalent (GA eq.) per kg of honey]. For evaluation of the antioxidant activity (AOA) three different methods were used, the ferric reducing antioxidant power (FRAP) assay, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and the 2,2′-azinobis(3-ethylbenzothiazolin)-6-sulfphonate (ABTS) assay. AOA was expressed in mg ascorbic acid equivalent (AA eq.) per kg of honey. The results indicated that TP and AOA in Czech honey varied greatly between the honey kinds, location and time of the harvest. Average TP ranged from 89.9 mg GA eq. kg^-1 in lime honey to 215.2 mg GA eq. kg⁻¹ in honeydew honey. Antioxidant activity determined by the DPPH, ABTS and FRAP methods was lowest in floral honeys. The highest values were obtained for honeydew and mixture honeys. ABTS and FRAP assays have been shown to be the optimal methods for AOA determination in honey. A positive linear correlation between AOA and TP was observed (in FRAP assay R² = 0.852). It indicates that phenolics are one of the main components responsible for antioxidant behaviour of honey. The obtained results support and extend complete knowledge about the content of bioactive phenolics and antioxidant activity in the Czech honeys.     

双甲脒胶体金免疫快速检测试纸条研制及其在蜂蜜中的应用

目的 基于胶体金免疫层析原理研制双甲脒快速检测试纸条,评价其在蜂蜜检测中的应用效果。方法 使用双甲脒-BSA偶联物作为人工抗原免疫小鼠获得单克隆抗体,制备胶体金微孔试剂,使用双甲脒-OVA偶联物和羊抗鼠IgG抗体分别作为试纸条的检测线和控制线,并对蜂蜜样品进行添加检测。结果研制的试纸条可以定性检测蜂蜜中双甲脒的残留,对蜂蜜中双甲脒的检测限为0.2mg/kg,检测用时约25min,,与双甲脒功能和结构相似的其他药物时无交叉反应,特异性较好。结论 本试验研制的试纸条适用于蜂场现场快速检测以及基层实验室中大量样本的筛查,为蜂蜜中抗生素残留控制提供有效监管手段。     

Evaluation of the phenolic content,antioxidant activity and colour of Slovenian honey

Honey samples from the seven most common honey types in Slovenia were screened for total phenolic content by the modified Folin–Ciocalteu method, for potential antioxidant activity using the ferric reducing antioxidant power (FRAP) assay and by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method for antiradical activity. In addition the colour characteristics of honey samples were analysed. The results of the study showed that total phenolic content, antioxidant activity and colour parameters differ widely among different honey types. Phenolic content expressed as gallic acid equivalent ranged from 44.8 mg/kg in acacia honey to 241.4 mg/kg in fir honey. Antioxidant activity was the lowest in the brightest acacia and lime honeys and the highest in darker honeys, namely fir, spruce and forest. The colour of the Slovenian honeys, analysed in this study was very variable and ranged from pale yellow to dark brown. Correlations between the parameters analysed were found to be statistically significant (p < 0.05).     

Hydroxymethylfurfuraldehyde and amylase contents in Australian honey

The quality of Australian honey samples (processed and unprocessed) was assessed using HPLC techniques. 5-Hydroxymethylfurfuraldehyde (HMF) was used as the main quality indicator. Sampling included four commercially-processed honeys (Australian rainforest, Beechworth, Homebrand and Leabrook) and three unprocessed (Banksia, Grey box and Mallee). All honey samples, except Leabrook and Beechworth, showed an initial HMF content less than the Codex Alimentarius and International Honey Commission standard (40 mg/kg). HMF contents in Leabrook and Beechworth were 50.8 ± 1.34 and 74.9 ± 2.34 mg/kg, respectively. Heating unprocessed honey at 85 °C for 2 min caused significant (p ? 0.05) increment in HMF contents. The amounts of HMF in Mallee samples increased from 34.0 ± 0.31 to 42.3 ± 0.37 mg/kg after 2 min at 85 °C. All honey samples showed amylase activity above the minimum limit (8 Gothes). The physiochemical properties of honey showed significant variations among samples. The results revealed also that heating was not the only factor influencing HMF formation in honey, but also honey composition, pH value and floral source can contribute to these variations. Consequently, the amount of HMF may be an insufficient sole indicator of honey quality.     

