Determination of β-agonists in pig feed,pig urine and pig liver using capillary electrophoresis with electrochemical detection

The fast separation capability of a capillary electrophoresis with electrochemical detection (CE–ED) system was demonstrated by determining five β-agonists including clenbuterol, metoprolol, ractopamine, isoxsuprine and salbutamol in several real samples. Factors influencing the separation and detection were examined and optimized. Under the optimum conditions, the five β-agonists could be well separated within 15 min at a separation voltage of 14 kV in a 100 mmoL H₃BO₃–Na₂B₄O₇ running buffer (pH 9.0). This method was successfully used in the analysis of clenbuterol, metoprolol, ractopamine, isoxsuprine and salbutamol in pig feed, pig urine and pig liver, providing a useful method for monitoring the illegal use of β-agonists from growth promotion in food-producing animals.     

Immunoaffinity chromatography purification and ultra-high-performance liquid chromatography-tandem mass spectrometry determination of four β-agonists in beef

A highly selective and sensitive method was developed for the simultaneous determination of four β-agonists (clenbuterol, salbutamol, ractopamine and terbutaline) in beef by immunoaffinity chromatography purification coupled to ultra-high-performance LC-MS/MS. The MS/MS conditions, ultra-high-performance LC mobile phase, injection solution, sample purification process and matrix effect were studied to optimise the operation conditions. The limits of detection (LODs) of the instrument for the studied β-agonists ranged from 0.20 to 0.25?µg?l^?1, and the LODs of the method for the studied β-agonists ranged from 0.20 to 3.00?µg?kg^?1 for beef. Calibration curves were constructed using a standard solution diluted with blank beef matrix. The linear ranges of the calibration curves ranged from 5 to 100?µg?kg^?1 and the coefficients of determination were >0.9942 (n?=?10) for all four β-agonists. Samples spiked at 5, 10 and 50?µg?kg^?1 showed recoveries >72% and RSDs <6.6%. The method is suitable for the simultaneous detection of four β-agonists at trace levels in beef.     

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.     

Immunoaffinity chromatography purification and ultra-high-performance liquid chromatography-tandem mass spectrometry determination of four β-agonists in beef

A highly selective and sensitive method was developed for the simultaneous determination of four β-agonists (clenbuterol, salbutamol, ractopamine and terbutaline) in beef by immunoaffinity chromatography purification coupled to ultra-high-performance LC-MS/MS. The MS/MS conditions, ultra-high-performance LC mobile phase, injection solution, sample purification process and matrix effect were studied to optimise the operation conditions. The limits of detection (LODs) of the instrument for the studied β-agonists ranged from 0.20 to 0.25 μg l(-1), and the LODs of the method for the studied β-agonists ranged from 0.20 to 3.00 μg kg(-1) for beef. Calibration curves were constructed using a standard solution diluted with blank beef matrix. The linear ranges of the calibration curves ranged from 5 to 100 μg kg(-1) and the coefficients of determination were >0.9942 (n = 10) for all four β-agonists. Samples spiked at 5, 10 and 50 μg kg(-1) showed recoveries >72% and RSDs <6.6%. The method is suitable for the simultaneous detection of four β-agonists at trace levels in beef.     

探索用液质串联联用法测定牛肉中的瘦肉精

目的:探索用液质串联联用法测定抽查牛肉中的瘦肉精含量。方法:酶解后的样品溶液,加入氘代克伦特罗与沙丁胺醇作为混合内标,过C18固相萃取小柱,用OasisMCX小柱收集,以3%的氨水甲醇溶液对MCX小柱进行洗脱后吹干,残余物溶于0.1%甲酸水溶液-甲醇溶液(95:5),以0.1%甲酸乙腈溶液和0.1%甲酸溶液为梯度流动相,上液质串联联用仪测定。结果:各β受体激动剂在0.5～5.0μg/kg浓度范围良好线性关系,平均回收率90.47%～102.58%,仪器定量限0.0543～0.2978μg/kg。结论:此方法极大排除了其他成分干扰,重现性好,专属性强,灵敏准确,可作检测牛肉中的瘦肉精残留量参考。     

