Development of an immunochromatographic assay for the β-adrenergic agonist feed additive zilpaterol

Zilpaterol is a β-adrenergic agonist feed additive approved in the United States to increase weight gain and improve feed efficiency of cattle. A zilpaterol immunochromatographic assay was developed as an economical and user-friendly rapid detection method for zilpaterol and validated using urine and tissue samples derived from animal studies. The assay sensitivity was 1.7–23.2 ng g^?1 or mL^?1 across a variety of feed and animal matrices and did not cross-react with clenbuterol or ractopamine. No sample pre-treatment of cattle and sheep urine was needed, but horse urine and feed required dilution; skeletal muscle required solvent extraction prior to testing. Of 32 incurred sheep urine samples tested, zilpaterol content was correctly identified in all but 2 samples. Horse urine containing >10 ng mL^?1 of incurred zilpaterol residue (n = 48) was correctly identified as zilpaterol positive. The assay correctly identified 0-day withdrawal sheep muscle samples as zilpaterol positive and the control and longer withdrawal day sheep muscle samples as negative. Zilpaterol was demonstrated to be stable in horse urine when stored at ?20°C for 7 years.     

Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereoisomers and bovine metabolites

Several commercially available immunoassay kits have been developed to detect the beta-adrenergic agonist clenbuterol HCl. Technical materials supplied with the kits do not generally report cross-reactivity with clenbuterol metabolites. Use of such kits to quantitate clenbuterol might lead to an overestimation of parent drug if metabolites were present. The objective of this study was to measure the cross-reactivity of clenbuterol metabolites with several commercially available clenbuterol immunoassays. Three clenbuterol-glucuronide conjugates, clenbuterol-sulphamate, 4-amino-3,5-dichloro-hippuric acid (clenbuterol-hippurate), and purified clenbuterol-stereoisomers were tested for cross-reactivity. The clenbuterol-sulphamate metabolite showed significant cross-reactivity (42-77%), but clenbuterol-hippurate showed very little competition (< 0.2%) towards clenbuterol. Clenbuterol-glucuronides had little (0.1-1.6%) cross-reactivity. In addition, (R)-, (S)-, and racemic clenbuterol were used to determine the stereospecificity of the kits. Both (R)- and (S)-clenbuterol competed for binding in two of the kits, however, in one kit the (S)-clenbuterol stereoisomer had an affinity 100 times greater than the (R)-stereoisomer. The presence of significant quantities of the sulphamate metabolite of clenbuterol in a biological matrix would cause an overestimation of the amount of parent clenbuterol. This study illustrates the inherent problems of using unvalidated immunoassays for quantitation purposes.     

盐酸克伦特罗时间分辨荧光免疫检测方法的建立

采用时间分辨荧光免疫分析(TRFIA)技术建立快速、高灵敏度的盐酸克伦特罗(CLB)全自动检测方法。以CLB-OVA包被96微孔板作为固相抗原,与游离CLB竞争限量的以Eu3+标记的抗CLB单克隆抗体,建立解离增强模式的荧光免疫分析体系检测CLB。该方法的灵敏度为0.03ng/mL,批内和批间变异系数分别为2.0%和8.9%,平均回收率为105.8%,与沙丁胺醇、莱克多巴胺的交叉反应率分别为0.96%和0.77%,结果表明,用TRFIA法检测CLB,灵敏度高、特异性强、稳定性好,具有良好的应用前景。     

Screening procedures for clenbuterol residue determination in raw swine livers using lateral-flow assay and enzyme-linked immunosorbent assay

Clenbuterol, which may cause symptoms of increased heart rate, muscular tremors, headache, nausea, and muscular cramps in patients, has been prohibited for consumption in many countries including the European Union, the United States, and China. A rapid lateral-flow strip assay was developed in our laboratory, and results obtained with this assay were compared with those obtained with a commercial enzyme-linked immunosorbent assay (ELISA) kit for the screening of clenbuterol in raw swine liver. A total of 128 swine livers were acquired from five local markets and prepared for analysis by the lateral-flow strip assay and ELISA. Analysis was completed in 10 min with the lateral-flow strip assay and in 90 min with the ELISA. In parallel with the ELISA, the rapid detection strip produced no false-negative results but had a false-positive rate of 6.3%. Cross-reactivity of the strip was assessed and was negative after tests with clenbuterol analogues such as terbutaline, salbutamol, ractopamine, ritodrine, and fenoterol. These data suggest that a lateral-flow strip assay can be used safely as a screening method as part of a clenbuterol residue surveillance program and should be a valuable tool in the food safety field, especially in developing countries.     