Raw Millefiori honey is packed full of antioxidants

Total polyphenols, flavonoids and antioxidant power of raw honey samples from two of the most common Italian varieties, i.e., Millefiori and Acacia, were evaluated. Phenolic content, expressed as caffeic acid equivalents, ranged from 12.5 to 17.5 mg/100 g and from 3 to 11 mg/100 g in Millefiori and Acacia honeys, respectively. All Millefiori samples exhibited the highest flavonoid concentration being between 1.23 and 2.93 mg catechin equivalents (CE)/100 g honey. Total flavonoids in 100 g Acacia honeys were in the range of 0.45–1.01 mg CE. Acacia honeys had lower total antioxidant power, as assessed by ferric reducing/antioxidant power assay, than Millefiori. The relationship between phenolic content and antioxidant power was discussed. Comparative experimental analysis was performed with an artificial honey and processed honeys. Raw Millefiori honey is rich in both amount and variety of antioxidant substances, and its inclusion in the diet may be recommended to complement other polyphenol sources.     

Contribution to the characterisation of honey-based Sardinian product abbamele: Volatile aroma composition,honey marker compounds and antioxidant activity

Sardinian abbamele is a typical product obtained from the honey recuperation from combs (traditional procedure) or by concentration of the honey diluted in water (industrial procedure). Seven abbamele samples were obtained to study the volatiles’ composition, the presence of honey marker compounds and their relationship with the production procedures. The long thermal treatment applied in abbamele production caused very high (1007.0–4405.8 mg/kg) HMF content (HPLC-DAD), while glucose and fructose amounts were quite similar to the honey ones (HPLC-RI). Total antioxidant activity (FRAP assay) of the samples ranged between 13.3 and 71.2 mmol Fe²⁺/kg, while antiradical activity (DPPH assay) ranged between 3.8 and 23.3 mmol TEAC/kg. Such high antioxidant values were linearly correlated with total phenol amount (1297.8–4469.5 mg GAE/kg) determined by Folin–Ciocalteau method. Thermally derived furan derivatives and terpenes were abundant among the headspace volatiles (HS-SPME), particularly limonene (0.5–76.0%) that probably originated from citrus rinds’ addition during abbamele production. GC and GC–MS analyses of USE isolates revealed HMF predominance as well as the honey marker compounds (if/when existing) such as methyl syringate (up to 49.2%), marker of Asphodelus microcarpus honey. High isophorone percentage (up to 30.9%) determined by HS-SPME followed by minor percentage of 4-ketoisophorone and norisoprenoids in one sample indicated Arbutus unedo L. honey use in the production. HPLC-DAD analysis confirmed the presence of specific honey markers: two samples showed high methyl syringate concentrations (150.4–120.1 mg/kg) while homogentisic acid and other specific markers of A. unedo honey were found in one sample. The compared GC–MS and HPLC-DAD data proved to be useful to obtain information about the use of specific honey in the production and to verify citrus addition.     

Flavonoid pattern of sage (Salvia officinalis L.) unifloral honey

The aim of the present paper was to determine the flavonoids in monofloral sage (Salvia officinalis L.) honey which is characteristic and specific for the area of Croatian coast and islands. For that purpose 38 sage honey samples from two production seasons were analysed. After specific pollen content determination, and analyses of selected physicochemical parameters which confirmed that samples are in compliance with national and international regulations and can be regarded as unifloral sage honeys, flavonoid fraction was isolated and analysed using RP-HPLC/DAD method. The HPLC analysis showed that all examined sage honey samples contain quercetin (3,3′,4′,5,7-pentahydroxyflavone), luteolin (3′,4′,5,7-tetrahydroxyflavone), kaempferol (3,4′,5,7-tetrahydroxyflavone), apigenin (4′,5,7-trihydroxyflavone), chrysin (5,7-dihydroxyflavone) and galangin (3,5,7-trihydroxyflavone), as well as p-coumaric (trans-4-hydroxycinnamic acid) and caffeic acid (3,4-dihydroxycinnamic acid). Total amount of identified flavonoids varied from 109.4 μg/100 g of honey to 589.9 μg/100 g of honey, with the average of 288.5 μg/100 g of honey. All analysed honey samples showed common and specific flavonoid profile which could be the basis for differentiating sage from other monofloral honeys.     

Simultaneous determination of four phenolic components in citrus honey by high performance liquid chromatography using electrochemical detection

A sensitive and accurate method for simultaneous separation and determination of four phenolic compounds, including caffeic acid, p-coumaric acid, ferulic acid, and hesperetin in Chinese citrus honey by high performance liquid chromatography using electrochemical detection (HPLC-ECD) has been established. Chromatographic separation was performed using a reversed phase column and methanol/4% (v/v) aqueous acetic acid as the mobile phase. The detection and quantification limits of the four compounds with ECD were 6–14 times greater than those obtained with diode-array detection (DAD). All calibration curves of the four phenolic compounds showed good linearity (r ? 0.9994) within the test ranges, 1.10–66 μg/ml, 0.35–70 μg/ml, 0.16–16 μg/ml and 0.03–10 μg/ml, respectively. The recoveries ranged from 98.9% to 100.3%. The extraction process was very simple, because of the dissolution of honey only involving water. Taken together, the application of ECD in honey determination leads to a significant improvement in the quantification of phenolic compounds, whereby paying the way for the establishment of a better quality control of citrus honey.     