Ultrasensitive and Specific Detection of Salbutamol in Swine Feed,Meat, and Urine Samples by a Competitive Immunochromatographic Test Integrated with Surface-Enhanced Raman Scattering

Salbutamol (SAL), a kind of β-agonist which can enhance the lean meat-to-fat ratio, has been inhibited as an additive used in animal feeds for livestock production in many countries due to its harmful effect to the consumers. In this study, an ultrasensitive and specific competitive immunochromatographic test (ICT) integrated with surface-enhanced Raman scattering (SERS) for the detection of SAL was described. The immunoprobe was prepared by immobilizing polyclonal antibody against SAL on the surface of Au@Ag nanoparticles in which the Raman reporter (4-mercaptobenzoic acid, MBA) had been sandwiched. After ICT procedures, the specific SERS signals generated from MBA on the test line of the ICT strip were measured for the quantitative determination of SAL. The assay was completed in 15 min. The IC₅₀ and the limit of detection (LOD) values of the assay for SAL were 0.028 ng mL^?1 and 3.0 pg mL^?1, respectively. There was no cross-reactivity (CR) of the assay with other three β-agonists (clenbuterol, phenylethanolamine A, and ractopamine), showing high specificity of the assay. Spiking experiments indicated that the average recoveries (n?=?3) of SAL from swine feed, meat, and urine samples were in ranges of 98.4–105.2 % with the relative standard deviations (RSDs) of 1.7–7.8 %. The results demonstrated that the proposed ICT was a feasible method for ultrasensitive and specific detection SAL in swine feed, meat and urine samples, and could be extended for the detection of other target analytes.     

高效液相色谱-串联质谱法测定卤肉中3种β-受体激动剂残留

建立卤肉中3种β-受体激动剂(克仑特罗、莱克多巴胺、沙丁胺醇)残留检测的高效液相色谱-串联质谱方法。样品经β-盐酸葡萄糖醛甙酶/芳基硫酯酶水解,MCX固相萃取柱净化,以乙腈-0.1%甲酸溶液作为流动相洗脱,采用电喷雾离子源,多反应监测方式进行采集,外标法定量。结果表明：3种β-受体激动剂在1～100ng/mL质量浓度范围内呈现良好的线性关系,R2均大于0.99,方法检测限均为0.25μg/kg,从0.5、1μg/kg和2μg/kg添加水平检测结果可以看出,方法平均回收率为80%～120%,相对标准偏差为1.0%～10.0%。     

β_2肾上腺素受体基因在Sf9细胞中的表达及纯化

通过密码子优化、昆虫杆状病毒表达系统(Bac-to-Bac)构建、表达条件筛选及镍柱亲和层析,研究β_2肾上腺素受体(β_2 adrenergic receptor,β_2AR)基因(β_2AR)在昆虫细胞Sf9中的高效表达及纯化策略。方法:人工合成改造后的β_2AR基因,将其克隆至转移载体p Fast Bac1中,构建重组杆状病毒表达质粒p Fast Bac1-β_2AR’,转染昆虫细胞Sf9,优化表达条件,采用镍离子亲和层析法纯化重组蛋白并进行活性鉴定。结果:适宜的表达条件为感染细胞使用的感染复数5、感染后表达时间48 h,Western blot分析显示在47 k D左右处出现清晰的特异性条带,与预期结果一致。纯化的受体蛋白纯度大于90%,活性鉴定结果显示该受体蛋白可特异性吸附盐酸克伦特罗、沙丁胺醇及莱克多巴胺3种β激动剂的酶标记物,OD值分别为0.983、0.947和0.912。结论:本研究实现了β_2AR受体在Sf9细胞中的表达,且纯化后的受体蛋白保持了较好的β激动剂亲和活性,为利用β_2AR受体开展β激动剂多残留快速检测技术提供了依据。     

