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Despite the presence of many studies on the role of mechanistic target of rapamycin ( mTOR) in cardiorenal tissues, the definitive role of mTOR in the pathogenesis of renal injury subsequent to ischaemia-reperfusion ( IR) remains unclear. The aims of the current study were to characterize the role of mTOR in normal kidney function and to investigate the role of mTOR activation in IR-induced kidney injury. In euvolemic anaesthetized rats, treatment with the mTOR inhibitor rapamycin increased blood pressure (121 ± 2 to 144 ± 3 mmHg; P<.05), decreased glomerular filtration rate ( GFR; 1.6 ± 0.3 to 0.5 ± 0.2 mL/min; P<.05) and increased urinary sodium excretion ( UNaV; 14 ± 1 to 109 ± 25 mmol/L per hour; P<.05). In rats subjected to IR, autophagy induction, p- mTOR expression and serum creatinine increased (1.9 ± 0.2 to 3 ± 0.3 mg/ dL; P<.05); treatment with rapamycin blunted p- mTOR expression but further increased autophagy induction and serum creatinine (3 ± 0.3 to 5 ± 0.6 mg/ dL; P<.05). In contrast, clenbuterol, an mTOR activator, blunted the effect of rapamycin on serum creatinine (4 ± 0.6 vs 2.3 ± 0.3 mg/ dL; P<.05), autophagy induction and p- mTOR expression. IR also increased 24 hour protein excretion (9 ± 3 to 17 ± 2 mg/day; P<.05) and kidney injury molecule-1 ( KIM-1) expression, and rapamycin treatment further increased KIM-1 expression. Clenbuterol exacerbated protein excretion (13 ± 2 to 26 ± 4 mg/day; P<.05) and antagonized the effect of rapamycin on KIM-1 expression. Histopathological data demonstrated kidney injury in IR rats that was worsened by rapamycin treatment but attenuated by clenbuterol treatment. Thus, mTOR signalling is crucial for normal kidney function and protecting the kidney against IR injury through autophagy suppression. [ABSTRACT FROM AUTHOR]

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, aβ2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy. [ABSTRACT FROM AUTHOR]

The aim of the current study was to examine the effects of clenbuterol injection into newly hatched chicks on both the abdominal fat pad tissue weight and the skeletal muscle weight during subsequent growth. Twenty-seven 1-day-old chicks were divided into two groups, receiving either a single intraperitoneal (i.p.) injection of clenbuterol (0.1 mg/kg body weight) or phosphate-buffered saline (PBS). Body weight gain, feed intake and feed conversion ratio were not affected by clenbuterol injection during the 5-week experimental period, while the abdominal fat pad tissue weight of the clenbuterol-injected chicks was lower than that of the control chicks at 5 weeks post-injection. Plasma non-esterified fatty acid concentrations were significantly increased in the clenbuterol-injected chicks, while plasma triacylglycerol concentrations did not differ. Additionally, the enzymatic activity of fatty acid synthase was lower in the liver of the clenbuterol-injected chicks. Conversely, the skeletal muscle weights were not affected by clenbuterol injection. These results suggest that a single clenbuterol injection into 1-day-old chicks decreases the abdominal fat pad tissue weight, but may not affect skeletal muscle weights during growth. © 2015 Japanese Society of Animal Science [ABSTRACT FROM AUTHOR]

