Determination of clenbuterol in pig liver by high-performance liquid chromatography with a coulometric electrode array system

A method has been developed to determine clenbuterol in pig liver using HPLC with coulometric electrode array system, for this compound can be irreversibly oxidized at high potentials by ordinary methods. Investigation into the effect of the pH of mobile phase on the retention factor and peak height of clenbuterol was made. The electrochemical behavior of clenbuterol at graphite electrodes was taken into account. Optimization of different extract conditions was also performed. The samples were pretreated using liquid-liquid extraction based on diethyl ether and the organic layer was evaporated to dryness. The residue was dissolved in mobile phase and monitored by an ESA electrochemical detector. Four electrodes in series were used for quantitation and the potentials of electrodes were set at 450, 600, 650 and 680 mV, respectively. Calibration curve showed good linearity and the detection limit of clenbuterol was 1.2 ng/g. This method developed using HPLC-ECD is reproducible, and sensitive enough for the determination of clenbuterol in pig liver. It is easy to perform.     

Supercritical fluid extraction of clenbuterol from bovine liver tissue.

The development of a supercritical fluid extraction procedure for the extraction of clenbuterol from bovine liver tissue is described. The procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol residues at the low ppb level. Method validation incorporating inter- and intra-assays was carried out on fortified liver tissue and showed good recoveries and low variations (RSD < 15%) for levels of clenbuterol of 0.5, 2 and 5 ppb. The developed procedure was shown to be successful for the determination of clenbuterol in incurred liver tissue. The effects of organic modifiers and of inherent sample moisture on analyte extractability are also presented.     

Supercritical fluid extraction (SFE) as a multi-residue extraction procedure for beta-agonists in bovine liver tissue

A supercritical fluid extraction procedure has been developed for the extraction of beta-agonists in bovine liver samples. The method is suitable for compounds of different beta-agonist classes: the substituted aniline-type compounds (e.g. clenbuterol) and the phenolic-type compounds (e.g. salbutamol), including conjugated forms of the latter. The developed procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol and salbutamol residues at the low ppb level. Addition of methanol modifier and removal of sample moisture are necessary for the extraction of more polar analytes such as salbutamol. Method validation incorporating intra- and inter-assays was carried out on fortified liver tissue and showed good recovery and low variation (RSD < 15%). An enzyme hydrolysis procedure was incorporated into the method for the deconjugation of conjugated residues. The developed procedure was shown to be successful for the determination of both clenbuterol and salbutamol in incurred liver tissue.     

同位素稀释质谱法测定猪肉中克伦特罗含量的国际比对APMP. QM-S6

建立了同位素稀释质谱法（IDMS）测定猪肉中克伦特罗含量的高准确度方法,并应用于亚太计量规划组织（Asia Pacific Metrology Programme,APMP）开展的国际比对“猪肉中克伦特罗含量测定”（APMP. QM-S6）。本研究考察了影响测定结果的喷雾电压值、流动相、色谱柱、提取条件、净化条件等主要因素并进行了优化,并对测定结果的不确定度进行了评定。结果表明:流动相组成以及pH值会影响克伦特罗的质谱响应以及最优的电喷雾电压值;样品溶剂会影响克伦特罗的色谱保留,甲醇溶剂会引起严重的溶剂效应,甚至导致峰分裂;克伦特罗易在固相萃取柱及亲水性滤膜上吸附,且吸附材料和滤膜上吸附的杂质有可能被洗脱,引起基质效应,干扰测定。提取效率最高的方法是采用0.1%（v/v）甲酸乙腈溶液为提取溶剂,通过匀浆进行提取。方法检出限（以信噪比大于3计）为0.2 μg/kg。比对样品中克伦特罗的含量为5.18 μg/kg±0.50 μg/kg（k=2）,比对结果与参考值等效一致,取得国际互认。该方法准确可靠,可为猪肉中克伦特罗的日常检测提供参考。     

A novel sorbent for the determination of clenbuterol in bovine liver.

The use of three C18 sorbents in matrix solid phase dispersion (MSPD) for the determination of clenbuterol in bovine liver fortified at 5 ng g-1 is described. MSPD grade C18 sorbents give rise to more efficient blending and packing of the material for subsequent washing and analyte elution in comparison with a non-MSPD grade C18 sorbent. Following enzymatic deconjugation of the liver extracts, radioimmunoassay is used as the method of determination. The mean recovery of clenbuterol with all sorbents is comparable and within the range 86-96% in two intra-assay studies (n = 3). The liver extracts in each case are highly coloured. The variation in recovery is observed to be lowest with MSPD grade C18 (end-capped). This sorbent was used in further studies to evaluate the use of solid phase extraction (SPE), post MSPD, with normal phase aminopropyl or mixed mode cation exchange columns for extract purification. The mean recovery of clenbuterol (n = 4, inter-assay study) following MSPD and normal phase SPE clean-up was 95 +/- 15% and 89 +/- 9% at fortification levels of 1 and 2.5 ng g-1, respectively.     