Development of a polyclonal indirect ELISA with sub-ng g−1 sensitivity for the analysis of clenbuterol in milk,animal feed,and liver samples and a small survey of residues in retail animal products

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with sub-ng g^?1 sensitivity for clenbuterol was developed. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All four antisera displayed high sensitivity with IC₅₀ and limit of detection (LOD) values lower than 0.9 and 0.03 ng ml^?1, respectively. The most sensitive ELISA was established with IC₅₀ and LOD values of 0.1–0.3 and 0.01–0.02 ng ml^?1, respectively, which are more than ten times lower than those reported in the literature. The cross-reactivity (CR) values of the four antisera with salbutamol, another frequently used β-agonist of similar molecular structure to clenbuterol, were estimated to be within 25–46%. No binding (CR < 0.01%) of nine other drugs which are frequently used in animal feeds was observed. The superior ELISA at optimal assay conditions was used for the analysis of five clenbuterol-fortified samples and the results were confirmed by high-performance liquid chromatography (HPLC). Acceptable recovery rates of 92.2–97.0% and intra-assay coefficients of variation of 1.3–5.3% were obtained. The proposed ELISA was highly correlated with HPLC (R ²= 0.9893, n = 5). Another sixteen samples were randomly collected from local markets and analysed by ELISA. The highest clenbuterol residues found in milk, feed, swine and chicken livers were 5.33, 64.04, 2.28 and 0.74 ng g^?1, respectively.     

Analysis of Multiple β-Agonist and β-Blocker Residues in Porcine Muscle Using Improved QuEChERS Method and UHPLC-LTQ Orbitrap Mass Spectrometry

The objective of the present study is to develop and optimize a simple, high-throughput method for determining 14 β-agonists (i.e., banbuterol, brombuterol, cimaterol, cimbuterol, clenbuterol, clenpeterol, clorpenaline, isoxsuprine, mabuterol, mapenterol, ractopamine, salbutamol, terbutaline, and tulobuterol) and two β-blockers (propranolol and penbutolol) in porcine muscle. The samples were pretreated by a modified quick, easy, cheap, efficient, rugged, and safe (QuEChERS) method, and analysis was carried out in a reversed phase HSS T3 C18 column using gradients of acetonitrile and 5 mmol L^?1 ammonium acetate (0.1 % formic acid) solution for elution. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-LTQ Orbitrap MS, resolution 60,000) was used for qualification and quantification of the 16 target compounds. Under optimized conditions, the limits of detection and quantification obtained ranged from 0.17 to 1.67 μg kg^?1 and from 0.56 to 5.00 μg kg^?1, respectively. Recoveries for spiking levels of 5.0, 10.0, and 20.0 μg kg^?1 ranged from 62.4 to 121.9 %, from 60.4 to 104.3 %, and from 66.5 to 121.3 %, respectively. The relative standard deviations obtained were lower than 20 % for all spiking levels assayed. The proposed method was applied successfully in sample analysis, and satisfactory results were obtained.     

Quick screening of priority β-agonists in urine using automated TurboFlow™-LC/Exactive mass spectrometry

