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。     

3种β-兴奋剂化学发光酶免疫试剂盒分析检测方法的评价

选取3种市售β-兴奋剂化学发光酶免疫分析检测试剂盒,从线性关系、灵敏度、选择性、检出限、准确度、重复性及变异性等方面进行实验,并经农业部1025号公告—18—2008中高效液相色谱—串联质谱法验证比较,初步建立了一套化学发光酶免疫分析检测试剂盒质量评价方法。结果表明:沙丁胺醇、盐酸克伦特罗、莱克多巴胺3种试剂盒在测定范围内线性关系良好,灵敏度分别为0.7、1.8、1.0 ng/mL,检出限分别为0.2、0.1、0.2 ng/mL,猪肝、猪肉和牛肉的回收率在81.9%~115.2%之间,重复性变异系数为3.3%~9.7%,批间变异系数为9.9%~16.5%,交叉反应率均符合产品说明书要求,可以用于猪肝、猪肉、牛肉样品中沙丁胺醇、盐酸克伦特罗、莱克多巴胺的残留检测。     

Detection and quantification of soy allergens in food: study of two commercial enzyme-linked immunosorbent assays

ABSTRACT:  The objective of this study was to evaluate the efficacy of 2 commercially available soy enzyme-linked immunosorbent assays (ELISA) and use them in detecting soy proteins in selected food commodities. Both ELISA kits exhibited high sensitivity. The determined limits of detection (LOD) (approximately 2 and <1 μg/mL for Tepnel Biosystems and Elisa Systems kits, respectively) were lower than those claimed by the manufacturer. Quantification range for both kits was, however, narrower and in a lower concentration range than defined by the kit providers. Our examination revealed a positive cross-reactivity with chickpea proteins and matrix interferences for both kits. The immunoreactivity of soy proteins, when tested by the Tepnel Biosystems kit, was partially reduced by papain and bromelain hydrolysis; it was significantly decreased by protein glycation (>47%). Nondenatured and nonheated soy protein isolate (SPI) samples were also significantly less antigenic than the treated ones.     

酶联免疫法检测猪肉中呋喃唑酮代谢物残留

通过检测猪肉中呋喃唑酮代谢物的残留以评价试剂盒的性能,采用酶联免疫法对猪肉中的呋喃唑酮代谢物残留量进行测定。结果表明:该方法的线性检测范围为0.025～2.025μg/L,最低检测限为0.1μg/kg,样本添加回收率为78.2%～100.3%,变异系数为4.7%～10.3%,与呋喃唑酮的交叉反应率为16.3%,与其他药物的交叉反应率均小于1%,且对实际样本的检测结果与液相色谱-串联质谱法基本一致。该法灵敏准确、重复性好、特异性高,适用于猪肉中呋喃唑酮代谢物残留的检测。     

Inter-laboratory validation study of five commercial ELISA test kits for the determination of peanut proteins in biscuits and dark chocolate.

The results of an inter-laboratory study with five commercially available peanut ELISA test kits to detect and quantify peanut residues in two food matrices (biscuit and dark chocolate) at four different concentrations (0-10 mg peanut kg(-1) matrix corresponding to about 0-2.5 mg peanut protein kg(-1) matrix) are reported. In general the five ELISA test kits evaluated could detect peanut protein in the two food matrices. In three cases, the study challenged the test kits beyond their intended use for quantification below the manufacturers' defined cut-off limits. Generally, all five ELISA test kits performed well in the concentration range 5-10 mg kg(-1) rather than in the low concentration range (2.0 or 2.5 mg kg(-1)). The variation in the found recoveries of peanut between the different test kits had a spread of 44-191% across all concentrations. The quantification characteristics between test kits differed significantly at the very low mg kg(-1) level. Two test kits performed well even at concentrations below 5 mg kg(-1) with reproducibilities of 27-36% for biscuits and 45-57% for chocolate.     

Cross-reactivity of some commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns to DON- and ZEN-conjugated forms and metabolites

Seven commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns (IACs) were tested for cross-reactivity to conjugated forms (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, DON-3-glucoside, DON-3-glucuronide, ZEN-glucosides, ZEN-glucuronide) and metabolites (de-epoxydeoxynivalenol, α-zearalenol, β-zearalenol) and nivalenol (NIV), using a semi-quantitative multi-mycotoxin ultra-performance liquid chromatography-tandem mass spectrometry method. The DON IACs showed cross-reactivity for nearly all DON derivatives tested. The ZEN IACs showed limited cross-reactivity to some of the ZEN derivatives. The IACs were evaluated for their potential use as sample clean-up for mycotoxins in serum.     