Combined solid‐phase microextraction and high‐performance liquid chromatography with ultroviolet detection for simultaneous analysis of clenbuterol,salbutamol and ractopamine in pig samples

A simple and sensitive method based on the combination of solid‐phase microextraction (SPME) and high‐performance liquid chromatography with ultroviolet detection was developed for the simultaneous determination of clenbuterol, salbutamol and ractopamine in pig samples. Parameters of the SPME procedure affecting extraction efficiency, such as the type of fiber, extraction time, extraction temperature, ion strength, pH of sample and stirring rate, were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.5–50 µg/L for clenbuterol and ractopamine, and 0.2–20 µg/L for salbutamol. The limits of detection were 0.1 µg/L for clenbuterol, 0.05 µg/L for salbutamol and 0.1μg/L for ractopamine, respectively. The averages of intra‐ and inter‐day accuracy ranged from 79.8 to 92.4%. The intra‐day and inter‐day precision were below 9.6% for the three analytes. This method exhibited the advantages of simplicity, rapidity and low solvent consumption, and was suitable for the monitoring of β₂‐agonists residue in pig samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative analytical quantitation of clenbuterol in biological matrices using GC-MS and EIA

A simple and sensitive procedure utilizing GC-MS for the identification and quantitation of clenbuterol in biofluids and tissues is described. This improved method utilizes trimethylboroxine for the derivatization of clenbuterol, requires only 1 mL/g of biological sample, and most importantly does not require an extra cleaning step for urine specimens prior to extraction. Linear quantitative response curves have been generated for derivatized clenbuterol over a concentration range of 5-200 ng/mL. The extraction efficiency at four representative points of the standard curve exceeded 90% in both specimen types (plasma and urine). Linear regression analyses of the standard curve in both specimen types exhibited correlation coefficients ranging from 0.997 to 1.000. The Limit of detection (LOD) and Limit of quantitation (LOQ) values for plasma specimens were determined to be 0.5 and 1.5 ng/mL respectively. For urine specimens, LOD and LOQ values were 0.2 and 0.7 ng/microL respectively. Percentage recoveries ranged from 91 to 95% for urine and 89 to 101% for plasma. Precision and accuracy (within-run and between-run) studies reflected a high level of reliability and reproducibility of the method. In addition to its reliability, sensitivity and simplicity, this modified procedure is more efficient and cost effective, requiring less time, only 1 mL of sample, and minimal amounts of extraction solvents. The applicability of the method for the detection and quantitation of clenbuterol in biological tissues of rats treated with the drug was demonstrated successfully. For comparative analysis of clenbuterol in plasma and liver samples, both GC-MS and enzyme immunoassay (EIA) methods are found to be suitable. Due to potential antibody-cross reactivity with EIA, the GC-MS method is the method of choice for most samples because of its specificity. However, the EIA method is considered the method of choice for analysis of clenbuterol found in concentrations below the limits of quantitation by GC-MS due to its sensitivity.     

Evaluation of methods aimed at complete removal of template from molecularly imprinted polymers.

Polymers imprinted with clenbuterol were used to study the influence of various post-polymerization treatments [e.g., thermal annealing, microwave assisted extraction (MAE), Soxhlet extraction and supercritical fluid template desorption] on the bleeding of residual template. The aim of the study was to reduce the bleeding to levels that would allow the use of the materials as affinity phases for extraction of clenbuterol from bovine urine at concentrations below 1 ng ml-1. After treatment, the clenbuterol imprinted polymers were packed into solid-phase extraction columns and the bleeding was estimated by quantifying the amount of template released in 10 ml of methanol-acetic acid (9 + 1 v/v). This was followed by an assessment of selectivity and recovery in comparison with non-treated material. The lowest bleeding level was found after MAE using 100% trifluoroacetic acid for 3 x 20 min at 100 degrees C. The collected eluate contained in this case 3 ng ml-1 of clenbuterol. The same material was subsequently used for the extraction of clenbuterol from spiked bovine urine. The resulting selectivity and recovery were lower compared with those obtained using the untreated material. A milder but still efficient method to reduce the bleeding level was found to be MAE with formic acid. In this case a bleeding level of 14 ng ml-1 was found after only a 1 h extraction time. In a second model system, using a polymer imprinted with L-phenylalanine anilide, the bleeding was reduced to a similar level by extensive on-line washing in good swelling solvents containing acid or base additives and after thermal annealing of the polymers in the dry state.     

