Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

BACKGROUND: Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the ß2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties. METHODS AND RESULTS: Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20 weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles. CONCLUSION: The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression.

A molecularly imprinted electrochemiluminescence nanoprobe based on complexes consisting of CdTe and multiwall carbon nanotube for sensitive determination of clenbuterol.

An electrochemiluminescence (ECL) nanoprobe was fabricated for the determination of clenbuterol (CLB). A molecularly imprinted polymer (MIP) film was coated on the surface of the glassy carbon electrode modified with CdTe-doped multiwall carbon nanotubes. The MIP film with CLB as the template molecule improves the selectivity of the nanoprobe, CdTe is used as ECL signal amplifier, and MWCNT works as the carrier. The ECL intensity is altered by elution and reabsorption of CLB. The possible reaction mechanism and experimental parameters of the nanoprobe are discussed. Under optimized conditions, the quenched ECL intensity and the CLB concentration have a linear relationship in the range 2.3 × 10-9 to 1.5 × 10-5 mol·L-1, and the detection limit is 1.0 × 10-9 mol·L-1 (S/N = 3). The nanoprobe was successfully applied to the determination of CLB in pork samples. Graphical abstract Schematic representation of the molecularly imprinted electrochemiluminescence nanoprobe based on complexes consisting of CdTe and multiwall carbon nanotube used to determinate clenbuterol.

A metal organic framework-functionalized monolithic column for enantioseparation of six basic chiral drugs by capillary electrochromatography.

Poly(glycidyl methacrylate)-co-(ethylene dimethacrylate) [poly(GMA-co-EDMA)] monoliths were used as a support to grow a zeolitic imidazolate framework-8 (ZIF-8) via layer-by-layer self-assembly. Pepsin, acting as as chiral selector, was covalently linked to the surface of the amino-modified ZIF-8 through the Schiff base method. The material was characterized by scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, Fourier transform infrared spectroscopy and elemental analysis. The pepsin-ZIF-8-poly(GMA-co-EDMA) column was utilized to the enantioseparation of the racemic forms of hydroxychloroquine (HCQ), chloroquine (CHQ), hydroxyzine (HXY), nefopam (NEF), clenbuterol (CLE) and amlodipine (AML). In comparison with a pepsin-poly(GMA-co-EDMA) monolithic column (without self-assembled ZIF-8 nanoparticles), the resolution is strongly enhanced (HCQ: 0.34 → 2.50; CHQ: 0.45 → 1.97; HXY: 0.39 → 1.43; NEF: 0.27 → 0.81; CLE: 0 → 0.81; AML: 0.16 → 0.72). Effects of self-assembly layers of ZIF-8, pepsin concentration, buffer pH values and applied voltage were investigated with hydroxychloroquine as the model analyte. The reproducibility of run-to-run, day-to-day and column-to-column were explored, and found to be satisfactory. Graphical abstractSchematic representation of capillary electrochromatography (CEC) systems with a pepsin-zeolitic imidazolate framework-8 (ZIF-8) modified poly(glycidyl methacrylate)-co-(ethylene dimethacrylate) [poly(GMA-co-EDMA)] monolithic column as stationary phases for separation of basic racemic drugs. ZIF-8 modified column was prepared via layer-by-layer self-assembly.

A fluorometric clenbuterol immunoassay using sulfur and nitrogen doped carbon quantum dots.

A fluorometric clenbuterol immunoassay is described that uses S- and N-co-doped carbon quantum dots as the fluorescent probe. Strongly fluorescent S/N-doped carbon quantum dots (S/N-CDs) were synthesized by hydrothermal method using fructose as the carbon precursor and L-cysteine as S/N sources. The S/N-CDs were characterized by transmission electron microscopy, energy dispersive spectroscopy and Fourier transform infrared spectroscopy (FTIR). Under 350 nm photoexcitation, they display strong purple fluorescence with an emission peak at 405 nm. In pH 4.0 solution, the amino groups (confirmed by FTIR) on the carbon quantum dots were coupled to clenbuterol antibody (Ab) by amine-amine coupling reaction to quench the fluorescence. If clenbuterol (Clen) is added, it binds to the Ab to generate a stable Ab-Clen immunocomplex and free S/N-CD. This causes the fluorescence of nanoprobe to be restored. The fluorescence of the system increases linearly in the 0.07-1.7 ng·mL-1 Clen concentration range. The probe of type S/N4-CD displays the best sensitivity. The detection limit is 23 pg·mL-1. Graphical abstract Schematic presentation of clenbuterol fluorometric immunoassay using sulfur and nitrogen doped carbon quantum dots.