Cross-reactivity of some commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns to DON- and ZEN-conjugated forms and metabolites

Seven commercially available deoxynivalenol (DON) and zearalenone (ZEN) immunoaffinity columns (IACs) were tested for cross-reactivity to conjugated forms (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, DON-3-glucoside, DON-3-glucuronide, ZEN-glucosides, ZEN-glucuronide) and metabolites (de-epoxydeoxynivalenol, α-zearalenol, β-zearalenol) and nivalenol (NIV), using a semi-quantitative multi-mycotoxin ultra-performance liquid chromatography-tandem mass spectrometry method. The DON IACs showed cross-reactivity for nearly all DON derivatives tested. The ZEN IACs showed limited cross-reactivity to some of the ZEN derivatives. The IACs were evaluated for their potential use as sample clean-up for mycotoxins in serum.     

Reproducibility of a commercially available kit utilizing enzyme-linked immunosorbent assay for determination of aflatoxin in peanut butter

The reproducibility of a commercially available kit utilising enzyme-linked immunosorbent assay (ELISA) for determination of total aflatoxins has been analysed. Peanut butter samples contaminated with aflatoxin at three concentrations (mean 5.8, 11.7 and 24.8 ng/g) were analysed on different occasions using four kits. Coefficients of variation at all concentrations ranged from 2.6% to 13.6% within replicate, 0.3% to 16.2% within plate and 6.7% to 10.2% between kits. Standard curve precision profiles showed a minimum coefficient variation (CV) of 2-4% and a working range (below 10% CV) covering almost the entire standard curve.     

Significance of xenobiotic metabolism for bioaccumulation kinetics of organic chemicals in Gammarus pulex

Bioaccumulation and biotransformation are key toxicokinetic processes that modify toxicity of chemicals and sensitivity of organisms. Bioaccumulation kinetics vary greatly among organisms and chemicals; thus, we investigated the influence of biotransformation kinetics on bioaccumulation in a model aquatic invertebrate using fifteen (14)C-labeled organic xenobiotics from diverse chemical classes and physicochemical properties (1,2,3-trichlorobenzene, imidacloprid, 4,6-dinitro-o-cresol, ethylacrylate, malathion, chlorpyrifos, aldicarb, carbofuran, carbaryl, 2,4-dichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, 4-nitrobenzyl-chloride, 2,4-dichloroaniline, and sea-nine (4,5-dichloro-2-octyl-3-isothiazolone)). We detected and identified metabolites using HPLC with UV and radio-detection as well as high resolution mass spectrometry (LTQ-Orbitrap). Kinetics of uptake, biotransformation, and elimination of parent compounds and metabolites were modeled with a first-order one-compartment model. Bioaccumulation factors were calculated for parent compounds and metabolite enrichment factors for metabolites. Out of 19 detected metabolites, we identified seven by standards or accurate mass measurements and two via pathway analysis and analogies to other compounds. 1,2,3-Trichlorobenzene, imidacloprid, and 4,6-dinitro-o-cresol were not biotransformed. Dietary uptake contributed little to overall uptake. Differentiation between parent and metabolites increased accuracy of bioaccumulation parameters compared to total (14)C measurements. Biotransformation dominated toxicokinetics and strongly affected internal concentrations of parent compounds and metabolites. Many metabolites reached higher internal concentrations than their parents, characterized by large metabolite enrichment factors.     

Quinoline and derivatives at a tar oil contaminated site: hydroxylated products as indicator for natural attenuation?