Production of “in house” reference materials for ELISA screening of bovine urine and liver samples for clenbuterol

Clenbuterol screening of bovines is done by analysis of urine, for monitoring living animals, and liver, for monitoring animals after slaughter. ELISA has generally been used as the main method for these purposes. Nevertheless, in Europe, methods must be validated according to Commission Decision (EC) 657/2007 criteria, i.e. by use of reference materials. Production of “in house” reference materials is a possibility, but the homogeneity, storage temperature, and period of stability of these materials must be investigated in the laboratory itself. This paper reports GC–MS evaluation of an “in-house”-produced batch of aliquots of bovine urine and liver, fortified with 10.0 ng/ml and 10.0 ng/g clenbuterol, respectively, and stored at ?20 °C and at ?60 °C. For urine stored for 20 weeks at ?20 °C and at 60 °C the stability of clenbuterol was proved at the 95% confidence level. For liver, however, it was demonstrated at the same confidence level that clenbuterol was highly unstable during storage for 20 weeks at either of the temperatures studied.     

Investigation of ractopamine molecularly imprinted stir bar sorptive extraction and its application for trace analysis of β2-agonists in complex samples

In this paper, a novel molecularly imprinted polymer (MIP) coated stir bar with ractopamine as template by glass capillary filling with magnetic core as substrate was prepared reproducibly. The ractopamine MIP coating was homogeneous and porous with the average thickness of 20.6 μm. The extraction apparatus for the stir bar was improved to avoid coating loss. The MIP-coated stir bar showed better extraction capacity and good selectivity than that of non-imprinted polymer (NIP) coated stir bar to ractopamine and its analogues. The extraction capacities of ractopamine, isoxsuprine, clenbuterol and fenoterol for MIP-coated stir bar were 3.3, 3.1, 2.8 and 2.4 times as much as that of the NIP coated stir bar, respectively. The MIP-coated stir bars could be used at least 40 times without apparent damage and kept in dried air for 8 months without reduce of extraction ability. A method for the determination of β₂-agonists in complex samples by MIP-coated stir bar sorptive extraction coupled with high-performance liquid chromatography (HPLC) was developed. The linear ranges were 0.5–40 μg/L for ractopamine and 1.0–40 μg/L for isoxsuprine and clenbuterol. The detection limits were within the range of 0.10–0.21 μg/L. The method was successfully applied to the analysis of β₂-agonists in spiked pork, liver and feed samples with the recoveries of 83.7–92.3%, 80.5–90.2% and 73.6–86.2%, respectively. The RSDs was within 2.9–8.1%. The method is very suitable for the determination of trace β₂-agonists in pork, liver and feed samples.     

Determination of beta-agonists in liver and retina by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of bromobuterol, cimaterol, clenbuterol, clenpenterol, hydroxymethylclenbuterol, isoxsuprine, mabuterol, ractopamine, ritrodrine, salbutamol, terbutaline, and tulobuterol residues in bovine liver and retina is reported. This procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on Oasis HLB solid-phase extraction cartridges, followed by determination of the residues by LC-tandem quadrupole MS using atmospheric pressure chemical ionization in the positive ion mode. Overall average recoveries ranged from 23 to 76% for liver and 34 to 77% for retina. The mean values for samples fortified at levels between 0.5-2.0 microg/kg (liver) and 5-20 microg/kg (retina) agreed within 98-118% of the spiked levels, with coefficients of variation ranging from 6 to 20%. The decision limits, CCalpha, ranged from 0.1 to 0.3 microg/kg for liver, 1-3 microg/kg for retina, and detection capabilities, CCbeta, from 0.2-0.5 microg/kg for liver and 2-5 microg/kg for retina.     

