Immunochromatographic Assay for Simultaneous and Quantitative Detection of 3-Methyl-Quinoxaline-2-Carboxylic Acid and Quinoxaline-2-Carboxylic Acid Residues in Animal Tissues Based on Highly Luminescent Quantum Dot Beads

Due to their potential adverse effects on human health, the use of carbadox and olaquindox in feedingstuffs was prohibited by the European Union since 1998. In this work, highly luminescent quantum dot beads (QBs) were synthesized by encapsulating CdSe/ZnS and used as novel fluorescent probes in the immunochromatographic assay (ICA) for simultaneous and quantitative determination of metabolites of olaquindox (3-methylquinoxaline-2-carboxylic acid, MQCA) and carbadox (quinoxaline-2-carboxylic, QCA). The fluorescence intensities of the test lines were recorded using a fluorescence strip reader. The 50% of inhibition for MQCA and QCA was shown to be 8.1 and 10.6 μg L^?1, respectively. The whole assay process can be accomplished within 10 min. The immunosensor was used to analyze spiked samples, and analyte intra- and inter-assay recovery rates ranged from 78.7 to 92.2% for MQCA and 80.6 to 95.8% for QCA, and coefficients of variation were all below 12%. The incurred tissues samples were assayed using both QB-based ICA and commercial ELISA kit and were further confirmed with LC-MS/MS. The QB-based ICA results exhibited good agreement with both commercial ELISA and LC-MS/MS methods.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for monitoring quinoxaline-2-carboxylic acid in the edible tissues of animals

The feed drug additive carbadox is a suspected carcinogen and mutagen. To monitor effectively residues of carbadox in the edible tissues of food-producing animals, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect quinoxaline-2-carboxylic acid, the marker residue of carbadox, was developed. Several haptens were synthesised and conjugated to the carrier protein. Nine female New Zealand white rabbits were immunised with the immunising conjugates to produce polyclonal antibodies according to the designed schemes of immunisation. The highly specific antibody that was very sensitive to N-butylquinoxaline-2-carboxamide with an IC₅₀ value of 7.75?µg?l^?1 was selected for the development of an ic-ELISA. The standard curves based on the N-butylquinoxaline-2-carboxamide matrix calibration ranged from 0.2 to 51.2?µg?l^?1. The decision limit and detection capability of the ic-ELISA were 0.60 and 0.83?µg?kg^?1 for liver and 0.68 and 0.79?µg?kg^?1 for muscle of swine, respectively. The recoveries were 57–108% with coefficients of variation of less than 20% when the quinoxaline-2-carboxylic acid was spiked into liver and muscle with the concentrations of 1.0–20.0?µg?kg^?1. Excellent correlations between the results of the ic-ELISA and an HPLC method (r?=?0.9956???0.9969) were observed for incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for screening residues of carbadox in the edible tissues of food-producing animals.     

Mass spectrometric analysis of muscle samples to detect potential antibiotic growth promoter misuse in broiler chickens

Mass spectrometric methods were developed and validated for the analysis in chicken muscle of a range of antibiotic growth promoters: spiramycin, tylosin, virginiamycin and bacitracin, and separately for two marker metabolites of carbadox (quinoxaline-2-carboxylic acid and 1,4-bisdesoxycarbadox), and a marker metabolite of olaquindox (3-methyl-quinoxaline-2-carboxylic acid). The use of these compounds as antibiotic growth promoters has been banned by the European Commission. This study aimed to develop methods to detect their residues in muscle samples as a means of checking for the use of these drugs during the rearing of broiler chickens. When fed growth-promoting doses for 6 days, spiramycin (31.4?µg?kg^?1), tylosin (1.0?µg?kg^?1), QCA (6.5?µg?kg^?1), DCBX (71.2?µg?kg^?1) and MQCA (0.2?µg?kg^?1) could be detected in the muscle 0 days after the withdrawal of fortified feed. Only spiramycin could consistently be detected beyond a withdrawal period of 1?day. All analytes showed stability to a commercial cooking process, therefore raw or cooked muscle could be used for monitoring purposes.     