克仑特罗食物中毒的酶联免疫法筛选与气-质联机确证研究

本文采用酶联免疫法筛选和气-质确证对克仑特罗食物中毒病因学诊断技术进行研究。组织样品经匀浆初提,液液萃取;生物材料经离心适当稀释,上酶标板显色进行筛选测定。克仑特罗浓度的自然对数与吸光度比值呈线性关系,线性方程Y=-22.078ln(X)+199.24,相关系数为0.9922。检出限为0.1μg/kg。在对猪肝、鸡肉和尿液样品基质的不同加标水平的回收率在50.5%～92.0%之间,相对标准偏差在12.3%～18.1%之间。中毒者食用中毒食品(酱猪肝)及血液和尿液的测定结果与气质联机法确证结果基本吻合,而且生物体液(特别是尿液在中毒7d内仍可以作为克仑特罗食物中毒的辅助病因学诊断材料。     

Effects of frozen storage on the recovery of clenbuterol from urine samples for official control.

The effects of storage under frozen conditions on clenbuterol recovery from spiked calf urine samples were studied. Urine samples contaminated with 10 micro g l(-1) clenbuterol and frozen at -15 degrees C were analysed every 15 days over 6 months. A single frozen contaminated urine sample was also thawed every 15 days over 3 months for the analysis of 10-ml aliquots, after which the remaining portion was frozen again (-15 degrees C). The presence of clenbuterol was determined by GC-MS. A gradual decline in clenbuterol recovery was observed from the first to the 180th day. It was only possible to recover 1.74 +/- 0.06 micro g l(-1) (17%) clenbuterol on the 180th day in samples stored at -15 degrees C. Likewise, clenbuterol totally disappeared from spiked urine samples that had been successively frozen and thawed over 3 months. The results were confirmed in a study performed on 2704 calf urine samples collected on farms and analysed by HPTLC and GC-MS. Of 73 positive samples, 61 had been frozen for <10 days.     

应用超高效液相色谱-四级杆-飞行时间质谱 鉴定克伦特罗在猪尿中的主要代谢产物

目的 应用超高效液相色谱/四级杆-飞行时间质谱技术分析鉴定了克伦特罗在猪尿中的代谢产物,并推测克伦特罗在猪体内的主要代谢途径。方法 按10 mg/kg bw的剂量,给猪口服灌食克伦特罗,分别采集给药前及给药后的尿液样品。应用超高效液相色谱/四级杆-飞行时间质谱技术对样品进行分析,采用MetaboLynx XS软件进行数据处理,共检测到6 种克伦特罗的代谢产物,并根据碎片离子信息进行了结构鉴定。结果 猪尿中克伦特罗的代谢产物包括4-N-羟基克伦特罗（4-N-OH-CLE）、4-硝基克伦特罗（4-NO2-CLE）、克伦特罗及4-N-OH-CLE的葡萄糖醛酸结合物（GLU-CLE和GLU-OH-CLE）等。结论 根据所检测到的代谢产物,克伦特罗在猪体内的代谢途径包括4-N-氧化和葡萄糖醛酸结合等。     

Evaluation of the illegal use of clenbuterol in Portuguese cattle farms from drinking water,urine, hair and feed samples

The recent discovery of clenbuterol contamination in Portuguese food led to the specific inspection of 16 cattle farms for β-agonists, involving the analysis of a total of 486 samples (78 feed, 106 drinking water, 168 urine and 134 hair). The samples were screened for the β-agonists: bromobuterol, cimaterol, clenbuterol, clenpenterol, clenproperol, hydroxymethylclenbuterol, mapenterol, salbutamol and terbutaline. Only clenbuterol was found in all analyzed matrices and the most likely method of illegal administration to animals was through drinking water. Of all samples analysed, 14.15% of drinking water were found positive in the range 0.03–3.80 mg l^?1 clenbuterol. Inclusion of hair samples in the Portuguese plan for clenbuterol residue control in live animals is discussed.     

