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 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.     

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.     

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。     

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.     

In vivo studies to highlight possible illegal treatments of rabbits with carbadox and olaquindox

For the treatment of rabbit dysentery and bacterial enteritis, veterinary practitioners often adopt veterinary medicinal products authorised for other food-producing species, but in some cases non-authorised drugs frequently used in the past, such as carbadox and olaquindox, might be illegally adopted. To verify the carbadox and olaquindox distribution and persistence in rabbit tissues, two independent in vivo studies were carried out. In the first study, 24 healthy rabbits received water medicated with carbadox at 100 mg l^?1 over a period 28 days, whereas in the second one, 24 healthy rabbits were administered water containing olaquindox at 100 mg l^?1. In each study rabbits were randomly assigned to four groups to be sacrificed respectively at 0, 5, 10 and 20 days from treatment withdrawal, for depletion studies. A control group of six animals was adopted for control and as a reservoir of blank tissues. Muscle and liver samples collected from each treated animal were stored at ?20°C pending the analysis. Sensitive and robust liquid chromatography-tandem mass spectrometry analytical methods were set up for the parent compounds and their main metabolites quinoxaline-2-carboxylic acid, desoxycarbadox and 3-methylquinoxaline-2-carboxylic acid to verify their residual. Data collected demonstrate that the combination of liver as target matrix, quinoxaline-2-carboxylic acid and 3-methylquinoxaline-2-carboxylic acid as marker residue and enzymatic digestion is strategic to evidence carbadox and/or olaquindox illegal treatments in rabbits, even 20 days after treatment withdrawal at concentration levels higher than 0.5 µg kg^?1. This findings suggests that liver should be proposed as target matrix for official control in national monitoring plan.     

Determination of carbadox metabolites, quinoxaline-2-carboxylic acid and desoxycarbadox, in swine muscle and liver by liquid chromatography/mass spectrometry

A sensitive and selective method using liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) for the determination of carbadox metabolites, quinoxaline-2-carboxylic acid (QCA) and desoxycarbadox (Desoxy-CDX), in swine muscle and liver has been developed. The LC separation was performed on a Cadenza CD-C18 column (10 cm x 2 mm i.d.) with a gradient system of 0.01% acetic acid-acetonitrile as the mobile phase at a flow rate of 0.2 mL/min. Negative ionization produced the [M-H]- molecular ion of QCA. On the other hand, the positive mode produced the [M+H]+ ion of Desoxy-CDX. The calibration graphs for QCA and Desoxy-CDX were rectilinear from 0.01 to 0.5 ng with selected ion monitoring (SIM). The drugs were extracted with 0.3% metaphosphoric acid-methanol (7:3), and the extracts were cleaned up on an Oasis HLB cartridge (60 mg) and by liquid-liquid extraction. The recoveries of QCA and Desoxy-CDX from swine muscle and liver fortified at 2.5 and 5 ng/g were 70.2-86.3%, and the detection limits were 1 ng/g for both drugs.     

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.     

鸡、鸭肉中金刚烷胺、金刚乙胺、索金刚胺间接竞争ELISA检测方法研究

目的:建立一种检测鸡肉、鸭肉中金刚烷胺、金刚乙胺、索金刚胺的间接竞争酶联免疫吸附方法(Indirect competitive enzyme-linked immunosorbent assay,ic-ELISA).方法:本研究基于间接竞争酶联免疫方法的原理,在酶标板微孔中预包被偶联抗原,样本中含有的金刚烷胺、金刚乙胺...     

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.     

SPE-HPLC-MS/MS法检测动物源性运动食品中喹乙醇及卡巴氧代谢物残留

建立一种快速、高效的固相萃取-高效液相色谱-串联质谱法（SPE-HPLC-MS/MS）测定动物源性运动食品中的3-甲基-喹喔啉-2-羧酸（MQCA）及喹恶啉-2-羧酸（QCA）的方法。样品前处理采用碱水解提取MQCA和QCA,经PAX固相萃取柱（60 mg/3 mL）净化后检测。以乙腈-0.1%甲酸溶液为流动相,在质谱检测器的多反应监测（MRM）模式下进行分析。结果表明,MQCA和QCA在0.1～50.0 ng/mL质量浓度范围内线性关系良好,相关系数R2为0.999 6～0.999 8,检出限均为0.05 μg/kg,定量限均为0.20 μg/kg。平均加标回收率为83.67%～96.08%,精密度试验结果相对标准偏差（RSD）为1.75%～3.10%。该方法具有前处理简单、净化效果好、灵敏度高和检测速度快的优点,适用于动物源性运动食品中的MQCA及QCA残留量的检测。     