Novel method for the rapid and specific extraction of multiple ß2 -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/H2 O. 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 ß2 -agonists residues in real food samples.

Rapid Determination of Clenbuterol in Pork by Direct Immersion Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry.

Direct immersion solid-phase microextraction (DI-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid analysis of clenbuterol in pork for the first time. In this work, a low-cost homemade 44 µm polydimethylsiloxane (PDMS) SPME fiber was employed to extract clenbuterol in pork. After extraction, derivatization was performed by suspending the fiber in the headspace of the 2 mL sample vial saturated with a vapor of 100 µL hexamethyldisilazane. Lastly, the fiber was directly introduced to GC-MS for analysis. All parameters that influenced absorption (extraction time), derivatization (derivatization reagent, time and temperature) and desorption (desorption time) were optimized. Under optimized conditions, the method offered a wide linear range (10-1000 ng g(-1)) and a low detection limit (3.6 ng g(-1)). Finally, the method was successfully applied in the analysis of pork from the market, and recoveries of the method for spiked pork were 97.4-105.7%. Compared with the traditional solvent extraction method, the proposed method was much cheaper and fast.

A Rapid Colorimetric Sensor of Clenbuterol Based on Cysteamine-Modified Gold Nanoparticles.

Demonstrated was a simple visual and rapid colorimetric sensor for detection of clenbuterol (CLB) based on gold nanoparticles (AuNPs) modified with cysteamine (CA) and characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-vis. The solution color from red to blue gray with increasing clenbuterol concentration resulted from the aggregation of AuNPs. The detection limit of clenbuterol is 50 nM by naked eyes. The selectivity of CA-AuNPs detection system for clenbuterol is excellent compared with other interferents in food. This sensor has been successfully applied to detect clenbuterol in real blood sample.

[Preparation of 1 µm non-porous C18 silica gel stationary phase for chiral-pressurized capillary electrochromatography].

Non-porous C18 silica gel stationary phase (1 µm) was prepared and applied to chiral separation in pressurized capillary electrochromatography (pCEC) for the enantioseparation of various basic compounds. The non-porous silica particles (1 µm) were synthesized using modified St6ber method. C18 stationary phase (1 µm) was prepared by immobilization of chloro-dimethyl-octadecylsilane. Using carboxymethyl-ß-cyclodextrin (CM-ß-CD) as the chiral additive, the pCEC conditions including the content of acetonitrile (ACN), concentration of buffer, pH, the concentration of chiral additive and flow rate as well as applied voltage were investigated to obtain the optimal pCEC conditions for the separation of four basic chiral compounds. The column provided an efficiency of up to 190,000 plates/m. Bupropion hydrochloride, clenbuterol hydrochloride, metoprolol tartrate, and esmolol hydrochloride were baseline separated under the conditions of 5 mmol/L ammonium acetate buffer at pH 4. 0 with 20% (v/ v) acetonitrile, and 15 mmol/L CM-ß-CD as the chiral additive. The applied voltage was 2 kV and flow rate was 0.03 mL/min with splitting ratio of 300:1. The resolution were 1.55, 2.82, 1. 69, 1. 70 for bupropion hydrochloride, clenbuterol hydrochloride, metoprolol tartrate, esmolol hydrochloride, respectively. The C18 coverage was improved by repeating silylation method. The synthesized 1 µm C18 packings have better mechanical strength and longer service life because of the special, non-porous structure. The column used in pCEC mode showed better separation of the racemates and a higher rate compared with those used in the capillary liquid chromatography (cLC) mode. This study provided an alternative way for the method of pCEC enantioseparation with chiral additives in the mobile phase and demonstrated the feasibility of micron particle stationary phase in chiral separation.

Automated headspace solid-phase microextraction and on-fiber derivatization for the determination of clenbuterol in meat products by gas chromatography coupled to mass spectrometry.

A method was developed for the determination of clenbuterol in meat using stable-isotope-dilution gas chromatography with mass spectrometry coupled with solid-phase microextraction and on-fiber derivatization. The samples were first homogenized with hydrochloric acid followed by protein deposition. After headspace solid-phase microextraction and on-fiber derivatization, the content of clenbuterol was measured with the aid of stable-isotope dilution. The condition of solid-phase microextraction was optimized by central composite design. The relative standard deviations, limit of detection, and recoveries for clenbuterol were 4.2-9.2%, 0.48 µg/kg, and 96-104%, respectively. The proposed method was satisfactory for analysis of real samples as compared with the Chinese standard method.