LC-MS-MS analysis of groundwater of a tar oil contaminated site showed the occurrence of the N-heterocycles quinoline and isoquinoline as well as their hydroxylated and hydrogenated metabolites. The concentrations of the hydroxylated compounds, 2(1H)-quinolinone and 1(2H)-isoquinolinone, were significantly higher than those of the nonsubstituted parent compounds. Therefore, exclusive quantification of the parent compounds leads to an underestimation of the amount of N-heterocycles present in the groundwater. Microbial degradation experiments of quinoline and isoquinoline with aquifer material of the site as inocculum showed the formation of hydroxylated and hydrogenated products under sulfate-reducing conditions, the prevailing conditions in the field. However, since analyses of seven tar products showed that these compounds are also primary constituents, their detection in groundwater is found to be a nonsufficient indicator for the occurrence of biological natural attenuation processes. Instead, the ratio of hydroxylated to parent compound (R(metabolite)) is proposed as a useful indicator. We found that 65-83% of all groundwater samples showed R(metabolite) for 2(1 H)-quinolinone, 1(2H)-isoquinolinone, 3,4-dihydro-2(1H)-quinolinone, and 3,4-dihydro-1(2H)-isoquinolinone, which was higher than the highest ratio found in tar products. With respect to the observed partition coefficient between tar oil and water of 3.5 for quinoline and isoquinoline and 0.3 for 2(1H)-quinolinone and 1(2H)-isoquinolinone, the ratio in groundwater would be approximately 10 times higher than the ratio in tar oil. When paying attention to these two parameters, 19-31% of groundwater samples exceed the highest tar oil ratio. This indicates that biological processes take place in the     

Effect of feed restriction on metabolites in cerebrospinal fluid and plasma of dairy cows

Endocrines and metabolites in the circulation act as long-term hunger or satiety signals in the brain during negative energy balance and play an important role in the control of feed intake. These signals also occur in the cerebrospinal fluid (CSF), which surrounds the hypothalamus and brainstem: 2 major centers of feed intake regulation. Thus CSF functions as a transport medium for fuel signals between blood and brain. The CSF metabolite concentrations are mainly under control of the blood-brain barriers, which provide specific carrier molecules facilitating the entry of substances required by the brain and protect the brain from factors that could impair neuronal function. The transport of small molecules such as amino acids (AA) across the blood-brain barriers may be limited by competing AA that share a common transporter for the uptake into brain. Consequently, CSF metabolite concentrations differ from those in blood. Thus it appears likely that central (CSF) rather than peripheral (blood) metabolites act as pivotal signals for the control of feed intake. However, the contribution of putative orexigenic and anorexigenic signals in CSF of cows has not been studied so far. Therefore, the aim of this study was to elucidate associations existing between both plasma and CSF metabolites, each in response to feed restriction-induced negative energy balance. Seven German Holstein dairy cows, between 87 and 96 DIM of the second lactation (milk yield, 27.9 L/d) were fed ad libitum (AL) for 4 d and CSF from the spinal cord and blood from the jugular vein was withdrawn before morning feeding at the fifth day. Subsequently, animals were feed restricted (R) to 50% of the previous AL intake for 4 d and CSF and plasma were collected at the ninth day. Body weight, feed intake, water intake, and milk production were determined. Thirty-one AA, β-hydroxybutyric acid, cholesterol, glucose, lactate, nonesterified fatty acids, urea, and osmolality were measured in both CSF and plasma, whereas free fatty acids and volatile fatty acids were determined in plasma only. Although plasma arginine (132%), leucine (134%), lysine (117%), nonesterified fatty acids (224%), and cholesterol (112%) increased, tryptophan and carnosine decreased (-33% and -20%, respectively) in R animals as compared with AL animals. In CSF, concentrations of these metabolites were not affected after R feeding, suggesting that these identified plasma metabolites have only little potential to contribute to central feed intake regulatory signaling in cows. By contrast, in CSF, serine, threonine, and tyrosine decreased (-20, -24, and -31%, respectively) after R feeding. Therefore, these 3 AA are potential centrally acting anorexigenic signals in cows.     

沙丁胺醇直接竞争ELISA 法快速测定

建立沙丁胺醇的直接竞争ELISA(dcELISA)快速检测方法,采用高碘酸钠法制备辣根过氧化物酶标记沙丁胺醇单克隆抗体,棋盘滴定法确定包被抗原质量浓度和抗体稀释倍数,通过单因素试验,考察反应体系中表面活性剂、离子浓度、甲醇体积分数、pH 值因素对dcELISA 性能的影响,确定最优检测条件,同时考察方法的特异性。结果表明：酶标抗体克分子比为2.1 时偶合物的滴度最高,最佳反应条件为包被抗原质量浓度1μg/mL,酶标抗体稀释2560 倍,0.01mol/L、pH7.4 的磷酸盐抗体稀释液(PBS)中含体积分数0.05% 的Tween-20、0.5mol/L NaCl溶液和体积分数5% 的甲醇时dcELISA 具有最高的灵敏度和最好的稳定性,所建立方法的IC50 为10.3ng/mL,检测限为0.049ng/mL,线性范围0.3～76.30ng/mL,批内变异系数13.8%,批间变异系数为22.38%,与克伦特罗交叉反应率为321.18%,与溴布特罗交差反应率为29.09%,与其他结构类似物没有明显交叉反应。本研究所建立的dcELISA 可用于沙丁胺醇和克伦特罗的多残留检测。     