A fully automated and validated 2D online solid-phase extraction (SPE) high-performance liquid chromatographic method was described for the direct simultaneous determination of clenbuterol (CLEN) and doxazosin (DOX) in spiked human plasma samples. Chromatographic separation of the analytes was achieved by column-switching. SPE column packed with restricted access material for depleting matrix and a reversed phase HPLC column for eluting analytes were used with UV detection on 250 nm. A 10mM acetate buffer (pH: 4) and acetonitrile mixture was used in gradient elution with 1 mL/min flow rate, as mass spectrometry friendly mobile phase. Calibration graphs were prepared for spiked plasma, and good linearity was achieved over a dynamic range of 75.0 ng/mL-200.0 µg/mL for CLEN and 37.5 ng/mL-100.0 µg/mL for DOX. Detection and quantification limits were 8.47 and 11.94 ng/mL, 25.66 and 36.18 ng/mL for CLEN and DOX, respectively. Online column-switching liquid chromatography technique combines the sample preparation by online SPE and separation steps in one allowing direct determination of drugs in biological fluids. This article could be assessed as a good example of combination of online sample preparation and separation, thus simultaneous determination of drugs in plasma samples. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This research outlines the application of an enzyme-linked immunosorbent assay (ELISA) for the analysis of clenbuterol in animal products. Our assay showed good sensitivity for clenbuterol (0.4 ng/g or 0.4 ppb) and low detection limit (0.09 ng/g or 0.09 ppb). A low cross-reactivity for other β-agonist drugs such as salbutamol, terbutaline, and epinephrine led to formatting an ELISA kit considered to have a high specificity for clenbuterol. A survey of Ho Chi Minh City pork market was conducted as part of the validation of our ELISA. ELISA results showed a surprisingly high value of contamination. However, it will be necessary to conduct a more statistically valid replicated survey with evaluation by other instrumental methods to obtain a definite conclusion. This ELISA kit will be used to monitor growth promoter residues in Vietnam's animal products. [ABSTRACT FROM AUTHOR]

A capillary immunochromatographic assay (CICA) using colloidal gold-labeled monoclonal antibodies was developed for semi-quantitative detection of clenbuterol (CLE) in this paper. This novel system fabricated in a competitive format was supported by glass capillary because of its easily modified, low cost, favorable optical properties. Here, two different-sized gold nanoparticles (13 and 26 nm) were prepared and corresponding assay conditions were optimized. By comparing the results between the two different-sized AuNPs, the 26-nm-AuNPs manifested higher sensitivities which the detection limit was estimated to be 0.35 ng/ml and the cut-off level with the naked eye of 2.5 ng/ml was observed. Using the optimized parameters, the intra-assay and inter-assay coefficients of variation (CVs) were ≤10.2 % and the recovery reached 70.8-115.5 % in pig urine, pork, beef, pork sausage and luncheon meat. The performance, including sample preparation, could be accomplished within 24 min. These results suggest that CICA holds promising use for sensitive, simple and low-cost on-site detection of CLE. [ABSTRACT FROM AUTHOR]

Background: Clenbuterol, a beta2-adrenergic receptor agonist, is used therapeutically to treat respiratory conditions in the horse. However, by virtue of its mechanism of action it has been suggested that clenbuterol may also have repartitioning affects in horses and as such the potential to affect performance. Clenbuterol decreases the percent fat and increases fat-free mass following high dose administration in combination with intense exercise in horses. In the current study, microarray analysis and real-time PCR were used to study the temporal effects of low and high dose chronic clenbuterol administration on differential gene expression of several skeletal muscle myosin heavy chains, genes involved in lipid metabolism and the β2-adrenergic receptor. The effect of clenbuterol administration on differential gene expression has not been previously reported in the horse, therefore the primary objective of the current study was to describe clenbuterol-induced temporal changes in gene expression following chronic oral administration of clenbuterol at both high and low doses. Results: Steady state clenbuterol concentrations were achieved at approximately 50 h post administration of the first dose for the low dose regimen and at approximately 18-19 days (10 days post administration of 3.2 μg/kg) for the escalating dosing regimen. Following chronic administration of the low dose (0.8 μg/kg BID) of clenbuterol, a total of 114 genes were differentially expressed, however, none of these changes were found to be significant following FDR adjustment of the p-values. A total of 7,093 genes were differentially expressed with 3,623 genes up regulated and 3,470 genes down regulated following chronic high dose administration. Of the genes selected for further study by real-time PCR, down-regulation of genes encoding myosin heavy chains 2 and 7, steroyl CoA desaturase and the β2-adrenergic receptor were noted. For most genes, expression levels returned towards baseline levels following cessation of drug administration. Conclusion: This study showed no evidence of modified gene expression following chronic low dose administration of clenbuterol to horses. However, following chronic administration of high doses of clenbuterol alterations were noted in transcripts encoding various myosin heavy chains, lipid metabolizing enzymes and the β2-adrenergic receptor. [ABSTRACT FROM AUTHOR]