Simultaneous determination of five quinoxaline-1,4-dioxides in animal feeds using an immunochromatographic strip

An immunochromatographic (ICG) strip was developed for the simultaneous quantitative determination of five quinoxaline-1,4-dioxides in animal feed. For this purpose, polyclonal antibodies (PcAb) with group-specific quinoxaline-1,4-dioxides were conjugated to colloidal gold particles as the detection reagent for ICG strips to test for quinoxaline-1,4-dioxides. This method achieved semi-quantitative detection of quinoxaline-1,4-dioxides within 5–10 min. The visual lower detection limits of the strip for quinocetone, cyadox, carbadox, mequindox and olaquindox were 10, 15, 15, 20 and 20 ng ml^?1, respectively. Using an ICG strip reader, the 50% inhibitions (IC₅₀ values) were calculated to be 9.1, 13.5, 16.6, 20.2 and 21.3 ng ml^?1 for quinocetone, cyadox, carbadox, mequindox and olaquindox, respectively. When used to analyse samples of animal feed, acceptable recovery rates of 77.5–99.5% and coefficients of variation (CVs) of 4.3–10.7% were obtained. Levels measured with the ICG strip for 10 spiked samples were confirmed by HPLC with a high correlation coefficient of 0.9965 (n = 10). In conclusion, the method was rapid and accurate for simultaneous determination of five quinoxaline-1,4-dioxides antibiotics in animal feed.     

Development and validation of an immunochromatographic assay for the rapid detection of quinoxaline-2-carboxylic acid,the major metabolite of carbadox in the edible tissues of pigs

A new method was developed for the determination of quinoxaline-2-carboxylic acid, the marker residue of carbadox, in the edible tissues of food-producing animals using a colloidal gold probe-based immunochromatographic assay. The highly specific polyclonal antibody (PcAb), which was very sensitive to N-butylquinoxaline-2-carboxylic acid (BQCA) with an IC₅₀ value of 2.38?ng?ml^?1, was selected for the development of an immunochromatographic assay (ICA). Only 5?min were required to perform this assay; it had a visual detection limit of 25?ng?g^?1 for quinoxaline-2-carboxylic acid. The results of the analysis of quinoxaline-2-carboxylic acid in animal tissues using the immunochromatographic assay showed good agreement with those obtained by HPLC. In conclusion, the method was rapid and accurate for screening residues of carbadox in the edible tissues of food-producing animals.     

Determination of olaquindox,carbadox and cyadox in animal feeds by ultra-performance liquid chromatography tandem mass spectrometry

Olaquindox, carbadox, and cyadox are chemically synthesised antibacterial and growth-promoting agents for animals. At high doses they may exert mutagenicity and hepatic and adrenal toxicities in animals. Regrettably, these substances are frequently abused or misused when added into animal feeds. Thus, developing a sensitive and reliable method for simultaneous determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds is crucially important for food safety monitoring. In this paper we optimised instrumental conditions, extraction solvents, solid phase extraction cartridges, and pH of the loading solvents on the Oasis HLB cartridge. Under the optimal conditions, mean recoveries ranged from 74.1 to 111%, and intra-day and inter-day variations were lower than 14.6% and 10.8%, respectively. The limits of quantification for olaquindox, carbadox, and cyadox were 0.05 mg kg^?1, 0.10 mg kg^?1, and 0.025 mg kg^?1, respectively. The proposed method uses ultra-performance liquid chromatography tandem mass spectrometry and is sensitive and reliable for the simultaneous determination of olaquindox, carbadox, and cyadox in three kinds of animal feeds (specifically, mixed feed, concentrated feed, and additive premixed feed). This method has good precision, high sensitivity, and good reproducibility, and thus it can be used for convenient and accurate determination of olaquindox, carbadox, and cyadox in different kinds of animal feeds.     