Eu3+ 标抗原盐酸克伦特罗的时间分辨荧光免疫检测

采用时间分辨荧光免疫分析(time-resolved fluoroimmunoassay,TRFIA)建立简便、快速、灵敏的盐酸克伦特罗(clenbuterol hydrochloride,CBL)检测方法。以自制盐酸克伦特罗单克隆抗体包被96 孔微孔板作为固相抗体,Eu3+ 标记抗原与样品中的CBL 竞争结合抗体,建立直接竞争荧光免疫分析体系。猪尿、组织样品添加回收率实验表明：方法灵敏度为0.02μg/L,样品回收率为91%～101%,与沙丁胺醇、莱克多巴胺等结构类似物的交叉反应率均小于1%。建立盐酸克伦特罗铕标抗原直接竞争时间分辨免疫检测方法,方法灵敏度高、特异性强,且操作简单,完全可以满足现有实际检测需求。     

Plasma calcium concentrations are decreased at least 9 hours before parturition in multiparous Holstein-Friesian cattle in a herd fed an acidogenic diet during late gestation

Calcium homeostatic mechanisms are challenged in periparturient multiparous dairy cattle due to the rapid transport of large amounts of calcium into the mammary gland associated with colostrogenesis, resulting in decreased plasma total calcium concentration ([Ca]). An unresolved issue is the timing of the decrease in plasma [Ca] relative to the time of parturition, with the consensus view being that plasma [Ca] does not decrease until after parturition. The objective of this study, therefore, was to characterize the change in plasma [Ca] over time in periparturient dairy cattle. Plasma and mid-stream urine samples were collected daily starting 3 d before calving from 104 periparturient Holstein-Friesian dairy cows in a herd fed an acidogenic total mixed ration during the late dry period. Mixed-models ANOVA and linear and multivariable regression analyses were conducted. Plasma [Ca] decreased in periparturient multiparous cattle (n = 70) but not in primiparous cattle (n = 34). Compared with mean values approximately 72 h before parturition ([Ca] = 2.32 mmol/L), mean plasma [Ca] in multiparous cattle first decreased at 9 h before parturition (2.13 mmol/L) and remained decreased for up to 48 h after parturition, with the lowest mean value (1.87 mmol/L) occurring at 28 h after parturition. Mean 24-h urine Ca excretion was calculated to decrease by 3.5 to 3.8 g in periparturient multiparous cattle. Regression analysis indicated that plasma [Ca] in the 12-h period before and 24-h period after parturition was strongly and negatively associated with age but was also negatively associated with milk production indices. We conclude that plasma [Ca] was decreased at least 9 h before parturition in multiparous dairy cattle fed an acidogenic diet in late gestation, and that calcium homeostasis was disrupted for 2 to 3 d around parturition.     

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.     

盐酸克仑特罗ELISA试剂盒的研制

在间接竞争酶联免疫吸附法的基础上,研制了一种可以检测尿样或食品中盐酸克仑特罗的试剂盒。ELISA检测方法的各个影响因素均通过实验进行了优化。该试剂盒具有良好的灵敏度,半抑制率(IC50)为0.6ng/ml,该抗体与肾上腺素等结构类似物的交叉反应率均小于0.3%,回收率为90%～104%,批内变异系数小于6%,批间变异系数小于10%,试剂盒在12个月保存期内稳定。可用于盐酸克仑特罗的快速定量检测。     

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 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.     

ELISA检测盐酸克伦特罗残留的方法学评价

以盐酸克伦特罗(clenbuterolhydrochloride)重氮化后分别连接到牛血清蛋白(BSA)和卵清蛋白(OVA)上制得免疫原BSA-CL和包被抗原OVA-CL。通过免疫兔获得含有多克隆抗体的血清,经硫酸铵沉淀、纯化,得到兔源抗CL的抗体,在此基础上建立了间接酶联免疫检测方法。实验结果表明,抗CL抗体最适稀释度为1:1000,羊抗兔酶联抗体(HRP-IgG)的最适稀释度为1:1500。该检测方法的检测灵敏度为1.452μg/L,线性检测范围为7.26～90.75μg/L。     

Quantification of free and protein-bound trans-resveratrol metabolites and identification of trans-resveratrol-C/O-conjugated diglucuronides - two novel resveratrol metabolites in human plasma