To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases. [ABSTRACT FROM PUBLISHER]

Increased activity of the noradrenergic system in the amygdala has been suggested to contribute to the hyperarousal symptoms associated with post-traumatic stress disorder (PTSD). However, only two studies have examined the content of noradrenaline or its metabolites in the amygdala of rats previously exposed to traumatic stress showing inconsistent results. The aim of this study was to investigate the effects of an inescapable foot shock (IFS) procedure (1) on reactivity to novelty in an open-field (as an index of hyperarousal), and (2) on noradrenaline release in the amygdala during an acute stress. To test the role of noradrenaline in amygdala, we also investigated the effects of microinjections of propranolol, a β-adrenoreceptor antagonist, and clenbuterol, a β-adrenoreceptor agonist, into the amygdala of IFS and control animals. Finally, we evaluated the expression of mRNA levels of β-adrenoreceptors (β1 and β2) in the amygdala, the hippocampus and the prefrontal cortex. Male Wistar rats (3 months) were stereotaxically implanted with bilateral guide cannulae. After recovering from surgery, animals were exposed to IFS (10 shocks, 0.86 mA, and 6 s per shock) and seven days later either microdialysis or microinjections were performed in amygdala. Animals exposed to IFS showed a reduced locomotion compared to non-shocked animals during the first 5 min in the open-field. In the amygdala, IFS animals showed an enhanced increase of noradrenaline induced by stress compared to control animals. Bilateral microinjections of propranolol (0.5 μg) into the amygdala one hour before testing in the open-field normalized the decreased locomotion observed in IFS animals. On the other hand, bilateral microinjections of clenbuterol (30 ng) into the amygdala of control animals did not change the exploratory activity induced by novelty in the open field. IFS modified the mRNA expression of β1 and β2 adrenoreceptors in the prefrontal cortex and the hippocampus. These results suggest that an increased noradrenergic activity in the amygdala contributes to the expression of hyperarousal in an animal model of PTSD. [ABSTRACT FROM AUTHOR]

The long-acting, highly lipophilic, β 2 -adrenoceptor agonist clenbuterol may represent a suitable therapeutic agent for the treatment of neuroinflammation as it drives an anti-inflammatory response within the CNS. However, clenbuterol is also known to increase the expression of IL-1β in the brain, a potent neuromodulator that plays a role in provoking sickness related symptoms including anxiety and depression-related behaviours. Here we demonstrate that, compared to the immunological stimulus lipopolysaccharide (LPS, 250 μg/kg), clenbuterol (0.5 mg/kg) selectively up-regulates expression of the central IL-1 system resulting in a mild stress-like response which is accompanied by a reduction in locomotor activity and food consumption in rats. We provide further evidence that clenbuterol-induced activation of the central IL-1 system occurs in a controlled and selective manner in tandem with its negative regulators IL-1ra and IL-1RII. Furthermore, we demonstrate that peripheral β 2 -adrenoceptors mediate the suppression of locomotor activity and food consumption induced by clenbuterol and that these effects are not linked to the central induction of IL-1β. Moreover, despite increasing central IL-1β expression, chronic administration of clenbuterol (0.03 mg/kg; twice daily for 21 days) fails to induce anxiety or depressive-like behaviour in rats in contrast to reports of the ability of exogenously administered IL-1 to induce these symptoms in rodents. Overall, our findings suggest that clenbuterol or other selective β 2 -adrenoceptor agonists could have the potential to combat neuroinflammatory or neurodegenerative disorders without inducing unwanted symptoms of depression and anxiety. [ABSTRACT FROM AUTHOR]