蛋白芯片法同时检测食品中克伦特罗与莱克多巴胺的残留量

建立了食品中克伦特罗和莱克多巴胺残留含量同时检测的微阵列蛋白芯片法。结果显示:克伦特罗的线性范围为0.07～1.2 ng/g;莱克多巴胺的线性范围为0.05～0.8 ng/g。猪肉、猪肝中上述两种物质的加标回收率为74%～132%,相对标准偏差均小于10%。将所建立的方法与HPLC-MS方法进行对比,两者检测结果一致。该方法简单、快速、通量高,可用于实际样品中克伦特罗和莱克多巴胺残留量的检测。     

Evaluation of matrix solid‐phase dispersion extraction for 11 β‐agonists in swine feed by liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of 11 β‐agonists (clenbuterol, salbutamol, ractopamine, terbutaline, fenoterol, cimaterol, isoxsuprine, mabuterol, mapenterol, clenproperol, and tulobuterol) in swine feed. This rapid, simple, and effective extraction method was based on matrix solid‐phase dispersion. The limit of quantification of clenbuterol, cimaterol, mabuterol, salbutamol, terbutaline, mapenterol, clenproperol, and tulobuterol was 1 μg/kg and that of ractopamine, fenoterol, and isoxsuprine was 2 μg/kg. The recoveries of β‐agonists spiked in swine feeds at a concentration range of 1–8 μg/kg were >83.1% with relative standard deviations <9.3%. This rapid and reliable method can be used to efficiently separate, characterize, and quantify the residues of 11 β‐agonists in swine feeds with advantages of simple pretreatment and environmental friendliness.     

质谱快速分析猪肉中痕量沙丁胺醇及克伦特罗

采用内部萃取电喷雾电离质谱( iEESI-MS)技术,在无需样品预处理的前提下,采用标准加入法直接对猪肉组织中沙丁胺醇与克伦特罗进行定性和定量分析。结果表明,本实验对猪肉组织中沙丁胺醇与克伦特罗具有较高的灵敏度,单个样品单一指标的检测时间少于30 s。在0.01~1000μg/kg浓度范围内,信号强度对数(Y)与浓度对数(X)具有较好的线性关系,定量限分别为6.2和9.8 ng/kg。本方法分析速度快、样本耗量少、灵敏度高,适用于猪肉中痕量沙丁胺醇与克伦特罗等“瘦肉精”的快速检测。     

Investigation of ractopamine‐imprinted polymer for dispersive solid‐phase extraction of trace β‐agonists in pig tissues

Ractopamine, as an alternative β‐agonist to clenbuterol, is more and more used as leanness‐enhancing agent in the swine industry. This work presents a new molecularly imprinted polymer (MIP) using ractopamine as template for dispersive solid‐phase extraction of trace ractopamine and the structural related β‐agonists in animal tissues. The binding properties and selectivity of MIP were investigated. High selectivity in polar environment was found, since the extraction capacity of ractopamine with the MIP was 4.5‐fold as much as that with the non‐imprinted polymer in acetonitrile. Cross‐selectivity investigation indicates that the MIP preferentially binds the template and then the structural analogues according to their molecular similarity. Thermodynamic and kinetic investigation was performed to interpret the specific adsorption and molecular recognition of the MIP for ractopamine. Standard free energy, standard enthalpy, and standard entropy were determined. Related information suggested that adsorption of ractopamine onto MIP was an exothermic, spontaneous process. The MIP can be applied as dispersive solid‐phase extraction material for enrichment of ractopamine, isoxsuprine, fenoterol and clenbuterol in complex samples before HPLC analysis. The method revealed detection limits of 0.20–0.90 μg/L, recoveries of 83.8–115.2 and 85.2–110.2% for the spiked pig muscle and pig liver, respectively, with the RSD from 2.5 to 8.8%.     

急性瘦肉精中毒检材的QuEChERS净化-液质联用快速确证方法

目前液相色谱串联质谱法测定动物源性食品中瘦肉精普遍采用酶水解提取、固相萃取净化的方式处理样品,该方法耗时长、分析成本高。 本研究采用酸水解和酶水解两种提取方式结合QuEChERS(Quick,Easy,Cheap,Effective,Rugged,Safe)净化和高效液相色谱串联质谱建立了动物性食品中特布他林、沙丁胺醇、莱克多巴胺和克伦特罗4种β2受体激动剂的快速定性确证和定量检测方法。 采用酶水解提取-QuEChERS净化处理样品,在电喷雾离子源正离子扫描(ESI+)和多反应监测(MRM)模式下,4种瘦肉精在1.0~20.0 μg/L浓度之间呈线性,线性相关系数均大于0.999,检出限和定量限分别为0.1和0.3 μg/kg,猪肉和牛肉样品中4种化合物的回收率为78.5%~112%,相对标准偏差RSD为2.8%~8.9%。 采用酸水解法代替酶水解提取时,尽管对于样品中莱克多巴胺测定结果稍偏低,但样品前处理时间大大缩短。 两种提取方法结合使用对于提高瘦肉精引起的食物中毒应急处置效率具有重要意义。     