Mass spectrometric analysis of muscle samples to detect potential antibiotic growth promoter misuse in broiler chickens

Mass spectrometric methods were developed and validated for the analysis in chicken muscle of a range of antibiotic growth promoters: spiramycin, tylosin, virginiamycin and bacitracin, and separately for two marker metabolites of carbadox (quinoxaline-2-carboxylic acid and 1,4-bisdesoxycarbadox), and a marker metabolite of olaquindox (3-methyl-quinoxaline-2-carboxylic acid). The use of these compounds as antibiotic growth promoters has been banned by the European Commission. This study aimed to develop methods to detect their residues in muscle samples as a means of checking for the use of these drugs during the rearing of broiler chickens. When fed growth-promoting doses for 6 days, spiramycin (31.4?μg?kg(-1)), tylosin (1.0?μg?kg(-1)), QCA (6.5?μg?kg(-1)), DCBX (71.2?μg?kg(-1)) and MQCA (0.2?μg?kg(-1)) could be detected in the muscle 0 days after the withdrawal of fortified feed. Only spiramycin could consistently be detected beyond a withdrawal period of 1?day. All analytes showed stability to a commercial cooking process, therefore raw or cooked muscle could be used for monitoring purposes.     

UPLC-MS/MS法测定金沙江水域鱼体中卡巴氧及喹乙醇代谢物

为建立一种白酒香型鉴别方法,以DB-WAX为色谱柱,样品加标后直接采用气相色谱（GC）法进样分析。结果表明,香气成分线性相关系数R2＞0.999,检测限＜5.11 mg/L,精密度试验结果的相对标准偏差（RSD）＜4.11%;平均回收率为80.20%～97.93%。采用偏最小二乘法（PLS）对不同香型白酒数据进行建模和预测,不同香型白酒样品在PLS得分图中能分开,利用训练集和验证集数据对模型进行验证,结果表明预测准确率较高,校正均方差（RMSEE）和预测均方差（RMSEP）值也说明PLS模型对不同白酒香型具有较强的预测性。该方法在判别白酒香型分析中具有较高的准确性。     

Development and validation of an immunochromatographic assay for the rapid detection of quinoxaline-2-carboxylic acid, the major metabolite of carbadox in the edible tissues of pigs

A new method was developed for the determination of quinoxaline-2-carboxylic acid, the marker residue of carbadox, in the edible tissues of food-producing animals using a colloidal gold probe-based immunochromatographic assay. The highly specific polyclonal antibody (PcAb), which was very sensitive to N-butylquinoxaline-2-carboxylic acid (BQCA) with an IC(50) value of 2.38 ng ml(-1), was selected for the development of an immunochromatographic assay (ICA). Only 5 min were required to perform this assay; it had a visual detection limit of 25 ng g(-1) for quinoxaline-2-carboxylic acid. The results of the analysis of quinoxaline-2-carboxylic acid in animal tissues using the immunochromatographic assay showed good agreement with those obtained by HPLC. In conclusion, the method was rapid and accurate for screening residues of carbadox in the edible tissues of food-producing animals.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for monitoring quinoxaline-2-carboxylic acid in the edible tissues of animals

The feed drug additive carbadox is a suspected carcinogen and mutagen. To monitor effectively residues of carbadox in the edible tissues of food-producing animals, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect quinoxaline-2-carboxylic acid, the marker residue of carbadox, was developed. Several haptens were synthesised and conjugated to the carrier protein. Nine female New Zealand white rabbits were immunised with the immunising conjugates to produce polyclonal antibodies according to the designed schemes of immunisation. The highly specific antibody that was very sensitive to N-butylquinoxaline-2-carboxamide with an IC(50) value of 7.75 μg l(-1) was selected for the development of an ic-ELISA. The standard curves based on the N-butylquinoxaline-2-carboxamide matrix calibration ranged from 0.2 to 51.2 μg l(-1). The decision limit and detection capability of the ic-ELISA were 0.60 and 0.83 μg kg(-1) for liver and 0.68 and 0.79 μg kg(-1) for muscle of swine, respectively. The recoveries were 57-108% with coefficients of variation of less than 20% when the quinoxaline-2-carboxylic acid was spiked into liver and muscle with the concentrations of 1.0-20.0 μg kg(-1). Excellent correlations between the results of the ic-ELISA and an HPLC method (r = 0.9956 - 0.9969) were observed for incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for screening residues of carbadox in the edible tissues of food-producing animals.     