Clenbuterol is a β-adrenergic agonist used as additive to increase the muscle mass of meat-producing animals. Previous studies were limited to evaluations of animal growth performance and determination of the residues. Several studies have focused on urine samples. Little information about the underlying molecular mechanisms that can explain Clenbuterol metabolism and promote energy repartition in animal muscle and fatty tissue is available. Therefore, this research aims to detect the metabolite variations in muscle and fatty tissue acquired from Chinese pigs fed with Clenbuterol using gas chromatography/mass spectrometry (GC/MS). Ten two-month old Enshi black pigs were fed under the same condition; five of which were fed with basic ration containing Clenbuterol for one month, whereas the other five pigs were fed only with basic ration. Muscle and fatty tissue were subjected to metabolomics analysis using GC/MS. Differences in metabolomic profiles between the two groups were characterized by multivariate statistical analysis. The muscle samples showed that 15 metabolites were significantly different in the Clenbuterol-treated group compared with the control group; 13 potential biomarkers were found in the fatty tissue. Most of the metabolites were associated with fatty acid metabolism and amino acid metabolism. Glycerol, phenylalanine, and leucine were the common metabolites between the muscle and fatty tissue. These metabolites may provide a new clue that contributes to the understanding of the energy reassignment induced by Clenbuterol. [ABSTRACT FROM AUTHOR]

Introduction and Aim: To understand health service access and needs of people who use performance and image enhancing drugs (PIED) in regional Queensland.Design and Methods: Semi-structured interviews were conducted with 21 people (n = 19 men) who reported the use of a range of PIEDs, including anabolic-androgenic steroids, human chorionic gonadotropin, growth hormone, clenbuterol, tamoxifen, insulin and peptides.Results: Participants reported accessing a range of services, including needle and syringe programs and pharmacies, for sterile injecting equipment. While PIEDs users attributed some stigma to needle and syringe programs, they were seen as an important service for injecting equipment. Participants reported receiving either positive care from health-care providers, such as general practitioners (GP), or having negative experiences due to the stigma attached with PIED use. Few participants reported disclosing their PIED use to their GP not only because of the concerns that their GP would no longer see them but also because they felt their GP was not knowledgeable about these substances.Discussion and Conclusion: Participants in the study reported no difficulty in accessing health services based on living in a regional area, with their concern focused more upon how they were viewed and treated by service staff. [Dunn M, Henshaw R, Mckay F. H. Do performance and image enhancing drug users in regional Queensland experience difficulty accessing health services? Drug Alcohol Rev 2016;35:377-382]. [ABSTRACT FROM AUTHOR]

Electrochemical immunosensors based PEDOT/MWCNT hybrid composite for detection of clenbuterol antibiotic has been developed. It shows promising properties as biosensors platform with combination of high conductive properties of poly(3,4-ethylenedioxythiophene) (PEDOT) and high surface area of carbon nanotube (CNT). Based on cyclic voltammetry (CV) measurement in potassium ferricyanide (K 3 [Fe(CN) 6 ]), lower peak potential separation (90.33 mV) and higher peak current (0.197 mA) were obtained compared with unmodified electrode (114.75 mV and 0.163 mA).This immunosensor, which was based on direct competitive method, was examined with standard clenbuterol hydrochloride for detection of clenbuterol through chronoamperometry at -0.1 V. [ABSTRACT FROM AUTHOR]

β-agonists are phenylethanolamines with different substituent groups on the aromatic ring and the terminal amino group which have the effect of nutrition redistribution and can accumulate in body tissues causing acute or chronic poisoning when consumed. Therefore, it is very important to establish a fast screening method for the detection of several kinds of β-agonists in food safety control. In this study, the aptamer-agonists (AP-Ago) has screened out by Isothermal Titration Calorimetric method. AP-Ago was a single-strand DNA with 22 base-pairs. The dissociation constant ( K d ) to phenylethanolamine (PHL) was 3.34×10 −5 mol L −1 . The AP-Ago based electrode was constructed by self-assembling on gold electrode. A label-free electrochemical aptasensor was then developed with AP-Ago-based gold electrode, which was sensitive to phenylethanolamine(PHL), clenbuterol (CLB), ractopamine (RAC), salbutamol (SAL) and procaterol (PRO). The detection limits were 0.04 ng/mL (RAC), 0.35 pg/mL (CLB), 1.0 pg/mL (PHL), 0.53 pg/mL (SAL) and 1.73 pg/mL(PRO), respectively, The detection time was 15 min. The reproductivity of the mentioned aptasensor is good with RSD of 2.09%. Comparing with ELISA and HPLC on β-agonists detection in actual sample, this aptasensor is advantage of fewer steps and fast screen-detection of these five β-agonists or their mixtures. This study suggests that the aptasensor can be developed to a rapid screening means with multi-β-agonists (may be one or more) in sample. [ABSTRACT FROM AUTHOR]