QuEChERS-HILIC-MS/MS测定禽蛋中利巴韦林及其代谢物残留量

建立了禽蛋中利巴韦林及其主要代谢物TCONH2、RTCOOH的QuEChERS提取净化并结合HILIC-MS/MS的检测方法。禽蛋样品经乙腈提取后,利用QuEChERS净化盐包(2 g无水硫酸钠、100 mg GCB、50 mg C18)净化后,浓缩,纯水复溶,水饱和正己烷除脂,过滤膜,在HILIC模式下进行检测,并利用利巴韦林同位素内标进行定量。在优化条件下,3种物质在2.00~100μg/L范围内线性关系良好,相关系数r不低于0.99,检出限为0.146~0.763μg/kg,定量限为0.438~2.26μg/kg。空白基质加标(5.00,10.0,50.0μg/kg)回收率为71.6%~97.3%,相对标准偏差(δ_RSD,n=6)为3.5%~8.6%。该方法操作简单快速,精确度和准确度较好,成本低,适用于禽蛋中利巴韦林及其代谢物残留量的快速定量检测。     

Immunoassay based on monoclonal antibodies versus LC-MS: deoxynivalenol in wheat and flour in Southern Brazil

An indirect competitive enzyme-linked immunosorbent assay (ELISA) method using a monoclonal antibody for deoxynivalenol (DON) detection in wheat and flour was standardised and validated (detection limit?=?177.1?μg?kg(-1)) and its performance was compared with LC-MS, quantification limit?=140?μg?kg(-1)). DON recovery ranged from 88.7% to 122.6% for wheat grain and from 70.6% to 139.3% for flour. Among the 38 wheat samples evaluated, DON was detected in 29 samples (76.3%) by ic-ELISA (281.6-12?291.4?μg?kg(-1)) and in 22 samples (57.9%) by LC-MS (155.3-9906.9?μg?kg(-1)). The 0.93 correlation coefficient between ic-ELISA and LC-MS data in 19 positive DON wheat samples demonstrated the reliability and efficiency of ic-ELISA. Results indicated that standardised ic-ELISA was suitable for DON screening in wheat samples and the need for continuous monitoring of mycotoxin levels in foodstuffs.     

Quinoxaline-2-carboxylic acid in pigs: criteria to distinguish between the illegal use of carbadox and environmental contamination

Carbadox cannot be used in food-producing animals within the European Union following the adoption of Commission Regulation EC 2788/98/EC. Monitoring of the longest remaining residue—quinoxaline-2-carboxylic acid (QCA)—is the most effective way of enforcing the prohibition on its use. The study was under taken to determine if QCA could be passed from pig to pig following the exposure of unmedicated animals to housing that had previously contained medicated animals. Drug-withdrawal studies were also carried out on medicated animals. Distinction between treated animals and those exposed to QCA might be required by competent national authorities to determine whether a positive result for QCA in tissue is truly 'violative'. Comparison of the ratio concentrations of QCA in tissues and body fluids was made to determine if they could be used as criteria for discrimination between illegally treated animals and environmental contamination.     

Tissue residues of clopidol (3,5-dichloro-2,6-dimethyl-4-pyridinol) in chickens in relation to withdrawal times

Broiler chickens were fed on a commercial diet containing 0.0125% clopidol for 34 days. They were killed 0-10 days after withdrawal of the premedicated feed and clopidol was determined in liver and muscle samples by a sensitive gas-liquid chromatography (g.l.c.) method. During the first two post-withdrawal days the clopidol concentration in the liver decreased rapidly from 7 to 0.5 mg/kg and the level in muscle declined from 3 to 0.1 mg/kg. There was little decline in the clopidol concentrations from days 2 to 10, the levels during this period being 0.2-0.8 mg/kg in liver and 0.05-0.2 mg/kg in muscle. In addition to the above experimental study, liver and muscle samples collected at a Swedish slaughterhouse from broiler chickens raised on clopidol-containing feed were analysed for residues of this drug. Large variations were found in the clopidol levels in broilers from different producers. The levels in the liver ranged from 0.05 to 8.0 mg/kg and those in muscle from 0.03 to 3.5 mg/kg. The present results emphasize the need to carry out field studies to check the levels of feed additive residues in edible tissues from chickens.     

