Multiresidue analysis of nine β‐agonists in animal muscles by LC‐MS/MS based on a new polymer cartridge for sample cleanup

A novel, sensitive, and reliable LC‐MS/MS method for multiresidue analysis of nine β‐agonists (salbutamol, terbutaline, cimaterol, fenoterol, clorprenaline, ractopamine, tulobuterol, clenbuterol, and penbuterol) in four farm animal muscles was developed. Muscle matrix was extracted with acetonitrile–10% sodium carbonate solution, and then was subjected to cleanup using a SPE cartridge packed with new polymer synthesized in acetone. Chromatographic separation of the components was performed on a Luna C₁₈ column using 0.1% of formic acid in water and acetonitrile. The mass spectrometer was operated in the positive electrospray mode. Good precision and accuracy were obtained for all analytes (except for fenoterol) at the spiked three levels of 1.0, 10, and 50 μg/kg. The decision limit and detection capability of nine β‐agonists ranged from 0.04 to 0.18 and 0.15 to 0.69 μg/kg, respectively. The method developed was successfully applied to the monitoring of nine β‐agonists in pork, beef, mutton, and chicken from Chinese markets.     

Selective Electrochemical Determination of Uric Acid in the Presence of Ascorbic Acid Using a Carbon Paste Electrode Modified with β‐Cyclodextrin

Uric acid (UA) was determined in the presence of ascorbic acid (AA) by using a carbon paste electrode modified superficially by a β‐cyclodextrin film (CPE/β‐CD). The surface carbon paste electrode was prepared applying a 30 cycles potential program and using a 1 M HClO₄+0.01 M β‐CD electrolytic solution. The UA and AA solutions were used to evaluate the electrode selectivity and sensitivity by cyclic voltammetric and amperometric methods. In these experiments the detection limit for UA was (4.6±0.01)×10^?6 M and the RSD calculated from the amperometric curves was 10%. From the data obtained it was possible to quantify UA in the urine and saliva samples. Selective detection of UA was improved by formation of an inclusion complex between β‐CD and UA. The results show that the CPE/β‐CD is a good candidate due to its selectivity and sensitivity in the UA determination in complex samples like the biological fluids.     

Determination of beta‐agonists in swine hair by μFIA and chemiluminescence

β‐Agonists are a group of illegal feed additives. In this paper, it was found that the light emission produced by the oxidation of luminol by potassium ferricyanide was enhanced by the β‐agonists (ractopamine, salbutamol, and terbutaline). Based on chemiluminescence phenomenon, a novel, rapid, and sensitive microflow injection analysis system on a microfluidic glass chip was established for determination of the β‐agonists. The chip was fabricated from two glass plates (64 mm × 32 mm) with microchannels of 200 μm width and 100 μm depth. The detection limits were achieved at 2.0 × 10^?8 mol/L of ractopamine, 1.0 × 10^?8 mol/L of terbutaline and 5.0 × 10^?7 mol/L of salbutamol. In this report, our method was applied for determination of the β‐agonists in swine hair from three different sources with satisfactory results.     

多壁碳纳米管和分子印迹膜修饰电极检测猪尿液中莱克多巴胺

采用原位热聚合技术,分别以多壁碳纳米管(MWCNs)和分子印迹膜(MIM)修饰丝网印刷电极(SPE),与多壁碳纳米管和非分子印迹膜(NIM)修饰的丝网印刷电极组合在一起,并将其组合的丝网印刷电极通过电极插口与便携式电导仪相连接,组装成检测莱克多巴胺残留的电导型传感器,优化检测条件,并建立了检测莱克多巴胺的标准曲线,测试了实际猪尿样中莱克多巴胺的含量.通过扫描电镜分析了该分子印迹膜的表征结构.结果表明,在莱克多巴胺分子印迹膜表面形成了大量印迹微孔.本传感器装置检测莱克多巴胺具有很高的灵敏度和特异性,检出限为0.033 mg/L,线性范围为0.33～8.0 mg/L,基于猪尿样的检测回收率达到91％～98％,可实现现场快速检测.     