Sensitive voltammetric sensor based on isopropanol-Nafion-PSS-GR nanocomposite modified glassy carbon electrode for determination of clenbuterol in pork.

In the present study, poly(sodium 4-styrenesulfonate) (PSS) functionalized graphene (GR) was synthesised via a simple one-step chemical reduction of exfoliated graphite oxides in the presence of PSS. Characterisation of as-made nanocomposite using Fourier transform infrared spectroscopy (FT-IR) and ultraviolet and visible spectroscopy (UV-vis) clearly demonstrate the successful attachment of PSS to graphene sheets. A novel clenbuterol (CLB) electrochemical sensor was fabricated based on isopropanol-Nafion-PSS-GR composite film modified glassy carbon electrode. In the Britton-Robinson buffer (pH 1.2), the sensor exhibited superior electrocatalytic activity towards the oxidation of CLB. Applying linear sweep voltammetry, a good linear relationship of the oxidation peak current with respect to concentrations of CLB cross the range of 7.5 × 10(-8)-2.5 × 10(-5)mol L(-1) and a detection limit of 2.2 × 10(-8) mol L(-1) were achieved. The proposed method was successfully applied for the determination of CLB in pork.

Possible contamination with clenbuterol from treated veal calves to untreated pen mates.

To investigate whether clenbuterol-treated calves could contaminate untreated pen mates, three animal experiments were performed. (1) One calf of a pen of five was treated with clenbuterol by injection (Ventipulmin injection, REG NL 2532, 2.5 mL/100 kg) twice a day for 10 days. (2) In two pens, one animal was treated with clenbuterol via oral administration (Ventipulmin syrup, REG NL 2532, 4 mL/125 kg) for 4 weeks. (3) In two pens, one animal was treated with clenbuterol via the milk (Ventipulmin, REG NL 2532, 2.5 mL/100 kg body weight) twice a day for 10 days. Here, the animal was set apart during treatment, cleaned and put back into the group. Levels of clenbuterol were analysed in hair and urine with LC-MS/MS. Clenbuterol administered by injection could not be transferred from treated to untreated calves. In the second experiment, all pen mates were found positive for clenbuterol in the hair. This contamination was probably due to licking the mouth of the treated animal or saliva from the treated animal spoiling the floor. In the third experiment, no pen mates were found positive for clenbuterol in the hair. Clenbuterol was found in the urine and hair of only treated animals.

[Development of a high-throughput suspension microarray technology for detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol].

OBJECTIVE: To establish a novel suspension microarray technology for the detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol (CAP, CL and E2). METHODS: The three conjugates that veterinary drug coupled with bovine serum albumin (BSA) were synthesized and identified by ultraviolet (UV) spectrophotometry and mass spectrum. The veterinary drug conjugates were immobilized on the polystyrene fluorescent microspheres/beads. There were competitive reactions between the veterinary drugs in the aqueous phase and that on the beads for combination with their specific biotinylated monoclonal antibodies. The optimum amount of the veterinary drug conjugates and the antibodies were optimized and selected. The detective standard curves were plotted. The specificity and the unknown samples were also determined by grouping according to different concentrations of the interferes and the samples. Meantime, the different microstructures of the surfaces of the beads were also observed by scanning electron microscope. RESULTS: Couplings were completed between small molecular veterinary drugs and BSA. The amounts of the three conjugates and the antibodies were optimized. The detective standard curves of the suspension array and their corresponding coefficients of determination (R2) were good (R2 > 0.99). The detection ranges of the three veterinary drugs were (40.00 - 6.25) x 10(5) ng/L, (50.00-7.81) x 10(5) ng/L and 1.00 x 10(3) - 7.29 x 10(5) ng/L respectively. Simultaneously, the specific detection of the suspension microarray was excellent and did not indicate significant cross-reactions. Errors between the found and the real are in the range of 8.09% - 17.03%. It can be considered that the relative standard deviations were relatively small. Successful couplings were also directly confirmed by the observation for microstructures of the surfaces of the beads by scanning of electron microscope and laid good foundation for the following responses. CONCLUSION: The high-throughput suspension microarray should provide a novel method for multi-analysis of the veterinary drugs and have a wide applicative prospects with simple operation, sensitive, rapid and low cost.

Simultaneous detection for three kinds of veterinary drugs: chloramphenicol, clenbuterol and 17-beta-estradiol by high-throughput suspension array technology.

