Development of an enzyme-linked immunosorbent assay based a monoclonal antibody for the detection of pyrethroids with phenoxybenzene multiresidue in river water

A sensitive and broad class selective direct competitive enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (McAb) has been described for the detection of pyrethroids with phenoxybenzene group. One monoclonal antibody, 2G(2)E(7), was obtained and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice. The assay with the most selectivity for the family pyrethroids with phenoxybenzene group was optimized. The IC(50) values of the optimized immunoassay were 1.8 μg L(-1) for deltamethrin, 1.5 μg L(-1) for cypermethrin, 2.0 μg L(-1) for fluvalinate and fenvalerate, 2.2 μg L(-1) for phenothrin, 2.4 μg L(-1) for flucythrinate, 3.0 μg L(-1) for fenpropathrin, and 5.0 μg L(-1) for permethrin. River water samples fortified with pyrethroids were analyzed with the ELISA to evaluate the accuracy of the assay. The recoveries of pyrethroids in spiked water samples ranged from 74 to 108%. The results indicate that the ELISA developed can accurately simultaneously determine pyrethroids with phenoxybenzene group in water samples.     

A novel green chemistry method for nonaqueous extraction and high-performance liquid chromatography detection of first-, second-, and third-generation tetracyclines, 4-epitetracycline, and tylosin in animal feeds

Although tetracyclines and macrolides are common additives for animal nutrition, methods for their simultaneous determination in animal feeds are nonexistent. By coupling an organic extraction and solid-phase extraction cleanup to a high-performance liquid chromatography separation and a nonaqueous postcolumn derivatization, we succeeded in detecting from 0.2 to 24.0 μg kg(-1) of tetracycline, oxytetracycline, chlortetracycline, doxycycline, tigecycline, and 4-epitetracycline in this complex and heterogeneous matrix. Minocycline and tylosin could also be detected with our procedure, but using UV spectrophotometry (1.5 ≤ LOD ≤ 1.9 mg kg(-1)). Linear responses with correlation coefficients between 0.996 and 0.999 were obtained for all analytes in the 0.5-10 mg kg(-1) concentration range. Average recoveries between 59 and 97% and between 98 and 102% were obtained for the tetracyclines and tylosin, respectively. Replicate standard deviations were typically below 5%. When this method was applied to 20 feeds marketed in Costa Rica, we detected labeling inconsistencies, banned mixtures of tetracyclines, and tetracycline concentrations that contravene international regulation.     

Residue depletion of tilmicosin in chicken tissues

A high-performance liquid chromatography (HPLC) method with detection at 290 nm was modified and validated for the determination of tilmicosin residues in broiler chicken tissues. The limits of detection (LOD) of the method were 0.01 microg/g for muscle and 0.025 microg/g for liver and kidney. Average recoveries ranged from 80.4 to 88.3%. Relative standard deviation values ranged from 5.2 to 12.1%. Residue depletion of tilmicosin in broiler chickens was examined after dosing over a 5-day period by incorporation of the drug into drinking water at 37.5 and 75.0 mg/L. Tilmicosin concentrations in liver and kidney were highest on day 3 of medication and on day 5 in muscle, in both low- and high-dose groups. The residue levels in both groups were significantly higher in liver than in kidney or muscle. A minimum withdrawal time of 9 days was indicated for residue levels in muscle, liver, and kidney tissues below the maximum residue level (MRL).     

Development of an immunoaffinity chromatography purification and ultra performance liquid chromatography tandem mass spectrometry method for determination of 12 sulfonamides in beef and milk

A highly selective and sensitive method was developed for the simultaneous determination of 12 sulfonamides in beef and milk by immunoaffinity chromatography purification coupled to ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The MS/MS conditions, UPLC mobile phase, injection solution, sample purification process, and matrix effect were studied to optimize the operating conditions. The limits of detection (LODs) of the instrument for the studied sulfonamides ranged from 0.4 to 2.0 μg L(-1), being 1.6-8.0 μg kg(-1) for beef and 1.8-6.4 μg kg(-1) for milk. The standard solution was diluted with blank beef or milk matrix for the construction of calibration curves, which had a linear range from 10 to 200 μg kg(-1) and regression coefficients higher than 0.990 (n=10) for all the studied sulfonamides. Samples spiked at 10, 20, and 100 μg kg(-1) showed recoveries above 70% and relative standard deviations below 10%.     