In this work, a portable label-free impedance immunosensor for the analysis of clenbuterol (CLB) was constructed by modifying L-cysteine ( L-cys) and glutaraldehyde (GA) on the surface of a screen-printed gold electrode (SPGE). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize electrochemical properties of the immunosensor. Under the optimized conditions, the immunosensor could detect CLB in the range of 1.0 × 10-1.0 mg/L ( R = 0.998), and the detection limit is 2.0 × 10 mg/L. The as-developed immunosensor can be applied in detecting CLB in real samples. The average recoveries in the spiked pork samples ranged from 92.4 to 104.6 %, and their relative standard deviations were 3.6-5.2 %. The testing results were consistent with those obtained from high-performance liquid chromatography method. The proposed immunosensor exhibits high sensitivity, specificity, and good stability. [ABSTRACT FROM AUTHOR]

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Clenbuterol (CB), a selective β2-adrenergic receptor (AR) agonist, induces muscle hypertrophy and counteracts muscle atrophy. However, it is paradoxically less effective in slow-twitch muscle than in fast-twitch muscle, though slow-twitch muscle has a greater density of β-AR. We recently demonstrated that Epac1 (exchange protein activated by cyclic AMP [cAMP]1) plays a pivotal role in β2-AR-mediated masseter muscle hypertrophy through activation of the Akt and calmodulin kinase II (CaMKII)/histone deacetylase 4 (HDAC4) signaling pathways. Here, we investigated the role of Epac1 in the differential hypertrophic effect of CB using tibialis anterior muscle (TA; typical fast-twitch muscle) and soleus muscle (SOL; typical slow-twitch muscle) of wild-type (WT) and Epac1-null mice (Epac1KO). The TA mass to tibial length (TL) ratio was similar in WT and Epac1KO at baseline and was significantly increased after CB infusion in WT, but not in Epac1KO. The SOL mass to TL ratio was also similar in WT and Epac1KO at baseline, but CB-induced hypertrophy was suppressed in both mice. In order to understand the mechanism involved, we measured the protein expression levels of β-AR signaling-related molecules, and found that phosphodiesterase 4 (PDE4) expression was 12-fold greater in SOL than in TA. These results are consistent with the idea that increased PDE4-mediated cAMP hydrolysis occurs in SOL compared to TA, resulting in a reduced cAMP concentration that is insufficient to activate Epac1 and its downstream Akt and CaMKII/HDAC4 hypertrophic signaling pathways in SOL of WT. This scenario can account for the differential effects of CB on fast- and slow-twitch muscles. [ABSTRACT FROM AUTHOR]

A novel single-isomer cyclodextrin derivative, heptakis {2,6-di-O-[3-(1,3-dicarboxyl propylamino)-2-hydroxypropyl]}-β-cyclodextrin (glutamic acid-β-cyclodextrin) was synthesized and used as a chiral selector in capillary electrophoresis for the enantioseparation of 12 basic drugs, including terbutaline, clorprenaline, tulobuterol, clenbuterol, procaterol, carvedilol, econazole, miconazole, homatropine methyl bromide, brompheniramine, chlorpheniramine and pheniramine. The primary factors affecting separation efficiency, which include the background electrolyte pH, the concentration of glutamic acid-β-cyclodextrin and phosphate buffer concentration, were investigated. Satisfactory enantioseparations were obtained using an uncoated fused-silica capillary of 50 cm (effective length 40 cm) × 50 μm id with 120 mM phosphate buffer (pH 2.5-4.0) containing 0.5-4.5 mM glutamic acid-β-cyclodextrin as background electrolyte. A voltage of 20 kV was applied and the capillary temperature was kept at 20°C. The results proved that glutamic acid-β-cyclodextrin was an effective chiral selector for studied 12 basic drugs. Moreover, the possible chiral recognition mechanism of brompheniramine, chlorpheniramine and pheniramine on glutamic acid-β-cyclodextrin was investigated using the semi-empirical Parametric Method 3. [ABSTRACT FROM AUTHOR]