Suspension array technology for simultaneous detection of three kinds of veterinary drugs, chloramphenicol (CAP), clenbuterol and 17-beta-estradiol has been developed. Conjugates of chloramphenicol and clenbuterol coupled with bovine serum albumin were synthesized and purified. Probes of suspension array were constituted by coupling the three conjugates on the fluorescent microspheres/beads and the microstructures of the beads' surface were observed by scanning electron microscopy which was a direct confirmation for the successful conjugates' coupling. The optimal addition of conjugates and the amounts of antibodies were optimized and selected, respectively. Standard curves were plotted and the coefficient of determination-R(2) was greater than 0.989 which suggested good logistic correlation. The detection ranges for the three veterinary drugs are 40-6.25x10(5) ng L(-1), 50-7.81x10(5) ng L(-1) and 1x10(3-)7.29x10(5) ng L(-1), respectively and the lowest detection limits (LDLs) of them are 40, 50 and 1000 ng L(-1), respectively. The suspension array is specific and has no significant cross-reactivity with other chemicals. Meanwhile, unknown samples were detected by suspension array and ELISA in comparison with each other. The errors between found and real for the detection of the unknown samples were relatively small to both of the two methods, whereas, the detection ranges of suspension array are broader and sensitive than that of the traditional ELISA. The high-throughput suspension array is proved to be a novel method for multi-analysis of veterinary drugs with simple operation, high sensitivity and low cost.

Development of a lateral-flow assay for rapid screening of the performance-enhancing sympathomimetic drug clenbuterol used in animal production; food safety assessments.

A lateral-flow assay that could provide visual evidence of the presence of clenbuterol in swine urine was developed. Colloidal gold was prepared and conjugated with anti-clenbuterol monoclonal antibody. Immunochromatographic test strips were produced, and then, 210 samples were tested on these strips. Analysis was completed in 10 min. Detection limit was 3 ppb of clenbuterol. Parallel GC-MS data indicated that clenbuterol rapid detection strip had no false negative. The false positive rate was 4.4%. Immunochromatographic strip has great applied value in the food safety field because it possesses benefits of sensitivity, stability, reproducibility, ease of use and inexpensive.

Simultaneous determination of Zilpaterol and other beta agonists in calf eye by gas chromatography/tandem mass spectrometry.

Adrenergic drugs for growth promotion have been outlawed in European meat production; however, molecules such as Ractopamine and Zilpaterol are licensed for feeding swine and cattle in the United States, Mexico, and South Africa. Analysis of bovine retinal extracts has recently shown considerable extension in the detection period following withdrawal. Previous studies demonstrated that residual concentrations of Clenbuterol and related substances in retinal tissue were > 100 ng/g at day 50 of withdrawal. A method was developed to identify and simultaneously quantify Clenbuterol-like substances with anilinic moieties and drugs with phenolic and catecholic moieties, such as Ractopamine and Zilpaterol, in retinal tissue. The method was validated according to SANCO/1805/2000. After extraction in 0.1 N HCl, samples were cleaned up on C18 non-endcapped solid-phase extraction columns and analyzed as trimethylchlorosilane derivatives by gas chromatography/tandem mass spectrometry, electron impact mode. At concentrations of agonists between 62.5 and 250.0 ng/g in bovine retina, mean recoveries ranged from 85.3 to 94.8%, repeatability was < 9.6%, and within-laboratory reproducibility was < 10.5%. The decision limits (CCalpha) were within the range of 66.3-70.4 ng/g, and the detection capability (CCbeta) varied from 73.9 to 79.8 ng/g. Results are discussed in terms of a multiresidue approach to improve reliability of the monitoring strategy.

Effects of nebulizing and drying gas flow on capillary electrophoresis/mass spectrometry.