Determination of multiresidues in rapeseed, rapeseed oil, and rapeseed meal by acetonitrile extraction, low-temperature cleanup, and detection by liquid chromatography with tandem mass spectrometry

A multiresidue method for determining pesticides in rapeseed, rapeseed oil, and rapeseed meal by use of liquid chromatography-tandem mass spectrometry is developed. Samples were extracted with acetonitrile or acidified acetonitrile and cleaned up by a 12 h freezing step. The recovery data were obtained by spiking blank samples at three concentration levels. The recoveries of 27 selected pesticides in rapeseed, rapeseed oil, and rapeseed meal were in the range of 70-118%, at the concentration level of 10 μg kg(-1), with intraday and interday precisions of lower than 22 and 27%, respectively. Linearity was studied between 2 and 500 μg L(-1) with determination coefficients (R(2)) of higher than 0.98 for all compounds in the three matrices. The limits of quantitation (LOQs) of pesticides in rapeseed, rapeseed oil, and rapeseed meal ranged from 0.3 to 18 μg kg(-1). The n-octanol-water partition coefficient showed more influence than water solubility in extracting pesticides by acetonitrile from matrices of high fat content. This method was successfully applied for routine analysis in commercial products.     

Production and characterization of monoclonal antibodies against the antibiotic tilmicosin

Monoclonal antibodies (Mabs) were developed that specifically bind tilmicosin. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) conjugates were used for the immunogen and plate coating antigen, respectively. The conjugates were synthesized by different methods, resulting in different linkages. Six hybridoma cell lines were isolated that produced Mabs that competed with tilmicosin, and have IgG1 isotype. The Til-1 and Til-5 Mabs had IC50 values for tilmicosin of 9.6 and 6.4 ng/well (48 and 32 ng/mL), respectively, and limits of detection at IC20 of 1.84 and 0.89 ng/well (9.2 and 4.45 ng/mL), respectively. The Mabs demonstrated high cross-reactivity to the macrolides containing 3,5-dimethylpiperidine at C20 and the amino sugar at C5. No cross-reactivity was observed for tylosin and other macrolides that did not contain 3,5-dimethylpiperidine. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the antibiotic tilmicosin by use of the developed Mabs. These Mabs may be excellent candidates for the determination and immunolocalization of tilmicosin.     

Determination of five macrolide antibiotic residues in eggs using liquid chromatography/electrospray ionization tandem mass spectrometry

A method using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of trace levels of five macrolide antibiotics (spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin) in eggs is presented. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity for both quantification and confirmation. Matrix-matched standard calibration curves were used to achieve the best accuracy of the method. A fully nested experimental design was used to study the measurement uncertainty arising from intermediate precision and trueness or proportional bias. The overall recoveries, that is, those determined by the nested experiments, of spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin at fortified levels of 60, 100, 200, and 300 microg/kg were 96.8, 98.2, 98.3, 98.8, and 95.4%, respectively. The LC/ESI-MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <1.0 microg/kg.     

Development of an indirect competitive ELISA for the detection of furazolidone marker residue in animal edible tissues

Due to its carcinogenicity and mutagenicity, furazolidone has been prohibited completely from being used in food animal production in the world since 1995. To monitor the illegal abuse of furazolidone, a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the determination of tissue-bound furazolidone metabolite 3-amino-2-oxazolidone (AOZ). The highly specific antibody was targeted for PAOZ, the benzaldehyde derivative of AOZ. The 50% inhibition values (IC 50) of 0.91 microg/L for AOZ was achieved with the most sensitive antibody Ab-B1 by altering ELISA conditions. In the ELISA, sample extraction and cleanup were performed by an is MAX cartridge following combined hydrolysis of the tissue-bound AOZ and derivatization of the homogenized tissues with benzaldehyde. The limits of detection (LOD) calculated from the analysis of 20 known negative tissue samples (swine liver, swine muscle, chicken liver, chicken muscle,and fish muscle) were 0.3-0.4 microg/kg (mean+3 SD). Recoveries of AOZ fortified at the levels of 0.4, 1, and 5 microg/kg ranged from 55.8 to 96.6% in the tissues. The coefficients of variation were less than 20% over the range of AOZ concentrations studied. The linear detection range was between 0.1 and 25.6 microg/L. Validation of the ELISA method with swine muscle and liver from furazolidone-treated pigs was carried out using HPLC, resulting in a similar correlation in swine muscle (r=0.99) and in swine liver (r=0.98). The results suggest that this ELISA is a specific, accurate, and sensitive method of detecting AOZ residues in animal edible tissues.     