A novel solid-phase microextraction method is reported that involved modification of the pores of hollow fibers with graphene oxide. The modified graphene oxide/hollow fiber was characterized by scanning electron microscopy and Raman spectroscopy and employed for the determination of ractopamine and clenbuterol in fortified beef. The repeatability of the method was satisfactory with intra-day and inter-day relative standard deviations between 2.5 and 5.2% for ractopamine and clenbuterol. Under the optimized conditions, the linear dynamic ranges of ractopamine and clenbuterol were from 0.05 to 2.5 µg/mL and 0.1 to 2.5 µg/mL, respectively. The limits of detection for ractopamine and clenbuterol were 0.01 and 0.03 µg/mL, and the limits of quantification were 0.03 and 0.08 µg/mL. The developed method was used for the determination of ractopamine and clenbuterol in fortified beef with average recoveries of 85.8–109.8% and 78.8–101.4%. [ABSTRACT FROM PUBLISHER]

A novel multiplexed immunochromatographic assay (ICA) based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of β-agonists, by utilizing ractopamine (RAC) and clenbuterol (CLE) as the models. Different from conventional multiplexed ICA methods which usually require two or more test lines, this strategy was developed for detection of two β-agonists by using only one test line on the nitrocellulose membrane. In this study, horseradish peroxidase and alkaline phosphatase were used as the signal probes to label RAC antibody and CLE antibody, respectively. The two CL reactions with flash type and glow type kinetics characteristics were triggered simultaneously by injecting the coreactants, then the signals for RAC and CLE detections were recorded at 3 s and 300 s after coreactants injection, respectively. Owing to the utilization of CL detection, this protocol showed ideal sensitivity for quantitation. Under the optimal conditions, the detection limits for RAC and CLE were 0.17 ng mL −1 and 0.067 ng mL −1 (S/N = 3), respectively. The whole assay process can be accomplished within 20 min without complicated sample pretreatment. The proposed method was successfully applied for the detection of RAC and CLE in spiked swine urine. It opens up a new pathway for designing a low cost, time-efficiency and multiplexed strategy for rapid screening and field assay. [ABSTRACT FROM AUTHOR]

A new kind of selective magnetic dispersive solid-phase sorbent based on multiple Fe 3 O 4 nanospheres as the core structure and molecular imprinted material as the shell structure was synthesized with tert -butylamine and 2-chloroaniline as the templates. The obtained multicore–shell-structured sorbent was spherical (diameter distribution 25–90 μm) with porous morphologies, thus incorporating strong magnetic properties and specific molecular recognition coupled with rapid adsorption and dynamic equilibrium. The sorbent was applied for rapid and selective screening of clenbuterol (CLB) in bovine urine samples. Good linearity was obtained in the range 1.25–200 ng mL −1 with the average recovery at three spiked levels ranging from 91.4% to 105.3%. The proposed method significantly improved the purification and extraction efficiency of CLB in urine samples and eliminated the effect of template leakage during quantitative analysis. [ABSTRACT FROM AUTHOR]

A rapid and simple high-performance liquid chromatography-UV method was developed for the separation and quantification of salbutamol, ractopamine, and clenbuterol in pork. A mixture of acetonitrile-formic acid-ammonium acetate was used as the mobile phase to separate three β-agonists on a C18 column with gradient. The effects of the addition of formic acid and ammonium acetate to mobile phases on the separation of β-agonists were investigated. These additives can greatly improve the resolution and sensitivity. Under the optimized chromatographic condition, this separation does not need extra sample preparation. Complete baseline separation of three β-agonists was achieved in < 20 minutes; the linear range is 0.2-50 µg/L with a correlation coefficient R² value of > 0.99. Excellent method reproducibility was found by intra- and interday precisions with a relative standard deviation of < 3%. The detection limit (S/N = 3) was found to be <0.05 µg/L; this method can be used for routine screening of the β-agonist residues in foods of animal origin before being identified by confirmatory methods. [ABSTRACT FROM AUTHOR]