Fluoride content of soft drinks,nectars, juices,juice drinks,concentrates, teas and infusions marketed in Portugal

A potentiometric method using a fluoride combination ion-selective electrode was validated and used to analyse 183 samples, including soft drinks, juices, nectars, juice drinks, concentrates, teas and infusions marketed in Portugal. The fluoride levels were higher in extract-based soft drinks, juice drinks and juice, with fluoride values of 0.86 ± 0.35, 0.40 ± 0.24 and 0.37 ± 0.11 mg l^?1, respectively. The lowest fluoride concentration was found in infusion samples (0.12 ± 0.01 mg l^?1), followed by teas and carbonated soft drinks with fluoride concentrations of 0.16 ± 0.12 and 0.18 ± 0.07 mg l^?1, respectively. Nectars, concentrates and juice-based drinks had similar fluoride concentrations of 0.33 ± 0.16, 0.29 ± 0.12 and 0.25 ± 0.14 mg l^?1, respectively. The fluoride concentrations in all these samples would only contribute intakes below the acceptable daily intake (ADI = 0.05 mg kg^?1 body weight day^?1), indicating that, individually, these beverages cannot induce fluoride toxicity in the population group of children.     

Simultaneous Determination of Cyadox and Its Metabolites in Chicken Tissues by LC-MS/MS

A specific and sensitive LC-MS/MS method was firstly established for the simultaneous extraction and determination of cyadox and its three main metabolites—1,4-bisdesoxycyadox, 4-desoxycyadox, and quinoxaline-2-carboxylic acid—in chicken muscle, liver, kidney, and fat tissues. Samples were subjected to extraction using ethyl acetate and followed by acetonitrile–chloroform (1:4, v/v) and further purified by Oasis mixed mode anion exchange SPE cartridge. Analysis was performed on a C₁₈ column by detection with MS in multiple-reaction monitoring mode. A gradient elution program with 0.1 % formic acid solution, acetonitrile, and 1 % formic acid (adjusted to pH 8 with ammonia) was performed at a flow rate of 0.2 mL min^?1. The correlation coefficients (r) for each calibration curve are higher than 0.99 within the experimental concentration range. The recoveries of the four target analytes at three spiking levels of 2.5, 25 and 250 μg kg^?1 were between 74.5 and 93.8 %, with relative standard deviations less than 12 %. The decision limits (CCαs) of the four analytes in chicken edible tissues ranged from 0.3 to 1.5 μg kg^?1, and the detection capabilities (CCβs) were below 2.3 μg kg^?1. The developed method demonstrated a satisfactory applicability in incurred chicken tissue samples.     

Determination of the exposure parameters that maximise the concentrations of the anaesthetic/sedative eugenol in rainbow trout (Oncorhynchus mykiss) skin-on fillet tissue

Studies were conducted to determine the anaesthetic/sedative concentrations and durations that would maximise anaesthetic/sedative residue concentrations in rainbow trout (Oncorhynchus mykiss) skin-on fillet tissue. Rainbow trout (167–404 g) were exposed to 50 mg l^?1 AQUI-S^® 20E (10% active ingredient, eugenol) in 17°C freshwater for durations up to 1440 min, 100 and 250 mg l^?1 AQUI-S^® 20E for durations up to 240 min, and 500 and 1000 mg l^?1 AQUI-S^® 20E for durations up to 90 min. Fish exposed to 100 mg l^?1 AQUI-S^® 20E for durations of 30, 60, 120 and 240 min had the greatest eugenol concentrations in the fillet tissue, 50, 58, 54 and 62 µg g^?1, respectively. All other exposure concentrations and durations resulted in significantly lower eugenol concentrations, i.e. all < 39 µg g^?1.     

Improvement of wine aromatic quality using mixtures of lysozyme and dimethyl dicarbonate,with low SO2 concentration

The use of sulphur dioxide (SO₂) in the treatment of foodstuffs presents some problems as it could lead to pseudo-allergies in some people. The aim of this research work was to study the addition of different preservative mixtures and their influence on the concentration of volatile compounds and sensorial quality in wine. To do so, vinifications were carried out using Garnacha must to which lysozyme, dimethyl dicarbonate (DMDC) and mixtures of these with SO₂ were added at different doses (25 and 50 mg l^?1). The results were compared with a control sample to which only SO₂ had been added (50 mg l^?1). In general, mixtures of SO₂ with lysozyme and DMDC favoured the formation of volatile compounds in the wines. Wines obtained from the mixtures of lysozyme and DMDC with 25 mg l^?1 of SO₂ had better sensorial quality than the wines obtained with 50 mg l^?1 as the only preservative used.     