Influence of industrial and alternative farming systems on contents of sugars, organic acids, total phenolic content, and the antioxidant activity of red beet (Beta vulgaris L. ssp. vulgaris Rote Kugel)

The contents of sugars, organic acids, total phenolic content, and the antioxidant activity were quantified in the flesh of red beet from conventional (CON), integrated (INT), organic (ORG), biodynamic (BD), and control farming systems using established methods. Significant differences were measured for malic acid, total phenolic content (TPC), and total antioxidant activity, where malic acid content ranged from 2.39 g kg(-1) FW (control) to 1.63 g kg(-1) FW (CON, ORG, and INT). The highest TPC was measured in BD and control samples (0.677 and 0.672 mg GAE g(-1), respectively), and the lowest in CON samples (0.511 mg GAE g(-1)). Antioxidant activity was positively correlated with TPC (r2=0.6187) and ranged from 0.823 μM TE g(-1) FW to 1.270 μM TE g(-1) FW in CON and BD samples, respectively, whereas total sugar content ranged from 21.03 g kg(-1) FW (CON) to 31.58 g kg(-1) FW (BD). The importance of sugars, organic acids, phenols, and antioxidants for human health, as well as for plant resilience and health, gained from this explorative study, is discussed and put into perspective.     

Quantitative analysis of tylosin in eggs by high performance liquid chromatography with electrospray ionization tandem mass spectrometry: residue depletion kinetics after administration via feed and drinking water in laying hens

Maximum residue limits (MRLs) have been established by the European Union when tylosin is used therapeutically. They are fixed at 200 microg/kg for eggs. A highly sensitive and selective quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method suitable for monitoring tylosin residues in eggs to determine its depletion kinetics was developed and validated. For sample pretreatment all samples were liquid-liquid extracted with citrate buffer (pH 5.0) and acetonitrile. Liquid chromatographic separation was carried out on a reversed phase C18 column employing a 0.5% formic acid/acetonitrile gradient system. The tylosin recovery in eggs at a concentration range from 1.0-400 microg/kg was >82% with relative standard deviations between 1.5 and 11.0%. In two experimental studies administrating tylosin via feed (final dosage: 1.5 g/kg) or drinking water (final dosage: 0.5 g/L), no residues above the MRL were found during and after treatment. Moreover, all samples were well below the actual MRL of 200 microg/kg. Therefore, our residue data suggest that a withholding period for eggs is not required when laying hens are treated with tylosin in recommended dosages via feed or drinking water. Keywords: Tylosin; residue; depletion; laying hen; withholding period; mass spectrometry.     

Determination of five macrolide antibiotic residues in honey by LC-ESI-MS and LC-ESI-MS/MS

Liquid chromatography-electrospray ionization mass spectrometry methods (LC-ESI-MS and LC-ESI-MS/MS) for the determination of five macrolide antibiotics including spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin in honey are presented. Macrolides were protonated to form singly and/or doubly charged pseudomolecular ions, depending on their chemical structures, in an electrospray positive ionization mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity. The recoveries, that is, determined by LC-ESI-MS/MS, of the five macrolides at fortified levels of 6, 16, 40, and 80 microg/kg ranged from 75.5 to 135.7% in light honey and from 42.1 to 111.0% in dark honey. The ion ratios obtained under MS/MS were key criteria to confirm the identity of macrolides in incurred samples. LC-ESI-MS/MS method detection limits of the five macrolides were <0.1 microg/kg.     

Ultratrace analysis of nine macrolides, including tulathromycin A (Draxxin), in edible animal tissues with minicolumn liquid chromatography tandem mass spectrometry

The analysis of nine macrolides is presented, including tulathromycin A (Draxxin), in beef, poultry, and pork muscle with a simple multiresidue extraction and analysis method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The sample preparation method involves extraction with acetonitrile and defatting with hexane followed by dilution of the extracts for analysis. Separation of the nine macrolides was performed using an Atlantis dC 18, 3 mum, 3.9 mm x 20 mm minicolumn (guard column). Detection was carried out with two multiple reaction monitoring experiments per macrolide. The method detection limits (MDLs) were based on three times standard deviation of eight repeat spikes at 3.0 ng/g of a mix of the nine macrolides in the various tissues. The MDLs and retention times for the macrolides were as follows: lincomycin, 0.19 ng/g (t R = 5.00 min); tulathromycin, 0.46 ng/g (t R = 5.63 min); spiramycin, 0.21 ng/g (t R = 6.06 min); pirlimycin, 0.10 ng/g (t R = 6.04 min); clindamycin, 0.16 ng/g (t R = 6.20 min); tilmicosin, 0.29 ng/g (t R = 6.38 min); erythromycin, 0.19 ng/g (t R = 6.62 min); tylosin, 0.10 ng/g (t R = 6.72 min); and josamycin, 0.09 ng/g (t R = 6.98 min). Precision at 25 ng/g (n = 4) ranged from 2.3 to 9.4% for the compounds from beef muscle. Of interest is the detection of incurred residues of tulathromycin A in edible calf tissue at 0.10-7 mug/g, which is presented here for the first time.     