Due to their late effects (increased muscle mass and reducing fat) clenbuterol is used by athletes and bodybuilders as an anabolic agent. According to the rules of the World Anti-Doping Agency, the B2 agonists in general are banned in sports. This article is an overview of its impact on health, adverse effects and toxicity. The doctors that treating teenagers who do body build in gorroad cycling or mountain, must have the basic knowledge of the risks involved clenbuterol when employed as ananabolic agent and its impact on health. [ABSTRACT FROM AUTHOR]

Potentimetric sensors based on molecularly imprinted polymers are reported for the selective determination of clenbuterol. The work involved the modification of multiwalled carbon nanotubes, the modification of chitosan with diethylene triamine pentaacetic acid, the synthesis of the clenbuterol-imprinted polymer, and the preparation of potentiometric sensors using the clenbuterol-imprinted polymer. The effect of pH, response time, lifetime, selectivity, reusability, reproducibility, accuracy, and stability of the sensors were characterized. The optimum pH was 6.0 for the prepared poly(vinyl chloride) membrane and carbon paste electrodes. The limit of detection of the poly(vinyl chloride) membrane electrode was 0.49 × 10−8 mM, with the linear dynamic range from 1.0 × 10−5to 1.0 × 10−9 mM. The response time for the membrane electrode was 2 min and the operational lifetime was 23 weeks. For the carbon paste electrode, the limit of detection was 0.91 × 10−8 mM, the linear dynamic range was from 1.0 × 10−4to 1.0 × 10−9 mM, the response time was 1 min, and the operational lifetime was 24 weeks. [ABSTRACT FROM AUTHOR]

A novel and ultra sensitive immunochromatographic assay sensor (ICA) based on up conversion phosphor (UCP) for quantitative detection of clenbuterol (CL) was developed. Monoclonal antibody against CL was labeled with UCP beads. The detection strategy is based on competitive immunoreaction between CL antibodies conjugated to UCP beads and CL or CL antigen on the UCP-ICA sensor. It enables ultra sensitive detection of CL in one single test without complicated sample preparation. Sensing results can be obtained within 10 min. Under optimized conditions, visual limit of detection ( v LOD) of UCP-ICA for CL was 0.1 ng/mL. Calculated LOD ( c LOD) for CL, as low as 0.01 ng/mL, could be achieved with the UCP-ICA sensor. Recoveries of CL in various sample matrixes ranged from 73.0% to 92.2% and relative standard deviations (RSD) were below 12%. The assay was evaluated with spiked and real samples and the results were compared with liquid chromatography-tandem mass. The developed novel assay method based on UCP could be a potential alternative format for on site and rapid detection of CL as well as other illegal drugs. [ABSTRACT FROM AUTHOR]

Background: Doping with anabolic androgenic steroids in sports has now developed to a widespread use of these agents among young people outside the sport. This is of major concern to the society. The purpose of the use is mainly for aesthetic reasons and is seen as a male phenomenon. But use also occurs in women where the knowledge is scarce. Our aim was to identify the pattern of doping agents in eight female cases and compare them with similar data from men.Methods: Eight female users were recruited through Anti-Doping Hot-Line, a national telephone counseling service on doping issues during the years 1998-2004. The use was confirmed with urine doping analysis at the Doping Laboratory. The characteristic of use, co-use of narcotics/other doping agents, exercise pattern, adverse-side effects, family history and reason to begin was evaluated.Results: The women used on average 1.9 different anabolic androgenic steroids and clenbuterol preparations. Ephedrine and growth hormone were co-used in five and one of the women, respectively. Three women reported co-use of narcotics (cannabis and cocaine). The average duration of anabolic agent use before contacting health care was 58 weeks (range 7-104). Side effects for anabolic androgenic steroids (n = 5) included voice changes, clitoral enlargement, body hair growth, whereas women using clenbuterol (n = 2) reported tachycardia and depression. All women except one had a man in close relationship encouraging them to begin with the doping agents.Conclusions: The use of doping agents in our eight women was different from that in male users. The women used less doping agents and were more prone to contact the health care, at an earlier stage, probably due to the adverse effects. The co-use with ephedrine, growth hormone and cannabis appeared to be in the same range as in men. This is the first study showing that a man in close relationship may motivate a woman to use anabolic agents. [ABSTRACT FROM AUTHOR]