Development of a fast MECK method for determination of 5-HMF in honey samples

In this study, 5-hydroxymethylfurfural (5-HMF) determination was carried out by a micellar electrokinetic capillary chromatography (MEKC) methodology, using caffeine as the internal standard (IS). The optimisation of the electrolyte composition was approached using a 3² full factorial design with a central point to study the MEKC electrolyte components. Inspection of the response surface indicated that the optimal electrolyte composition was 5 mmol L⁻¹ sodium tetraborate (STB, pH 9.3) containing 120 mmol L⁻¹ sodium dodecyl sulphate (SDS). Under optimal CE conditions, separation of the investigated substance was achieved in less than 0.7 min. Quality parameters, such as linearity (R² > 0.99), precision (RSD < 5.41%), detection and quantification limits (3.37 and 11.24 mg kg⁻¹ for honey samples) and recovery (96.37–99.56%). The proposed methodology was successfully applied to the analysis of 5-HMF in honey samples. The analytical performance of this method makes it suitable for implementation in food laboratories for the routine determination of 5-HMF in honey samples.     

Development of nanocolloidal gold based immunochromatographic assay for rapid detection of transgenic vegetative insecticidal protein in genetically modified crops

Vegetative insecticidal protein (Vip) is now being used for transgenic expression in several crops; conferring resistance against lepidopteron pests. A rapid, single step, sensitive and specific immunochromatographic (IC) strip test for the detection of recombinant Vip-S protein in the transgenic samples was developed. Polyclonal rabbit anti-Vip-S IgG conjugated to nanocolloidal gold served as a probe to detect Vip protein in test samples. The detection limit for the developed IC strip was 100 ng/ml (100 ppb) and on addition of gold enhancer the sensitivity increased to 1 ng/ml (1 ppb) of Vip-S protein. The assay was validated with transgenic brinjal samples. The assay time was less than 10 min, suitable for rapid on-site testing. No cross-reactivity was observed with other transgenic plant proteins employed for pest and weed management, i.e. Cry1Ac, Cry1Ab, and CP4-EPSPS. This on-site test offers rapid screening for a genetically modified crops having relatively new transgene (vip) entering the global market.     

Performance characteristics of the Duopath® cereus enterotoxins assay for rapid detection of enterotoxinogenic Bacillus cereus strains

The Duopath® Cereus Enterotoxins test (Merck KGaA) is a newly developed gold-labeled lateral flow immunoassay for the detection of Bacillus cereus enterotoxins. The test uses monoclonal antibodies (MAbs) against the L₂ component of hemolysin BL (Hbl) and NheB of the non-hemolytic enterotoxin (Nhe), respectively. The inclusivity and exclusivity of the assay was tested using 44 B. cereus, B. cereus group and Bacillus spp. strains. Apart from the B. mycoides type strain the results were in full agreement with those obtained by other immunological and molecular biological methods. The detection limit of the assay was 6 ng/ml for NheB and 20 ng/ml for the Hbl-L₂-component, respectively. Using artificially and naturally contaminated food samples (n = 76) the assay was positive after 18-24 h enrichment if at least 10² enterotoxin producing B. cereus/g were present. After 30 h enrichment samples contaminated with as low as 1 enterotoxin producing B. cereus/g gave positive results. In addition, testing of suspected colonies for enterotoxin production is possible. The assay is easy to perform and results can be clearly read without instrumentation.     

Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle,chicken muscle,egg, fish,milk and kidney

A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1 μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5 μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10 μg/kg in kidney and 20 μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015 μg/kg, and the detection limits were 1.5 μg/kg in pig muscle and 6 μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r²) which was greater than 0.9.     

Analysis of multi-pesticide residues in the foods of animal origin by GC–MS coupled with accelerated solvent extraction and gel permeation chromatography cleanup

A new analytical method was developed to simultaneously determine residues of 109 pesticides (including isomers) in the foods of animal origin. Acetonitrile was selected for accelerated solvent extraction (ASE) for effectively extracting the pesticides from the fatty samples. The cleanup was performed with an automated gel permeation chromatography (GPC) cleanup system. The prepared samples were analysed with GC–MS in the selected ion monitoring mode (SIM) using one target and two qualitative ions for each analyte. Chlorpyrifos-d₁₀ was used as an internal standard. The lowest limit of detection was 0.3 μg kg⁻¹ for some pesticides. The recoveries and relative standard deviations (RSDs) were checked by spiking untreated samples with pesticides at 0.05, 0.1 and 0.2 mg kg⁻¹. The average recoveries of most pesticides were from 62.6% to 107.8%. The precision values expressed as RSD were all ?20.5% (n = 6). Good linearity (r ? 0.99) was observed between 0.05 and 1.5 μg mL⁻¹.     