Gas chromatographic determination of N-nitrosamines, aromatic amines, and melamine in milk and dairy products using an automatic solid-phase extraction system

A reliable analytical method was presented for the simultaneous determination of six N-nitrosamines, nine aromatic amines, and melamine in milk and dairy products using gas chromatography coupled with mass spectrometry. The sample treatment includes the precipitation of proteins with acetonitrile, centrifugation, solvent changeover by evaporation, and continuous solid-phase extraction for cleanup and preconcentration purposes. Samples (5 g) containing 0.15-500 ng of each amine were analyzed, and low detection limits (15-130 ng/kg) were achieved. Recoveries for milk and dairy products samples spiked with 1, 10, and 50 μg/kg ranged from 92% to 101%, with intraday and interday relative standard deviation values below 7.5%. The method was successfully applied to determine amine residues in several milk types (human breast, cow, and goat) and dairy products.     

Dispersive cleanup of acetonitrile extracts of tea samples by mixed multiwalled carbon nanotubes, primary secondary amine, and graphitized carbon black sorbents

A method for analysis of 37 pesticide residues in tea samples was developed and validated and was based on reversed-dispersive solid-phase extraction (r-DSPE) cleanup in acetonitrile solution, followed by liquid chromatography-electrospray tandem mass spectrometry determination. Green tea, oolong tea, and puer tea were selected as matrixes and represent the majority of tea types. Acetonitrile was used as the extraction solvent, with sodium chloride and magnesium sulfate enhancing partitioning of analytes into the organic phase. The extract was then cleaned up by r-DSPE using a mixture of multiwalled carbon nanotubes, primary secondary amine, and graphitized carbon black as sorbents to absorb interferences. Further optimization of sample preparation and determination allowed recoveries of between 70% and 111% for all 37 pesticides with relative standard deviations lower than 14% at two concentration levels of 10 and 100 μg kg(-1). Limits of quantification ranged from 5 to 20 μg kg(-1) for all pesticides. The developed method was successfully applied to the determination of pesticide residues in market tea samples.     

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC??) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 μg/kg, alternariol at 1-13 μg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.     

Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum

Ractopamine hydrochloride is a beta-adrenergic leanness-enhancing agent approved for use in swine in the United States. Depletion of ractopamine and its metabolites from animal tissues, urine, and serum is of interest for the detection of illegal use. The objectives of this study were to measure the residues of ractopamine in swine incurred samples after treatment with dietary ractopamine for 28 consecutive days. An efficient and sensitive analytical method was developed for the detection of parent ractopamine and its metabolites in swine tissues, urine, and serum by HPLC-FLD. After extraction, enzymatic digestion, and solid-phase cleanup of the samples, ractopamine residues were determined by liquid chromatography (LC) with fluorescence detector. The limits of detection (LOD) for tissues, urine, and serum were 1 ng g(-1), 0.5 ng mL(-1), and 0.5 ng mL(-1), respectively. Recoveries ranged from 70.5 to 94.5% for samples fortified at 1-50 ng g(-1) or ng mL(-1). Sixty pigs were fed twice daily for 28 consecutive days with feeds containing 18 mg kg(-1) ractopamine HCl. The residue concentrations in urine, liver, and kidney were 650.06 ng mL(-1), 46.09 ng g(-1), and 169.27 ng g(-1), respectively, compared with those in muscle, fat, and serum (4.94 ng g(-1), 3.28 ng g(-1), and 7.48 ng mL(-1), respectively) at the feeding period of 7 days. The residue concentrations at withdrawal period of 0 days in all edible tissues were lower than tolerance values established by the FDA and MRL values listed by the JECFA. These data support the withdrawal time of 0 days established by the FDA for ractopamine used as feed additive in swine.     