This study was focused on examining the influence of gas flow parameters on capillary electrophoresis/mass spectrometry (CE /MS) performance using sheath-liquid CE /MS interfaces. The effects of nebulizing and drying gas velocity and drying gas temperature on CE separation and MS detection sensitivity were systematically determined. Nebulizing gas velocity was observed to be a critical parameter in the optimization of CE /MS method, since it affected both MS detection sensitivity, and also CE separation efficiency for one interface design tested. Better detection sensitivity was obtained when the nebulizing gas velocity was increased. However, high velocity of the nebulizing gas flow can cause a hydrodynamic bulk flow inside the CE capillary, thus clearly increasing the apparent mobility and decreasing the resolution obtained for the compounds studied. Increasing the drying gas velocity or temperature did not affect the apparent mobility or the separation efficiency and the temperature could be increased to achieve the optimal detection sensitivity in the CE /MS analysis. For comparison, the effects of nebulizing gas flow were studied using a different design of the coaxial sheath-liquid CE /MS interface, and in this case better detection sensitivity but no effect on CE separation efficiency was observed with increased nebulizing gas velocity. These different effects of nebulizing gas flow on the CE bulk flow were concluded to result from pressure differences at the tip of the CE capillaries for the different CE /MS interface arrangements. It is therefore recommended that the cross-sectional dimensions of the fused-silica and steel capillaries, and the gas streamlines, should be optimized when CE /MS interfaces are built. Moreover, the effect of gas flow on CE separation should be studied when optimizing the CE /MS operation parameters.

Development of clenbuterol reference materials: lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 1. Preparation, homogeneity and stability.

Within the EU Standards, Measurement and Testing Program (SMT) two clenbuterol reference materials (RMs) were developed. Since clenbuterol readily accumulates and is slowly depleted from pigmented tissues such as the retina, homogenized eye liquid content is the most sensitive tissue for the detection of clenbuterol misuse. Therefore, both of the RMs were produced from bovine eye matrix: a negative control--RM 673 eye reference material, clenbuterol free (<0.50 microg/kg eye matrix) and a positive--RM 674 eye reference material containing clenbuterol (approximately 10 microg/kg eye matrix). Eyes were sampled from 103 German Simmental cattle and the inner liquid content was homogenized to a wet homogenized liquid content (HLC). This clenbuterol negative pool was divided into two sub-pools, one of which was spiked with clenbuterol to a final concentration of 10 microg clenbuterol/kg HLC. Of each pool exactly 2.0 +/- 0.01 g (+/- 0.5%) portions were weighed into 790 containers. Lyophilization of the 1,580 containers was performed in one batch. Parameters for the filling of containers, dry matter content, and residual moisture were in accordance with EU requirements. A three-year stability study and two homogeneity studies at various storage temperatures (-60 degrees C, -20 degrees C, +4 degrees C, +20 degrees C, and +37 degrees C) were performed. Low variation was observed within all of the homogeneity studies, proving that each of the RMs were homogeneous and that this was independent of storage temperature and storage time. In the stability studies, measured clenbuterol concentrations remained constant for RM 673 under the detection limit at 0.15 +/- 0.01 microg clenbuterol equivalent/kg HLC (n = 110) and were also constant for RM 674 at 11.21 +/- 0.15 microg clenbuterol/kg HLC (n=150; measured as duplicates). These studies demonstrate that clenbuterol-containing and clenbuterol-free RMs in bovine eye matrix can be successfully produced. Based on the results described above, it is concluded that both RMs may be suitable as candidates for certification.

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.

Positive chemical ionization and tandem mass spectrometric fragmentation for the gas chromatographic analysis of beta-agonists using the ion trap technique.

A procedure is described for the determination of three characteristic beta-agonists (clenbuterol, terbutalin and salbutamol) based on the formation of the corresponding protonated molecules and related collisional experiments. Quantification was carried out on selected collisional fragments and the reproducibility of the relative abundances of these fragments was estimated. The performance of the method was tested on bovine urine samples spiked at the lowest level of 0.2 ng ml(-1) in each of the chosen compounds.

Determination of clenbuterol residues in bovine hair by using diphasic dialysis and gas chromatography-mass spectrometry.

A method for the determination of clenbuterol (4-amino-3,5-dichloro-alpha[(tert.-butylamino)methyl]-benzyl alcohol hydrochloride) in hair of living cows has been developed. Hair samples were digested in an alkaline medium. The diphasic dialysis technique is a semi-permeable membrane technology developed for the direct extraction of relatively low-molecular-mass analytes such as clenbuterol. In this case, we used sodium citrate buffer to homogenize the digested hair, dichloromethane was used as the extraction solvent at 37 degrees C, and stirring was applied at 150 rpm for 4 h. The analysis was carried out using gas chromatography-mass spectrometry. The calibration curve for clenbuterol in hair was linear in the range from 12.5 to 400 ng g(-1). The detection limit of clenbuterol was 5 ng g(-1) and the quantification limit was 12.5 ng g(-1), in hair. A good inter-day reproducibility was obtained (R.S.D. = 7.08%). The repeatability and intra-day reproducibility (50 ng g(-1) of hair, n = 10) show R.S.D.s of 7.1 and 9.5%, respectively.