This paper describes a method for the determination of priority β-agonists in urine based on a fully automated sample preparation procedure using an online TurboFlow? chromatography clean-up step and determination with Orbitrap? mass analyser technology. The principle of the method was the enrichment of the β-agonists after enzymatic hydrolysis overnight on a small column packed with a special stationary phase (TurboFlow?) while flushing away sample matrix and interfering compounds. Thereafter, the analytes were transferred onto an analytical column and detected by liquid chromatography/high-resolution mass spectrometry in full-scan mode at a resolution of R?=?50,000 FWHM (full width at half maximum) and in higher energy collisional dissociation (HCD) scan mode at a resolving power of 10,000 FWHM. The optimisation of each step of the method, such as selection of the TurboFlow? and analytical column as well as sample loading and elution parameters were performed using a standard solution containing salbutamol, clenbuterol and mabuterol at a concentration of 100?μg?l(-1). The developed automated sample preparation significantly improved the throughput and efficiency of the previously used screening method and it resulted in a considerable reduction in analysis time. Validation experiments including 24 β-agonists in urine gave decision limits (CCα) between 0.05 and 0.35?μg?l(-1). The repeatability of analyses for urine samples spiked at 0.5?μg?l(-1) was within the range of 5-26% and recoveries for all compounds were within 89-107%.     

Detection of clenbuterol in beef meat,liver and kidney by mid‐infrared spectroscopy (FT‐Mid IR) and multivariate analysis

Mid‐infrared spectroscopy (FT‐Mid IR) coupled with multivariate analysis was used to predict clenbuterol in beef meat, liver and kidney. A SIMCA model was also developed to discriminate between pure (beef meat, liver and kidney) and spiked with clenbuterol samples (beef meat‐clenbuterol, liver‐clenbuterol and kidney‐clenbuterol). The best models to predict clenbuterol concentrations were obtained using the partial least squares algorithm (PLS) with a R² > 0.9 and SEC and standard error of prediction <0.296 and 0.324, respectively. The SIMCA model used to discriminate pure and spiked with clenbuterol samples showed 100% correct classification rate. Methods detection limit was 2 μg kg^?1. FT‐Mid IR coupled with chemometrics could be a simple and rapid screening tool for monitoring clenbuterol in beef meat, liver and kidney implicated in food poisoning. This method could be use for screening purposes.     

Development of a Competitive Indirect Enzyme-Linked Immunosorbent Assay for Screening Phenylethanolamine A Residues in Pork Samples

Phenylethanolamine A (PEA), a new alternative β-agonist, has been illegally used in farming to promote the muscle growth in food-producing animals. In this study, a sensitive and convenient competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for determination of PEA residues in pork samples. The produced antibody was highly specific to PEA and exhibited a negligible cross-reactivity toward some other β-agonists. The developed technique was characterized by the limit of detection below 0.08 μg kg^?1 and the IC₅₀ value of 0.93 pmol mL^?1 (0.32 ng mL^?1). Validation of the technique was done using artificially spiked and naturally contaminated pork samples. The recoveries ranged from 79.6 to 112.6 % for the samples spiked at levels of 0.1–5 μg kg^?1 with the variation coefficients below 15 %. The analysis of naturally contaminated samples showed that the obtained data corresponded with the data obtained by the LC-MS/MS. The developed ciELISA was shown to be a feasible highly sensitive and specific screening tool for PEA residue analysis.     

Predicted intake of trace elements and minerals via household drinking water by 6-year-old children from Krakow,Poland. Part 5: Zinc

Zinc (Zn) exposure in pre-school children via household drinking water collected by a double sampling method (morning, evening) was evaluated in a sample of the Polish population. Zn concentration was measured by flame atomic absorption spectrometry. Rural and suburban Krakow sites were non-distinguishable in respect of Zn concentrations. However, significantly lower Zn was found in urban as compared with non-urban sites [geometric mean (95% confidence interval) 0.14 (0.01–1.95) mg l^?1 versus 0.52 (0.03–10.2) mg l^?1, p < 0.001.] Zn levels in water standing overnight in pipelines were higher in all sites by 0.36 mg l^?1 on average, but observed really contaminations were higher. The Zn limit based on the taste and colour of drinking water (3 mg l^?1) was exceeded in 1% and 10% of households from urban and non-urban sites, respectively. The Zn intake predictions for evening water samples for 6-year-old children averaged between 2% and 9% of the recommended dietary allowance (RDA, 10 mg day^?1) for urban and non-urban sites, respectively. Mean Zn intake prediction for the exceedance fraction was 64% of RDA. In conclusion, overnight contamination of drinking water from in-house pipelines was significant and common to all sites investigated. Secondly, drinking water can be considered a significant contributor to dietary Zn intake by children in non-urban sites and may shift the population borderline of deficiency.     