浙中红壤油菜田供钾特性和钾肥用量研究

通过多点田间试验研究红壤性水田油菜供肥特性和钾肥施用量 .1 8个田间试验施钾油菜籽增产幅度为 7.5～ 90 .8kg/亩 ,无钾区相对产量变幅为 48.2～ 93.1 % .1mol/LNH4OAc提取的土壤速效钾含量与无钾区相对产量之间达极显著相关 ,相关系数为 0 .6 44 9 (n =1 8) .土壤速效钾的“临界水平”为80mg/kgK ,能满足 95 %相对产量的“足够水平值”为 1 5 0mg/kgK ,系统研究平衡施肥法中各参数的确定 ,并用平衡施肥法和施肥曲线确定油菜钾肥合理用量 ,两种方法具有可比性     

Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of olaquindox residues

A rapid immunochromatographic lateral flow test strip of competitive format has been developed for the specific determination of olaquindox (OLA) residues in pig urine and muscle tissues. The sensitivity of the test strip was found to be 1.58 ± 0.27 μg/kg and 1.70 ± 0.26 μg/kg of OLA in pig urine and muscle tissues, and the lower detection limit was 0.27 ± 0.08 μg/kg and 0.31 ± 0.07 μg/kg respectively. For negative pig urine and muscle samples spiked with 4, 12, and 36 μg/kg, the recovery range was 83.0-94.0% and 78.8-87.4% and the coefficient of variation scope [CV (%)] was 3.17-7.41% and 4.66-7.64% respectively. Parallel analysis of OLA samples from pig urine and muscle tissue showed comparable results from the test strip and HPLC. Each test requires 5-8 min, and the test strip can provide a useful screening method for quantitative, semiquantitative, or qualitative detection of OLA residues.     

Determination and depletion of residues of carbadox, tylosin, and virginiamycin in kidney, liver, and muscle of pigs in feeding experiments

The results of residue determinations of the growth promotors carbadox, tylosin, and virginiamycin in kidney, liver, and muscle from pigs in feeding experiments are described as well as the analytical methods used. Residues of the carbadox metabolite quinoxaline-2-carboxylic acid were found in liver from pigs fed 20 mg/kg in the diet with a withdrawal time of 30 days. No residues were detected in muscle with zero withdrawal time. The limit of determination was 0.01 mg/kg for both tissues. No residues of virginiamycin and tylosin were found in pigs fed 50 and 40 mg/kg, respectively, in the diet, even with zero withdrawal time. Residues of tylosin of 0.06 mg/kg and below were detected in liver and kidney from pigs fed 200 or 400 mg/kg and slaughtered within 3 h after the last feeding.     

Deoxynivalenol, deoxynivalenol-3-glucoside, and enniatins: the major mycotoxins found in cereal-based products on the Czech market

Fusarium toxins, Alternaria toxins, and ergot alkaloids represent common groups of mycotoxins that can be found in cereals grown under temperate climatic conditions. Because most of them are chemically and thermally stable, these toxic fungal secondary metabolites might be transferred from grains into the final products. To get information on the commensurate contamination of various cereal-based products collected from the Czech retail market in 2010, the occurrence of "traditional" mycotoxins such as groups of A and B trichothecenes and zearalenone, less routinely determined Alternaria toxins (alternariol, alternariol monomethyl ether and altenuene), ergot alkaloids (ergosine, ergocryptine, ergocristine, and ergocornine) and "emerging" mycotoxins (enniatins A, A1, B, and B1 and beauvericin) were monitored. In a total 116 samples derived from white flour and mixed flour, breakfast cereals, snacks, and flour, only trichothecenes A and B and enniatins were found. Deoxynivalenol was detected in 75% of samples with concentrations ranging from 13 to 594 μg/kg, but its masked form, deoxynivalenol-3-β-d-glucoside, has an even higher incidence of 80% of samples, and concentrations ranging between 5 and 72 μg/kg were detected. Nivalenol was found only in three samples at levels of 30 μg/kg. For enniatins, all of the samples investigated were contaminated with at least one of four target enniatins. Enniatin A was detected in 97% of samples (concentration range of 20-2532 μg/kg) followed by enniatin B with an incidence in 91% of the samples (concentration range of 13-941 μg/kg) and enniatin B1 with an incidence of 80% in the samples tested (concentration range of 8-785 μg/kg). Enniatin A1 was found only in 44% of samples at levels ranging between 8 and 851 μg/kg.