Synthesis of β‐Cyclodextrin‐2,4‐dihydroxyacetophenone‐phenylhydrazine and Its Application

A novel host reagent of β‐cyclodextrin‐2,4‐dihydroxyacetophenone‐phenylhydrazine(β‐CDP‐DHPH) was synthesized and characterized by IR and ¹H NMR spectra. A highly selective and sensitive spectrofluorimetric determination of trace amounts of cadmium was proposed based on the reaction between Cd²⁺ and β‐CDP‐DHPH at pH 10.0. The molar ratio of β‐CDP‐DHPH to Cd²⁺ was 1:1. The linear range of this method was 0.56‐120 μg·L^?;1 with a detection limit of 0.20 μg·L^?;1. The interferences of 39 common ions in the determination of cadmium were investigated, and the results showed that the host reagent had a quite high selectivity. This method was rapid and simple in determination of trace amounts of cadmium in mineral, tap and river water.     

New sulfonate composite functionalized with multiwalled carbon nanotubes with cryogel solid‐phase extraction sorbent for the determination of β‐agonists in animal feeds

A new mixed‐mode cation‐exchange sulfonate composite functionalized with multiwalled carbon nanotubes with polyvinyl alcohol cryogel was fabricated and used for the first time as a solid‐phase extraction sorbent for the determination of β‐agonists in animal feeds. Feed samples were extracted with 0.20 M phosphoric acid and methanol (1:4, v/v) using ultrasonication, cleaned‐up using the developed sorbent to which the β‐agonists bound then finally eluted with 5.0% ammonia in methanol and analyzed by high‐performance liquid chromatography. Various parameters that affected the extraction efficiency were optimized. Under the optimal conditions, the developed sorbent strongly interacted with β‐agonists by cationic exchange and hydrophobic and hydrophilic interactions, that provided a high extraction efficiency in the range of 92.8 ± 3.7–104.4 ± 2.3% over a range of 0.04–2.0 mg/kg for salbutamol and ractopamine, and 0.40–8.0 mg/kg for clenbuterol. The relative standard deviations were less than 6.0%. The developed method was successfully applied for the determination of β‐agonists in various types of animal feed and effectively reduced any matrix interference.     

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.     

Determination of β2-Agonists in Porcine Urine by Molecularly Imprinted Solid Phase Extraction Followed Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Detection

A novel, sensitive, and robust method has been developed to detect 9 β₂-agonists in porcine urine to monitor illegal use of β₂-agonists in swine rearing. The method based on the molecular imprinted polymer (MIP) rapid extraction followed ultra-performance liquid chromatography coupled tandem mass spectrometry (UPLC-MS/MS) detection. The cleaning efficiency of MIP cartridges was demonstrated by comparing with common ion exchange solid phase extraction. The presented method was validated in accordance with the European Commission Decision 2002/657/EC. The linearity, decision limit (CCα), detection capability (CCβ), recovery, precision, robustness, and stability were studied in detail. CCα and CCβ values were from 0.006 ng/mL to 0.03 ng/mL and from 0.02 ng/mL to 0.08 ng/mL, respectively. The mean recoveries and repeatability varied from 68.8% to 94.2% and from 2.8% to 10.1%. The proposed method was applied to test 170 porcine urine samples from the Shaanxi province in China and two urine samples were confirmed as clenbuterol positive and the concentrations of clenbuterol in positive urine samples were about 0.08 ng/mL and 0.1 ng/mL, respectively. The developed method was demonstrated to be more sensitive and robust for the determination of 9 β₂-agonists in porcine urine. The method was proven to be simple and easy in operation with high selectivity and good reproducibility.     

Polymer monolith microextraction online coupled to hydrophilic interaction chromatography/mass spectrometry for analysis of β2‐agonist in human urine

An analytical method based on online combination of polymer monolith microextraction (PMME) technique with hydrophilic interaction LC (HILIC)/MS is presented. The extraction was performed with a poly(methacrylic acid‐co‐ethylene glycol dimethacrylate) monolithic column while the subsequent separation was carried out on a Luna silica column by HILIC. After 1:1 v/v dilution with 20 mM phosphate solution at pH 7.0 and centrifugation, urine sample was directly used for extraction. After optimization, 85% ACN (containing 0.3% formic acid v/v) was used for rapid online elution, which was also the mobile phase in HILIC to avoid band broadening during separation or carry‐over that was usually observed in PMME‐RP LC system. Online automation of extraction and separation procedures was realized under the control of a program in this study. The developed method was applied to rapid and sensitive monitoring of three β₂‐agonist traces in human urine. The LODs (S/N = 3) of the method were found to be 0.05–0.09 ng/mL of β₂‐agonists in urine. The recoveries of three β₂‐agonists spiked in five different urine samples ranged from 79.8 to 119.8%, with RSDs less than 18.0%.     