β2肾上腺素受体基因的改造及在无细胞合成体系中的表达

依据大肠杆菌密码子偏好性,设计合成β2肾上腺素受体（β2 adrenergic receptor,β2AR）基因序列,应用大肠杆菌无细胞系统对其进行高效表达,经负载镍离子的顺磁颗粒MagneHis? Ni-Particles纯化后,对受体蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）和活性鉴定。结果表明：改造后的β2AR基因密码子适应指数（codon adaptation index,CAI）为0.96,GC含量从58%降低到46.17%,更有利于该基因在大肠杆菌系统中的表达。表达体系中优化后的Mg2＋浓度为22?mmol/L,此时表达量为1 250 μg/mL。SDS-PAGE分析显示,纯化蛋白在47?kDa左右出现清晰的特异性条带,与预期结果一致,纯度大于90%。直接酶受体分析检测结果显示,当纯化受体蛋白1∶500稀释包被时,该重组受体与盐酸克伦特罗、沙丁胺醇及莱克多巴胺的酶标记物结合的OD值分别为0.976、0.836和0.728,亲和活性依次降低。β2AR蛋白在无细胞体系中的成功表达,为研发基于受体的β激动剂多残留快速检测技术提供了理论支持。     

Simultaneous and confirmative detection of multi-residues of β2-agonists and β-blockers in urine using LC-MS/MS/MS coupled with β-receptor molecular imprinted polymer SPE clean-up

A liquid chromatography–linear ion-trap spectrometry (LC-MS³) method using β-receptor molecular-imprinted polymer (MIP) solid-phase extraction (SPE) as clean-up was developed to determine simultaneously and confirmatively residues of 25 β₂-agonists and 21 β-blockers in urine samples. Urine samples were subjected to enzymatic hydrolysis by β-glucoronidase/arylsulphatase, and then extracted with perchloric acid. Sample clean-up was performed using β-receptor MIP SPE. A Supelco Ascentis^® express Rp-Amide column was used to separate the analytes, and MS³ detection used an electrospray ionisation source in positive-ion mode. Recovery studies were carried out using blank urine samples fortified with the 46 analytes at the levels of 0.5, 1.0 and 2.0 μg l^–1. Recoveries were obtained ranging from 60.1% to 109.9% with relative standard deviations (RSDs, n = 7) from 0.5% to 19.4%. The limits of detection (LODs) and limits of quantitation (LOQs) of the 46 analytes in urine were 0.02–0.18 and 0.05–0.60 μg l^–1, respectively. As a result of the selective clean-up by MIP SPE and MS³ detection of the target drugs, the sensitivity and accuracy of the present method was high enough for monitoring β₂-agonist and β-blocker residues in urine samples. Satisfactory results were obtained in the process of the determination of positive urine samples.     

羊奶及奶粉中27种β-受体激动剂类药物残留UPLC-MS/MS检测方法

采用超高效液相色谱串联质谱建立羊奶及奶粉中吡布特罗、丙卡特罗、瑞普特罗、克伦普罗、奥达特罗等27种β-受体激动剂类药物残留检测方法。样品经过1%甲酸乙腈提取、Oasis PRi ME HLB柱净化后,经Acquity UPLC BEH C18（100 mm×2.1 mm,1.7μm）分离,以甲醇和0.1%甲酸水溶液为流动相进行梯度洗脱,多通道MRM信号采集模式,27种β-受体激动剂类药物能在10.5 min内出峰完好,在1.0¹00.0μg/L浓度范围内,27种β-受体激动剂类药物线性良好,相关系数均在0.993以上;羊奶中最低定量限均低于0.5μg/kg,通过0.5、2.5、5.0μg/kg三个浓度的加标回收实验表明,回收率为65.7%¹19%,批内变异系数为0.79%¹0.4%,批间变异系数为2.13%¹5.7%。奶粉中最低定量限均低于2.0μg/kg,通过2.0、10.0、20.0μg/kg三个浓度的加标回收实验表明,回收率为60.5%¹09%,批内变异系数为1.91%¹2.4%,批间变异系数为3.06%¹7.2%。所建立方法为一种高通量检测羊奶及奶粉中β-受体激动剂类药物残留确证分析方法。      