超高效液相色谱-荧光检测法测定禽肉组织中乙氧酰胺苯甲酯残留量

建立超高效液相色谱-荧光检测法测定禽肉组织中乙氧酰胺苯甲酯（ethopabate,ETP）残留量的分析方法。样品用乙腈提取,采用C18色谱柱（2.1 mm×100 mm,1.8 μm）,乙腈-水（30∶70,V/V）为流动相,流速0.3 mL/min,柱温30 ℃,进样量5.0 μL,采用荧光检测器,在激发波长272 nm、发射波长394 nm条件下测定。结果表明：ETP在5～500 ng/mL质量浓度范围内呈良好的线性关系（R2=0.999 7）;检出限为2 μg/kg,定量限为5 μg/kg;平均回收率为83.0%～91.5%,相对标准偏差为1.6%～2.9%。本方法适用于检测禽肉组织中ETP残留量。     

Distribution of chloramphenicol to tissues,plasma and urine in pigs after oral intake of low doses

Toxic effects of chloramphenicol in humans caused the ban for its use in food-producing animals in the EU. A minimum required performance level (MRPL) was specified for chloramphenicol at 0.3 μg kg^–1 for various matrices, including urine. In 2012, residues of chloramphenicol were found in pig urine and muscle without signs of illegal use. Regarding its natural occurrence in straw, it was hypothesised that this might be the source, straw being compulsory for use as bedding material for pigs in Sweden. Therefore, we investigated if low daily doses of chloramphenicol (4, 40 and 400 μg/pig) given orally during 14 days could result in residues in pig tissues and urine. A dose-related increase of residues was found in muscle, plasma, kidney and urine (showing the highest levels), but no chloramphenicol was found in the liver. At the lowest dose, residues were below the MRPL in all tissues except in the urine. However, in the middle dose, residues were above the MRPL in all tissues except muscle, and at the highest dose in all matrices. This study proves that exposure of pigs to chloramphenicol in doses occurring naturally in straw could result in residues above the MRPL in plasma, kidney and especially urine.     

Development of Monoclonal Antibodies and Indirect Competitive Enzyme-Linked Immunosorbent Assay Kits for the Detection of Clenbuterol and Salbutamol in the Tissues and Products of Food-Producing Animals

To better monitor the residues of clenbuterol and salbutamol in edible tissues and products of animals treated with these compounds, a monoclonal antibody (mAb) against the β-agonists was prepared, and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on this antibody was also developed. The hapten of salbutamol was synthesized and conjugated to carrier proteins via different linkers. Female Balb/c mice were immunized with the hapten-protein conjugates to produce monoclonal antibodies using the standard fusion procedures. After fusion and four cloning cycles, eight hybridomas were isolated and of which one clone, 6E5, that has the isotype IgG1, was selected for the present study. The cross-reactivities of the mAb 6E5 towards salbutamol, clenbuterol, mabuterol, cimaterol, clenproperol, mapenterol, tuoloterol and terbutaline were determined as 104.5, 100, 100, 83.6, 71.9, 44.2, 6.3 and 2.3%, respectively. The limit of detection for salbutamol and clenbuterol in edible animal tissues was 0.21 and 0.57 μg kg^?1, respectively. The recoveries ranged from 55.4 to 122.0%, and the CVs were less than 19.6%. When used with spiked or incurred samples, or in a field trial, the established ic-ELISA has demonstrated a consistent performance in various biological matrices, suggesting this is a sensitive, accurate and low-cost analytical method.     

Simultaneous determination of lincomycin and spectinomycin residues in animal tissues by gas chromatography-nitrogen phosphorus detection and gas chromatography-mass spectrometry with accelerated solvent extraction

A new multi-dimensional analytical method using gas chromatography-nitrogen phosphorus detection (GC-NPD) and gas chromatography-mass spectrometry (GC-MS) was developed for qualitative and quantitative measurement of lincomycin and spectinomycin residues in food animal tissues. This method is based on a new extraction procedure using accelerated solvent extraction (ASE). The analytes were extracted by phosphate buffer with trichloroacetic acid deproteinization and clean-up by C?? solid-phase extraction (SPE) adding dodecanesulfonic acid sodium salt as an ion-pair reagent. The eluted fraction was evaporated and derivatised with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) for GC-NPD analysis and GC-MS confirmation. Parameters for extraction pressure, temperature and cycle of ASE, clean-up, derivatisation and analysis procedure were optimised. The method was validated in muscle, kidney and liver of swine, bovine with a low concentration (limit of quantification) of 16.4 and 21.4 μg kg?1 for these two analytes using GC-NPD. For GC-MS, the limits of quantification were 4.1 and 5.6 μg kg?1, respectively. Spiked recoveries from levels of 20 to 200 μg kg?1 were found to be between 73% and 99% with a relative standard deviation (RSD) of less than 17% in GC-NPD. For GC-MS, levels from 5 to 20 μg kg?1 had between 70% and 93% with an RSD of less than 21%. This rapid and reliable method can be used for the characterisation and quantification of residues of lincomycin and spectinomycin in animal tissues.     

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.