Simultaneous determination of β2‐agonists in human urine by fast‐gas chromatography/mass spectrometry: method validation and clinical application

A fast screening protocol was developed and validated for the simultaneous determination of 15 β₂‐agonists in human urine (bambuterol, cimbuterol, clenbuterol, fenoterol, formoterol, isoproterenol, mapenterol, metaproterenol, procaterol, ractopamine, ritodrine, salbutamol, salmeterol, terbutaline, tulobuterol). The overall sample processing includes deconjugation with enzyme hydrolysis, liquid–liquid extraction, followed by derivatization of the extract and detection of β₂‐agonists trimethylsilyl‐derivatives by fast‐gas chromatography/electron impact–mass spectrometry (fast‐GC/EI‐MS). Sample extraction and derivatization were optimized with the purpose of improving recoveries and reaction yields for a variety of analytes with different structures simultaneously, while keeping the procedure simple and reliable. Validation parameters were determined for each analyte under investigation, including selectivity, linearity, intra‐ and inter‐assay precision, extraction recoveries and signal to noise ratio (S/N) at the lowest calibration level. Fast‐GC/MS sequences, based on the use of short columns, high carrier‐gas velocity and fast temperature ramping, allow considerable reduction of the analysis time (7 min), while maintaining adequate chromatographic resolution. The overall GC cycle time was less than 9 min, allowing a processing rate of 6 samples/h. High MS‐sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. The method was successfully tested on real samples arising from clinical treatments. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct quantification of 11‐nor‐Δ9‐tetrahydrocannabinol‐9‐carboxylic acid in urine by liquid chromatography/tandem mass spectrometry in relation to doping control analysis

An accurate and precise method for the quantification of 11‐nor‐Δ⁹‐tetrahydrocannabinol‐9‐carboxylic acid (THCA) in urine by liquid chromatography/tandem mass spectrometry (LC/MS/MS) for doping analysis purposes has been developed. The method involves the use of only 200 µL of urine and the use of D₉‐THCA as internal standard. No extraction procedure is used. The urine samples are hydrolysed using sodium hydroxide and diluted with a mixture of methanol/glacial acetic acid (1:1). Chromatographic separation is achieved using a C8 column with gradient elution. All MS and MS/MS parameters were optimised in both positive and negative electrospray ionisation modes. For the identification and the quantification of THCA three product ions are monitored in both ionisation modes. The method is linear over the studied range (5–40 ng/mL), with satisfactory intra‐and inter‐assay precision, and the relative standard deviations (RSDs) are lower than 15%. Good accuracy is achieved with bias less than 10% at all levels tested. No significant matrix effects are observed. The selectivity and specificity are satisfactory, and no interferences are detected. The LC/MS/MS method was applied for the analysis of 48 real urine samples previously analysed with a routine gas chromatography/mass spectrometry (GC/MS) method. A good correlation between the two methods was obtained (r² > 0.98) with a slope close to 1. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of ionic liquid as additive in determination of three β‐agonists by capillary electrophoresis with amperometric detection

CE coupled with amperometric detection method was developed using ionic liquid 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (EMImBF₄) as additive for the simultaneous detection of clenbuterol (CLB), terbutaline (TER), and ractopamine (RAC) in feed. The effects of detection potential, concentration of EMImBF₄, pH, and concentration of the running buffer, separation voltage as well as injection time on the separation and detection of these three β‐agonists were investigated in detail. Under the optimum conditions: the detection potential at 1.05 V, 50 mmol/L Tris‐HAc at pH 8.0 with 0.6% (v/v) EMImBF₄, electrokinetic injection 6 s at 16 kV and separation voltage at 16 kV, a baseline separation for these three analytes could be achieved within 11 min. Introduction of EMImBF₄ into the running buffer resulted in significant improvement in separation selectivity and enhancement in peak currents for those β‐agonists, especially for TER and RAC, which could not be separated in the running buffer without additive. The method exhibited wide linear range with LOD (S/N = 3) of 2, 1, and 2 nmol/L for CLB, TER, and RAC, respectively. The precision was determined in both intraday (n = 5) and interday (n = 3) assays, and the RSDs for both migration time and peak current were less than 6%. The proposed method was also applied to analyze β‐agonists in feed sample.     