Toxicokinetic model describing bioconcentration and biotransformation of diazinon in Daphnia magna

A toxicokinetic model for Daphnia magna , which simulates the internal concentration of the insecticide diazinon, its detoxification product 2-isopropyl-6-methyl-4-pyrimidinol, and its active metabolite diazoxon, is presented. During in vivo exposure to diazinon with and without inhibition of cytochrome P450 by piperonyl butoxide, the parent compound as well as its metabolites were quantified with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in extracts of D. magna . Rate constants of all relevant toxicokinetic steps were obtained by modeling the time course of the internal concentrations with a multicomponent first-order kinetics model. When cytochrome P450 was inhibited, the kinetic bioconcentration factor (BCF) of diazinon increased from 17.8 to 51.0 mL·g(ww)(-1). This clearly indicates that diazinon is biotransformed to a high degree by cytochrome P450 in D. magna . The dominant elimination step of diazinon was shown to be its oxidative dearylation to pyrimidinol (62% of total elimination) with a corresponding rate constant of 0.16 h(-1). In contrast, oxidative activation to diazoxon with a rate constant of 0.02 h(-1) amounted to only 8% of the total elimination. During exposure to diazinon, the active metabolite diazoxon could be detected only in very low concentrations (approximately 0.5% of the parent compound), presumably due to a very fast reaction with the target site acetylcholinesterase. During the exposure experiments (no feeding of daphnids), an exponential decline of the lipid content in D. magna with a first-order rate constant of 0.013 h(-1) was observed. For short exposure times (≤ 24 h), this had only a minor influence on the determined TK parameters. Such a TK model containing detailed biotransformation processes is an important tool for estimation of the toxic potential of chemicals, particularly, when active metabolites are formed inside an organism.     

Aerobic and anaerobic in vitro testing of feed additives claiming to detoxify deoxynivalenol and zearalenone

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by fungi of the genus Fusarium which frequently contaminate maize and grain cereals. Mycotoxin-contaminated feed endangers animal health and leads to economic losses in animal production. Several mycotoxin elimination strategies, including the use of commercially available DON and ZEN detoxifying agents, have been developed. However, frequently there is no scientific proof of the efficacy of such adsorbents and degrading products. We therefore tested 20 commercially available products claiming to detoxify DON and/or ZEN either by biodegradation (4 products) or a combination of degradation and adsorption (16 products) under aerobic and anaerobic conditions at approx. pH 7. Under the applied conditions, a complete reduction of DON and consequent formation of the known non-toxic metabolite DOM-1 was exclusively observed in samples taken from the anaerobic degradation experiment of one product. For all other products, incubated under aerobic and anaerobic conditions, a maximum DON reduction of 17% after 72 h of incubation was detected. Aerobic and anaerobic incubation of only one tested product resulted in complete ZEN reduction as well as in the formation of the less-toxic metabolites DHZEN and HZEN. With this product, 68–97% of the toxin was metabolised within 3 h. After 24 h, a ZEN reduction ≥ 60% was obtained with four additional products during aerobic incubation only. Six of the 20 investigated products produced α- and/or β-ZEL, which are metabolites showing similar oestrogenic activity compared to ZEN. Aerobic and anaerobic degradation to unknown metabolites with unidentified toxicity was obtained with 10 and 3 products, respectively. The results of our study demonstrate the importance of in vitro experiments to critically screen agents claiming mycotoxin detoxification.     