Cadmium and lead in female cattle livers and kidneys from Vojvodina,northern Serbia

ABSTRACT

Concentrations of cadmium (Cd) and lead (Pb) were determined in livers (n = 52) and kidneys (n = 52) of female cattle (345–2717 days old) from dairy farms in the region Vojvodina. Cd and Pb were analysed by inductively coupled plasma-optical emission spectrometry, after microwave digestion. Cd and Pb concentrations did not exceed the Serbian and European maximum set limits in any sample. The Cd concentrations in the livers and kidneys ranged from 0.033 to 0.151 mg kg^?1 wet weight and from 0.055 to 0.510 mg kg^?1 wet weight, respectively. The corresponding Pb concentrations were 0.015-0.159 mg kg^?1 wet weight and 0.021-0.196 mg kg^?1 wet weight, respectively. Mean Cd and Pb concentrations were significantly lower (p < 0.001) in the liver (0.072 and 0.053 mg kg^?1 wet weight) than in the kidney (0.190 and 0.075 mg kg^?1 wet weight). There were good correlations between Cd in liver and Cd in kidney, Pb in liver and Pb in kidney, Cd level and age and Pb level and age in both tissues.     

Tissue deposition and residue depletion of melamine in fattening pigs following oral administration

The adulteration of animal feed as well as milk products with melamine has led to concerns about the ability to establish appropriate withdrawal intervals to ensure food safety. Two experiments were conducted in this study. The first was to investigate the deposition and depletion of melamine in blood and tissues of pigs exposed to adulterated feed with high doses of melamine. A total of 500 or 1000 mg kg^–1 melamine was added to the diet for fattening pigs (initial BW = ±60.24 kg). Melamine residues were detected in tissues (brain, duodenum, liver, heart, muscle and kidney) by LC-MS/MS. Dose-dependent effects were found between melamine residual concentration and its dose in feed. Five days after the withdrawal of melamine from the diets, the residue concentration in tissues fell below 2.5 mg kg^–1. In the second experiment, blood samples were taken at different time points from fattening pigs (BW = 100 kg) fed with adulterated feed with 1000 mg kg^–1 of melamine for 42 days. Results from the pharmacokinetics analysis showed that it would take 83 h for the melamine level in plasma depleting to the safe level of 50 ng ml^–1 after an expose of 1000 mg kg^–1 melamine contaminated feed for 42 days.     

Silver migration from nanosilver and a commercially available zeolite filler polyethylene composites to food simulants

Polyethylene composites containing Agion^TM commercial silver ion filler at three different percentage fill rates (0.5, 1.0 and 2% w/w) and polyethylene composites containing laboratory produced silver nanoparticles (Agnps) at two different percentage fill rates (0.1 and 0.5% w/w) underwent migration tests according to Commission Regulation (EU) No. 10/2011. Migrated silver in the two simulants (acidified water with 3% acetic acid and distilled water) was quantified using two techniques: inductively coupled atomic emission spectroscopy (ICPAES) and Hach Lange spectroscopy. The former had higher sensitivity with mean silver migration from Agion composites (n = 12) ranging from < 0.001 to 1.50 × 10^?2 mg l^–1. Mean silver migration from Agnps composites ranged from 4.65 × 10^?2 to 0.38 mg l^–1 and 8.92 × 10^?2 and 5.15 × 10^?2 mg l^–1 for Hach Lange spectrophotometry and ICPAES, respectively. Both percentage fill rate in the composite and the simulant type, as factors, were found to be significant in both silver migration from Agion (p < 0.0001 and < 0.01, respectively) and Agnps (p < 0.05 and < 0.01, respectively). Transmission electron microscopy (TEM) imagery showed differences in size distributions and morphology of particles (shape and degree of agglomeration) before and after migration. PE composites containing 0.5% Agion, simulating contact with non-acidic foods, was the only scenario that did not exceed the permitted migration level of non-authorised substances given in EU 10/2011. This study illustrates the need for careful engineering of the composite filler system to conform to limits with cognisance of food pH and percentage fill rate.     