Novel method for the rapid and specific extraction of multiple β2‐agonist residues in food by tailor‐made Monolith‐MIPs extraction disks and detection by gas chromatography with mass spectrometry

A quick and specific pretreatment method based on a series of extraction clean‐up disks, consisting of molecularly imprinted polymer monoliths and C₁₈ adsorbent, was developed for the specific enrichment of salbutamol and clenbuterol residues in food. The molecularly imprinted monolithic polymer disk was synthesized using salbutamol as a template through a one‐step synthesis process. It can simultaneously and specifically recognize salbutamol and clenbuterol. The monolithic polymer disk and series of C₁₈ disks were assembled with a syringe to form a set of tailor‐made devices for the extraction of target molecules. In a single run, salbutamol and clenbuterol can be specifically extracted, cleaned, and eluted by methanol/acetic acid/H₂O. The target molecules, after a silylation derivatization reaction were detected by gas chromatography‐mass spectrometry. The parameters including solvent desorption, sample pH, and the cycles of reloading were investigated and discussed. Under the optimized extraction and clean‐up conditions, the limits of detection and quantitation were determined as 0.018–0.022 and 0.042–0.049 ng/g for salbutamol and clenbuterol, respectively. The assay described was convenient, rapid, and specific; thereby potentially efficient in the high‐throughput analysis of β₂‐agonists residues in real food samples.     

Determination of clenbuterol in urine as its cyclic boronate derivative by gas chromatography-mass spectrometry

A rapid and reliable gas chromatographic-mass spectrometric method for the determination of clenbuterol in urine is described. Penbutolol was used as internal standard. Four derivatization procedures have been tested, of which 1-butaneboronic acid gave the best results. The method includes extraction of the alkalinized urine (3 ml) with tert.-butyl methyl ether-n-butanol (9:1), derivatization with 1-butaneboronic acid (15 min at room temperature), and analysis in the selected-ion monitoring mode of the derivatives of clenbuterol at m/z 243, 327 and 342 and of penbutolol at m/z 342 and 357. The detection limit is 0.5 ng/ml and the recovery better than 90%.     

A fast immunoassay for the screening of beta-agonists in hair.

Hair has been shown to be an excellent site for the accumulation of clenbuterol residues. Compared with other matrices, hair sampling is very easy and this might result in large numbers of samples. In this study, a simple digestion-extraction procedure was combined with a sensitive clenbuterol ELISA, which resulted in an easy screening procedure suitable for the detection of at least four beta-agonists. Hair from untreated cows (n = 40) resulted in low blank levels (0.9 +/- 0.7 and 0.5 +/- 0.2 ng g-1 for black and white hair, respectively). The detection limits for clenbuterol, bromobuterol, mapenterol and mabuterol were determined as 1-1.5 ng g-1 for white and 3-4 ng g-1 for black hair. The accumulation of mabuterol and mapenterol in black and white hair from treated calves was demonstrated by GC-MS. The screening assay is not suitable for the detection of cimbuterol (owing to the low extraction efficiency) and for salbutamol and terbutaline (owing to the low cross-reactivity of the antibodies used for the ELISA and the low extraction efficiency). Black hair samples from cows treated with clenbuterol were still found to be positive (> 5 ng g-1) at 23 weeks after treatment. The fast screening procedure is a powerful means to detect and track the illegal use of clenbuterol, bromobuterol, mabuterol and mapenterol in animal production.     

The use of nonendcapped C18 columns in the cleanup of clenbuterol and a new adrenergic agonist from bovine liver by gas chromatography-tandem mass spectrometry analysis

More specific official methodology is needed to survey the illegal use of clenbuterol in animal production plus the synthesis of new compounds that currently elude routine analytical methods. The identification of a new adrenergic agonist, N1-(2-(4-amino-3,5-dichlorophenyl)-2-hydroxyethyl)-N1-isopropyl-propanamide (known as compound A) in animal feed has prompted studies to verify if the existing cleanup procedures developed for clenbuterol are really effective. This study considers the ion-exchange mechanism on cyanopropyl (CN), sulfonic cation exchange (SCX), mixed phase (MPH) (C8 + SCX), and nonendcapped C18 (C18NE) solid-phase extraction (SPE) columns. Results indicate that compound A (by contrast with clenbuterol) is not efficiently retained on the CN, SCX, and MPH SPE columns (recovery <10%). This finding thus leads to the development of a gas chromatography-tandem mass spectrometry procedure based on C18NE SPE that is able to purify both agonists from bovine livers spiked at 0.5, 1.0, and 2.0 ppb with a mean recovery of 93% for clenbuterol and 92% for compound A.