Mass balance of metolachlor in a grassed phytoremediation system

Metolachlor is a point-source pollutant at agrochemical dealerships in the Midwest, as well as a non point-source contaminant of surface waters caused by runoff. Prairie grasses have been used in filter strips to control runoff and are also useful for phytoremediation; however, little is known about the fate of metolachlor and its metabolites within a grassed system. Effects of uptake by prairie grasses on the formation and fate of degradation products are not known. In this study, [U-ring-14C]metolachlor was added to enclosed systems to determine the fate of the parent compound and its metabolites in soil and plants. Mineralization and volatilization were monitored over the 97 day study and found to be 1.05 and 0.2%, respectively, for vegetated systems. At the end of the study, soil and plant material was evaluated for the presence of parent metolachlor and selected metabolites, as well as bound residues. Metolachlor ethane sulfonic acid was the dominant metabolite in soil and plant tissue. Over 7% of applied radioactivity was taken up by the grasses, and plant uptake/metabolism appeared to be the main mechanism for phytoremediation of metolachlor. Vegetation significantly reduced the amount of metolachlor in soil by 9%, indicating potential success as a remediation tool.     

Selective enzyme-linked immunosorbent assay for triclosan. Application to wastewater treatment plant effluents

A sensitive and highly selective enzyme-linked immunosorbent assay was developed for triclosan, one of the most common bactericides in personal care products, which is also considered as an emerging contaminant, given its presence in the effluents of sewage and wastewater treatment plants. From synthesized haptens, polyclonal rabbit antibodies against triclosan were raised. The best ELISA immunoassay was based on an antibody-coated format, yielding a detection limit of 0.03 microg L(-1), an I50 of 3.85 microg L(-1), and a dynamic range from 0.22 to 42.16 microg L(-1), with little or no cross-reactivity (< 10%) to similarly structured compounds, including its metabolite methyltriclosan (CR < 6%). The assay was applied as a screening method to quantify triclosan in surface water and in wastewaters. After C18 solid-phase extraction, nanogram per liter concentrations were determined in effluents of urban wastewater treatment plants. The satisfactory recoveries achieved as well as the agreement between immunochemical and chromatographic methods (GC-MS) indicate its potential for either screening or laboratory quantification.     

Production of optically active ketoprofen by direct enzymatic esterification

For the production of optically active ketoprofen, enzymatic resolution of racemic ketoprofen in an organic solvent has been accomplished via enantioselective esterification. Pharmacologically inactive (R)-ketoprofen is converted into the corresponding (R)-ester by this method. Enantioselectivity in lipase-catalyzed resolution of racemic ketoprofen was mainly dependent on the sources of lipase, alcohol moiety, organic solvent, and water content. Ethanol was used as the alkyl donor and the optimum water content required for highly efficient enzymatic resolution was determined to be 0.1-0.15% (v/v), which was maintained using salt hydrates such as Na2SO4 x 10H2O. (S)-Ketoprofen could be obtained with high enantioselectivity (E=15) in n-hexane supplemented with ethylene dichloride (20% (v/v)) using commercially available Candida antarctica lipase (Novozym 435).     

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。     

气相色谱-负化学源-质谱法测定茶叶中的氟虫腈及其代谢物

建立气相色谱-负化学源-质谱法测定茶叶中氟虫腈及其代谢物：氟甲腈、氟虫腈砜和氟虫腈亚砜。采用乙腈提取茶叶中的氟虫腈及其代谢物，并通过正己烷液液分配后，经PSA-SPE柱净化。采用气相色谱-负化学源-质谱法进行氟虫腈及其代谢物的检测，外标法定量。结果表明，茶叶中的氟虫腈及其代谢物在16 min内被分离。方法中4种目标物的线性范围为0.002～0.200?μg/mL，相关系数R²>0.999，检出限(R_SN=10)为0.002 mg/kg，相对标准偏差低于5%。在添加量为0.002、0.010 mg/kg和0.020 mg/kg时，加标回收率在96.33%～102.50%之间。本方法线性范围广、准确性高、重复性好，可以快速准确地对市售茶叶中氟虫腈及其代谢物进行痕量检测分析。     

Current trends in rapid tests for mycotoxins

There are an ample number of commercial testing kits available for mycotoxin analysis on the market today, including enzyme-linked immunosorbent assays, membrane-based immunoassays, fluorescence polarisation immunoassays and fluorometric assays. It can be observed from the literature that not only are developments and improvements ongoing for these assays but there are also novel assays being developed using biosensor technology. This review focuses on both the currently available methods and recent innovative methods for mycotoxin testing. Furthermore, it highlights trends that are influencing assay developments such as multiplexing capabilities and rapid on-site analysis, indicating the possible detection methods that will shape the future market.