A method for the determination of total and reduced methimazole in various biological samples

A simple, rapid, reproducible and sensitive method based on HPLC with ultraviolet detection was developed for the determination of methimazole (MMI) in animal tissues and plasma samples. Under the optimum experimental conditions, the calibration curves for MMI were linear in the tested range 0.5–20 mg kg^?1 tissue sample (mg l^?1 plasma) with correlation coefficients better than 0.99. The performance of the proposed method was tested for the determination of MMI levels in brain, liver, thyroid gland and plasma of MMI-treated hens, as well as in their eggs and embryos. The proposed method reduces time and simplifies the sample preparation procedure.     

Quantitative Profiling of Volatile and Phenolic Substances in the Wine Vernaccia di Serrapetrona by Development of an HS-SPME-GC-FID/MS Method and HPLC-MS

A headspace solid-phase microextraction coupled to gas chromatography with mass spectrometry and flame ionization detection (HS-SPME-GC-MS/FID) method has been developed for quantifying the main volatiles of the Italian sparkling red wine Vernaccia di Serrapetrona, leading to establish that the divinylbenzene/carboxen/polydimethylsiloxane-coated (gray) fiber, an equilibration time of 15 min, and an extraction time of 15 min at a temperature of 35 °C are the conditions representing the best compromise in terms of sensitivity and time expense, to carry out the analyses. Among the volatiles quantified, 2-phenylethanol, 3-methylbutyl acetate, and ethyl esters levels resulted to be above their odor thresholds, thus being probably responsible for the aroma of this wine with their positive attributes. Phenolic compounds, namely gallic acid, p-coumaric acid, caffeic acid, (+)-catechin, and (+)-epicatechin, were also quantified using high-performance liquid chromatography-MS, thus obtaining an overall characterization of molecules fundamental for both the sensory and the healthy characteristics of the wine. The total phenolic content was found to be in the range of 19.25–61.67 mg l^?1 with the most abundant compound being gallic acid (7.44–25.78 mg l^?1). Main differences in volatile and phenolic compounds between samples were discussed and analyzed by chemometric techniques as principal component analysis.     

Analysis of Fluopicolide and Propamocarb Residues on Tomato and Soil Using QuEChERS Sample Preparation Method in Combination with GLC and GCMS

Persistence of fluopicolide and propamocarb in tomato was studied following three applications of a combination formulation of Infinito 68.75 SC (fluopicolide 6.25 %?+?propamocarb 62.5 %) at 1500 and 3000 mL ha^?1 by 7 days interval, starting the spray at fruit development stage. QuEChERS method included extraction of sample with ethyl acetate and cleanup of dispersive solid-phase extraction was used for the determination of fluopicolide and propamocarb residues on tomato and soil. Residues of fluopicolide and propamocarb in tomato were estimated by gas–liquid chromatography and gas chromatograph–mass spectrometry, respectively. Half-lives for fluopicolide were found to be 2.58 and 2.31 days, whereas for propamocarb these values were observed to be 1.49 and 2.08 days at single and double the application rates, respectively. Residues of fluopicolide dissipated below its limit of quantification (LOQ) of 0.01 mg kg^?1 after 7 and 10 days at single and double the application dosage, respectively. Similarly, residues of propamocarb took 5 and 7 days to reach LOQ of 0.10 mg kg^?1, at single and double dosages, respectively. Soil samples collected after 15 days of the last application did not show the presence of fluopicolide and propamocarb at their detection limit of 0.01 and 0.10 mg kg^?1, respectively. The initial deposit of residues of propamocarb and fluopicolide for both the dosages were below the prescribed codex maximum residue limit values of 2 and 1 mg kg^?1, respectively. Therefore, a 1-day waiting period was suggested to reduce human health risks before consumption of tomato fruits.