The polyphenol trans-resveratrol (t-RES) is present as t-RES-3-O-beta-D-glycoside, termed piceid, in several plant-derived foods. Although data on the metabolism and on in vivo effects of t-RES have been reported, quantitative data on the metabolites formed after dietary intake of t-RES or piceid are still lacking. In this study, 85.5 mg of piceid per 70 kg of body weight (bw) were administered to healthy volunteers in a bolus dose. t-RES metabolites formed in plasma and urine were identified and quantified by LC-MS/MS, NMR, and HPLC-DAD analysis using chemically synthesized t-RES conjugate standards. In addition, the amount of t-RES metabolites bound noncovalently to plasma proteins was determined for the first time in humans. The metabolites identified and quantified were t-RES-3-sulfate, t-RES-3,4'-disulfate, t-RES-3,5-disulfate, t-RES-3-glucuronide and t-RES-4'-glucuronide, with t-RES-sulfates being the dominant conjugates in plasma and urine. Besides these metabolites, two novel t-RES-C/O-conjugated diglucuronides have been identified and quantified in plasma and urine. Moreover, it could be shown that up to 50% of the plasma t-RES-3-sulfate, t-RES-disulfates, and the novel t-RES-C/O-diglucuronides were bound to proteins. Total recovery of the dietary administered piceid in urine ranged between 13.6 and 35.7%.     

高效液相色谱内标法测定动物饲料中盐酸克伦特罗

以茶碱为内标物,建立高效液相色谱内标法测定禽畜饲料中盐酸克伦特罗的检测方法。样品经三氯乙酸溶液提取,经过Oasia HLB固相萃取小柱（3 mL/60 mg）处理,再用体积分数为5%的甲醇溶液冲洗后,采用Nova-Pak C18色谱柱（3.9 mm×150 mm×5 μm）分离,以乙腈-28 g/L磷酸二氢钠缓冲溶液（25:75,V/V）为流动相进行洗脱,流速0.8 mL/min,检测波长210 nm。结果表明,茶碱在该条件下能够与盐酸克伦特罗分离完全,可以作为测定盐酸克伦特罗的内标物。盐酸克伦特罗的加标回收率为97.8%～99.2%,精密度试验结果的相对标准偏差（RSD）为0.13%,表明该方法操作简单、准确度高、精密度良好,可用于饲料中盐酸克伦特罗含量分析。     

微型丝网印刷电极快速检测克仑特罗的方法

研制能用于96 孔酶标板快速检测的微型丝网印刷电极,并建立克仑特罗的电化学分析方法。通过间接竞 争免疫反应与计时电流法相结合实现克仑特罗的残留量的定量测定。实验中考察并优化免疫反应与酶催化反应的 条件。结果表明：在优化的条件下,克仑特罗标准溶液质量浓度在0.025～2.0 μg/L范围内呈现良好线性关系（r为 0.999 2）。采用本实验方法检测猪肉和猪尿中克仑特罗的结果与商品化ELISA试剂盒一致。本方法检测限分别为 0.18 μg/L和0.05 μg/L,比ELISA试剂盒检测限低一个数量级。     

Clenbuterol residues in pig muscle after repeat administration in a growth-promoting dose

The aim of this study was to determine the level of clenbuterol residues in muscle tissue of pigs after repeat administration in a growth-promoting dose. An anabolic dose of clenbuterol (20 μg/kg body mass per day) was administered orally to experimental group (n = 12) for 28 days, whereas control animals (n = 3) were left untreated. Clenbuterol treated pigs were randomly sacrificed (n = 3) on days 0, 3, 7 and 14 of treatment discontinuation and clenbuterol residues determined in muscle tissue. Determination of residual clenbuterol was by enzyme-linked immunosorbent assay (ELISA) as a screening method and liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmation method. The highest clenbuterol content in the muscle of treated animals was recorded on day 0 of treatment cessation (4.40 ± 0.37 ng/g) and significantly (p < 0.05) exceeded the maximum residue limit (MRL) of 0.1 ng/g. On day 3 of withdrawal, it was 0.49 ± 0.22 ng/g and on day 7 0.10 ± 0.02 ng/g (at MRL); on day 14 of treatment discontinuation, clenbuterol content was below the limit of detection (< 0.1 ng/g) in all samples. Administration of clenbuterol as a growth promoter in pig production could lead to residues in meat for human consumption up to 7 days after treatment discontinuation.     

糖化酶活力测定方法研究

烟用酶制剂中糖化酶的活力是烟草工业生产中的重要指标。建立了3,5-二硝基水杨酸（DNS）测定糖化酶活力的方法。结果表明,在碱性条件下,糖化酶水解液与DNS反应产物的最大吸收波长为480 nm,线性范围为1.30～171.5 U/mL,加标回收率为99.8%～103.4%,RSD为0.7%～2.9%,检出限为1.58 U/mL,该方法可用于烟用酶制剂样品中糖化酶的测定,与标准方法相比,其准确性符合要求,且具有简便、快速的特点。