Dispersive liquid–liquid microextraction for the determination of organochlorine pesticides residues in honey by gas chromatography-electron capture and ion trap mass spectrometric detection

A simple dispersive liquid–liquid microextraction (DLLME) protocol for the determination of 15 organochlorine pesticides residues in honey is proposed. The selected pesticides were separated using gas chromatography and detected by electron capture (ECD) or ion trap mass spectrometry (GC-IT/MS). Several parameters affecting the extraction efficiency namely type and volume of organic extraction solvent, type and volume of disperser solvent, sample pH, ionic strength, extraction time and centrifugation speed were systematically investigated. The final DLLME protocol involved the addition of 750 μL acetonitrile (disperser) and 50 μL chloroform (extraction solvent) into a 5 mL aqueous honey solution followed by centrifugation. The sedimented organic phase (chloroform) were analysed directly by GC-IT/MS or evaporated and reconstituted in acetonitrile prior to the GC-ECD analysis. The analytical performance of the GC-ECD and GC-IT/MS methods was compared and discussed. Under the selected experimental conditions, the enrichment factors varied between of 36 and 114. The limits of detection (LOD) were in the range of 0.02–0.15 μg L⁻¹ (0.4–3 ng g⁻¹) for GC-ECD and 0.01–0.2 μg L⁻¹ (0.2–4 ng g⁻¹) for GC-IT/MS which is adequate to verify compliance of products to legal tolerances. The proposed method was applied to the analysis of the selected organochlorine pesticides residues in various honey samples obtained from Greek region. Mean recoveries were ranged from 75% to 119% while the precision was better than 20% in both methodologies.     

The antioxidant activity of teas measured by the FRAP method adapted to the FIA system: Optimising the conditions using the response surface methodology

This study proposes a FRAP assay adapted to FIA system with a merging zones configuration. The FIA system conditions were optimised with the response surface methodology using the central composite rotatable design. The optimisation parameters studied were: the carrier flow rate, the lengths of the sample and reagent loops, and reactor length. The conditions selected in accordance with the results were: carrier flow rate of 1.00 ml/min, length of the loops 18.2 cm and length of the reaction coil 210.1 cm. The detection and quantification limits were, respectively, 28.6 and 86.8 μmol/l Fe²⁺, and the precision was 1.27%. The proposed method had an analytical frequency of 30 samples/h and about 95% less volume of FRAP reagent was consumed. The FRAP assay adapted to the FIA system under the optimised conditions was utilised to determine the antioxidant activity of tea samples.     

Residues of antibiotics and sulfonamides in honeys from Basque Country (NE Spain)

BACKGROUND: The aim of this work was to ensure that Label Basque market honey is free of veterinary residues. RESULTS: A total of 567 Basque honey samples were previously analyzed with the respective Charm II system—68 samples were presumptive positive for sulfonamides (SA‐s), 24 samples for tetracyclines (TC‐s), and no positive samples for chloramphenicol (CAP) (<0.3 µg kg^?1) residues. The residues were mostly confirmed by liquid chromatography fluorescence detection (LC‐FD) and tandem mass spectrometry (LC‐MS/MS), according to the latest European Union criteria for the analyses of veterinary drug residues (2002/657/EC). These techniques confirmed that 19 of the 68 samples, presumptive contaminated with SA‐s, contained sulfathiazole (STZ) residues at levels from 20 to 210 µg kg^?1, and the 24 samples presumptive contaminated with TC‐s, were also confirmed, showing tetracycline (TC) levels from 15 to 920 µg kg^?1. Linearity range, decision limit (CCα), detection capability (CCβ), precision and reproducibility were also determined. CONCLUSION: Residues of veterinary drugs were confirmed in a very limited number of honey samples: sulfathiazole (3.40%) and tetracycline (4.22%). This work reports the advantages of the Charm II assay, but also its limitations, detecting SA‐s in most (87.7%) of the heather (Erica vagans) honey samples. The false positives detected in this honey were assumed to be of an unknown compound that has not been confirmed as a drug residue. Until now, no studies have been performed to find out if other heather honeys of different geographical origins give similar false positives for SA‐s. Copyright © 2008 Society of Chemical Industry