pCEC coupling with ESI‐MS for the analysis of β2‐agonists and narcotics using a poly‐(1‐hexadecene‐co‐TMPTMA) monolithic column

A pressure‐assisted CEC with ESI‐MS based on poly(1‐hexadecene‐co‐trimethylolpropane trimethacrylate) monolithic column for rapid analysis of two β₂‐agonists and three narcotics was established in this article. After the organic polymer‐based monolithic column was prepared by an in‐situ polymerization procedure, a systematic investigation of the pressure‐assisted CEC separation and ESI‐MS detection parameters was performed. Baseline separation of the studied analytes could be obtained using the solution containing 75% ACN v/v and 20 mmol/L ammonium acetate with pH 8.0 as running buffer, when applying separation voltage of 20 kV and assisted pressure of 5 bar. Under the optimized conditions, two β₂‐agonists and three narcotics could be completely resolved and accurately determined within 15 min. Finally, the proposed method was successfully used for real urine samples detection.     

Newborn screening for 3‐hydroxy‐3‐methylglutaric aciduria using direct analysis in real‐time mass spectrometry

A novel method utilizing ambient thermal desorption ionization with a direct analysis in real‐time source integrated with mass spectrometry (DART‐MS) was established and applied to the rapid analysis of 3‐hydroxy‐3‐methylglutaric (3‐HMG) acid in the neonatal urine. Instrument parameter settings were optimized to obtain high sensitive and accurate determination of 3‐HMG acid. The use of helium gas heated to temperature of 400°C was observed to permit deprotonation, 3‐HMG acid producing an abundant (M‐H)^? (m/z 161) in the negative ion mode. The calibration curve was determined to be linear over the range of 0.05‐5 mg/L, with the correlation coefficient r = 0.9988 and the relative standard deviations (n = 6) in the range of 1.5‐11.8%. The limit of detection was 0.002 mg/L, and the limit of quantitation was 0.007 mg/L. The recoveries ranged from 88.0% to 123.1%. Four urine samples from patients and four simulated urine samples were investigated. The results of DART‐MS were in agreement with the values determined using established methods at the hospitals. The proposed method demonstrated significant potential in the application of the high‐throughput screening in newborn screening.     

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%.     

Analysis of β‐blockers in groundwater using large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection and liquid chromatography time‐of‐flight mass spectrometry

Atenolol, nadolol, metoprolol, bisoprolol and betaxolol were simultaneously determined in groundwater samples by large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection (LVI‐LC‐LC‐FD) and liquid chromatography‐time‐of‐flight mass spectrometry (LC‐TOF‐MS). The LVI‐LC‐LC‐FD method combines analyte isolation, preconcentration and determination into a single step. Significant reductions in costs for sample pre‐treatment (solvent and solid phases for clean up) and method development times are also achieved. Using LC‐TOF‐MS, accurate mass measurements within 3 ppm error were obtained for all of the β‐blockers studied. Empirical formula information can be obtained by this method, allowing the unequivocal identification of the target compounds in the samples. To increase the sensitivity, a solid‐phase extraction step with Oasis MCX cartridge was carried out yielding recoveries of 79–114% (n=5) with RSD 2–7% for the LC‐TOF‐MS method. SPE gives a high purification of β‐blockers compared with the existing methods. A 100% methanol wash was allowed for these compounds with no loss of analytes. Limit of quantification was 1–7 ng/L for LVI‐LC‐LC‐FD and 0.25–5 ng/L for LC‐TOF‐MS. As a result of selective extraction and effective removal of coextractives, no matrix effect was observed in LVI‐LC‐LC‐FD and LC‐TOF‐MS analyses. The methods were applied to detect and quantify β‐blockers in groundwater samples of Almería (Spain).     

Magnetic molecularly imprinted polymers for the determination of β‐agonist residues in milk by ultra high performance liquid chromatography with tandem mass spectrometry

A simple, accurate, and highly sensitive analytical method was developed in this study for the determination of nine β‐agonists in milk. In this method, a new magnetic adsorbent of molecularly imprinted polymers/magnetic nanoparticles prepared by simple physical blending was adopted, which enabled magnetic solid‐phase extraction. Thus, the resultant material can be separated from the solvent rapidly and conveniently by a magnet. Two kinds of molecularly imprinted polymer/magnetic nanoparticles materials were fabricated, and the characteristics of materials such as the ratio, pH, amount, desorption, and regeneration were investigated. The analytes were quantified by ultra high performance liquid chromatography coupled to an electrospray ionization tandem mass spectrometer operating in multiple reaction monitoring modes. The detection limit of the method was 0.003–0.3 μg/L, and the detection capability was 0.01–0.3 μg/L. The recoveries of these compounds were 65.7–114% at three spiked levels. Reproducibility represented by relative standard deviation was 11.2% or less. The method was successfully applied to the screening of real samples obtained from local markets and confirmation of the suspected target analytes.     