超高效液相色谱-串联质谱方法同时测定猪肉中7种β-受体激动剂残留量

建立同时测定猪肉中苯乙醇胺A、盐酸克伦特罗、沙丁胺醇、莱克多巴胺、特布他林、西马特罗和氯丙那林7种β-受体激动剂残留量的超高效液相色谱-串联质谱方法（UPLC-MS/MS）。样品采用1%甲酸-乙腈一次性振荡提取,用β-受体激动剂专用固相萃取柱净化、多反应监测（MRM）、内标法定量。结果表明：7种β-受体激动剂检出限均为0.2μg/kg,定量限为0.5μg/kg。空白猪肉中添加1、2、10μg/kg水平,7种β-受体激动剂总体平均回收率72.2%～116.9%,总体相对偏差均小于2.5%。该方法不需要酶解,分析速度快,灵敏度高,重现性好,各项技术指标均满足国内外相关法规要求,可用于猪肉中7种β-受体激动剂残留的快速检测。     

Prevalence of arcobacter species in drinking water, spring water, and raw milk as determined by multiplex PCR

The aim of this study was to investigate the incidence of Arcobacter species in water sources and raw milk from healthy animals in Kayseri, Turkey. A total of 175 samples of drinking water (n = 100), spring water (n = 25), and raw milk (n = 50) were examined. Arcobacter species were isolated using the membrane filtration technique. Overall, 7 (4%) of the 175 samples yielded Arcobacter spp.: 3 (3%) drinking water samples, 1 (4%) spring water sample, and 3 (6%) raw milk samples. Two species of Arcobacter were recovered from the seven positive samples: Arcobacter butzleri, Arcobacter skirrowii, and A. butzleri plus A. skirrowii found in 3 (1.7%), 2 (1.1%), and 2 (1.1%) samples, respectively. Our study is the first to report the isolation of both A. butzleri and A. skirrowii together from drinking water and is the first report of Arcobacter in milk from healthy cows in Turkey. Based on these findings, the presence of Arcobacter species in environmental waters and raw milk may pose a potential hazard for human health.     

Development of a label-free electrochemical immunosensor based on carbon nanotube for rapid determination of clenbuterol

We have fabricated a label-free electrochemical immunosensor for the detection of clenbuterol, a kind of β-agonist. Clenbuterol was covalently linked to multi-wall carbon nanotubes (MWCNTs) through a two-step process using 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide and N-hydroxysulfo-succinimide as crosslinkers. The clenbuterol-MWCNT conjugates were cast on a glassy carbon electrode. Cyclic voltammetry and differential pulse voltammetry were employed to monitor the fabrication steps of immunoreaction system using the redox probe of K₃Fe(CN)₆. In the presence of monoclonal antibody against clenbuterol, the redox peak current of [Fe(CN)₆]^3−/4− was decreased, presumably due to that antibody in solution could adsorb on the electrode surface modified clenbuterol-MWCNT conjugates. The selected monoclonal antibody showed very high sensitivity and specificity for clenbuterol, and was used for the detection and quantitative determination of clenbuterol in solution with a competitive mechanism. This approach provided a detection limit of 0.32 ng mL⁻¹. Accurate detection of clenbuterol in spiked animal feeds was demonstrated by comparison with conventional ELISA assays and LC–MS method.