分子印迹固相萃取-高效液相色谱法测定饲料中莱克多巴胺

以莱克多巴胺为模板分子,丙烯酰胺为功能单体,乙二醇二甲基丙烯酸酯为交联剂,合成了对莱克多巴胺具有高选择性的分子印迹聚合物。考察了甲醇、乙腈、丙酮和氯仿-甲醇与三乙胺构成致孔剂合成的聚合物性能及其形貌特征。通过正交试验优化的聚合反应配方为: 1.0 mmol莱克多巴胺,4.0 mmol丙烯酰胺,20.0 mmol乙二醇二甲基丙烯酸酯,6.0 mL乙腈-三乙胺(30:1, v/v), 50.0 mg偶氮二异丁腈。建立的基于分子印迹固相萃取-高效液相色谱测定饲料试样中莱克多巴胺的方法,在0.50～100 mg/L质量浓度范围内有良好的线性关系(r=0.9994);饲料试样中1.0、10及100 mg/kg 3个添加水平的莱克多巴胺平均回收率大于80%;批内、批间测定的相对标准偏差小于10%;检出限(信噪比为3)达到0.1 mg/kg。该方法灵敏、可靠,用于饲料等复杂基质中莱克多巴胺检测的效果优于相关标准分析方法。     

Aptamer based ultrasensitive determination of the β-adrenergic agonist ractopamine using PicoGreen as a fluorescent DNA probe

The authors describe an aptamer based assay for determination of ractopamine (RAC) by using PicoGreen (PG) as a fluorescent probe specific for dsDNA. In the absence of RAC, the aptamer forms a duplex structure with a complementary sequence that results in enhanced PG fluorescence. Upon binding to RAC, the aptamer undergoes a structural switch. This reduces the number of DNA duplexes formed and causes a reduction of fluorescence intensity of PG as measured at excitation/emission wavelengths of 480/520 nm. Under optimized conditions, the dynamic calibration plot covers the 50 pM to 50 μM concentration range, with a 50 pM detection limit. This meets the safety supervision regulations of the European Commission in terms of residue limits of RAC in food. The method displays high selectivity over other β-adrenergic agonists including clenbuterol, dopamine and salbutamol. The assay was successfully applied to samples of swine urine at spiking levels of 7.4 nM, 22.2 nM and 37 nM. Average recoveries ranged from 95 to 110%, with an RSD of <1.5%. The method is expected to represent a promising tool for simple, rapid and sensitive on-site detection of RAC in animal products.

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Graphical abstract An aptamer based fluorescent assay for determination of ractopamine was developed with a dynamic range of 50 pM to 50 μM. The average recovery from spiked urine samples ranged from 95 to 110%, with an RSD of <1.5%.

     

A Novel Electrochemical Sensor Based on Molecularly Imprinted Solid-Phase Extraction for Rapid Determination of Ractopamine in Pig Urine

A new absorbent (polymer) for solid-phase extraction of ractopamine (RAC) was synthesized on the multi-walled carbon nanotubes (MWCTs) using grafting technique and surface imprinting methods. The superficial characteristics of the polymer were studied by scanning electron microscopy (SEM) and UN spectrometry was applied to investigate the static and kinetic adsorption capacity of the new absorbent. After the experimental conditions for the solid-phase extraction of RAC were optimized, a sensing system for the determination of RAC was established by connecting the solid-phase extraction column with a portable amperometric sensing system. The SEM study showed that numbers of imprinted micro-pores were exhibited on the surface of the imprinted polymers, and the absorption experiments indicated that the molecularly imprinted absorbent possessed satisfactory kinetics for the adsorption of ractopamine. The current response of the amperometric sensor demonstrated a linear correlation to the concentration of RAC over the range of 50 to 450 nM (r = 0.998) and the detection limit was 15 nM. Satisfactory sensitivity and stability was also presented under the optimized experimental conditions. The recoveries of RAC samples reached 87.3–94.8% in urine sample.