The development of highly sensitive detection of -agonists compound by using surface plasmon resonance (SPR) immunoassay functionalized alkanethiols monolayer on gold surface was reported here. For the construction of robust SPR sensor surface, the illegal compound (as antigen) was immobilized onto gold succinimide-terminated monolayer to perform amide-coupling reaction. Structural characteristic of monolayer is an important issue for further application of selt-assembled monolayer (SAM)-based molecular electronics. This study focuses on the relation between structure of alkanethiols SAM of different chain length and its sensitivity of the detection. The surface structure is characterized by electrochemical methods and STM. The target compound in this research is clenbuterol as farmers used illegally to increase their profit gain. For the detection of clenbuterol, indirect competitive inhibition method was used. In this work, the SPR senses the dielectric constant change at the interface from the binding of the unreacted antibody to antigen-immobilized on the sensor surface. By this method, clenbuterol can be detected within ppt level and only requires 5min per sample. Furthermore, shorter chain alkanethiol sensor surface produces higher sensitivity in the detection process. [ABSTRACT FROM AUTHOR]

A nanocomposite consisting of zinc sulfide quantum dots and polyaniline (ZnSQD@PANI) was placed on a gold electrode along with antibody against clenbuterol to give an amperometric immunosensor for clenbuterol. Compared to the use of pristine PANI, the electrode modified with the ZnSQD@PANI nanocomposite adsorbs clenbuterol antibody much better and therefore exhibits higher sensitivity to clenbuterol. The biosensor, when operated at a working potential of 0.21 V (vs. Ag/AgCl), displays a detection limit as low as 5.5 pg⋅mL and works over the 0.01 to 10 ng⋅mL concentration range. Related species such as salbutamol and ractopamine, urine components such as urea and uric acid, and the ions Ca(II), Na(I), and K(I) do not interfere. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

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 fiberwas 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, themethod offered awide linear range (10-1000 ng g-1) and a lowdetection limit (3.6 ng g-1). Finally, themethodwas successfully applied in the analysis of pork fromthe 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. [ABSTRACT FROM AUTHOR]

Being the most used and preferred β2-agonist, clenbuterol (CLB) has been reported to be illegally abused in animal production and breeding as it improves growth rate while inhibiting fat synthesis and deposition leading to buildup of lean muscle. Due to its potential hazard to human health, causing food poisoning that can be fatal, it has been forbidden as a growth promoter for food-producing animals in most countries including China. Therefore, frequent monitoring of CLB abuse is necessary for consumer safety against its residues. We therefore report development of a monoclonal antibody and lateral flow test strip for sensitive detection of CLB in urine samples. In this investigation, male BALB/C mice were first immunized for antibody production. Thereafter, gold nanoparticles were utilized for antigen and antibody immobilization during construction of the strip. Under optimized conditions, the cut-off limit of the test strip was found to be 2.5 and 5 ng/g in phosphate-buffered saline (PBS) and urine using the wet method. When the dry method was used for CLB detection, the cut-off limit of the test strip in PBS and urine was found to be 3 ng/g. Each test was evaluated within 6 min. The data obtained show that the strip developed is sensitive and has an advantage of fast test results and simplicity and hence can be adopted for screening of CLB residues in urine samples for mitigation of CLB residue effects to human health. [ABSTRACT FROM AUTHOR]

We aimed to evaluate whether living tissues such as urine, plasma and hair were suitable for monitoring clenbuterol (CL) abuse after its subchronic administration of a growth-promoting dose to the Chinese Simmental beef cattle. Eight male, white and red pied Chinese Simmental beef cattle were involved in the experiment, and the CL dose was 16 μg/kg BW/day. Liquid chromatography tandem mass spectrometry (LC-MS-MS) was used to determine CL residues in different tissues, and the addition of D9-clenbuterol internal standard was applied to increase determination accuracy. The recovery of plasma, urine, hair and in vivo tissues was 88.5-114.2, 83.9-114.3, 88.6-116.9 and 85.3-121.7%, respectively. The results showed that CL residue concentrations in the plasma, on Days 14 after withdrawal and later, were lower than the limit of detection (LOD) (0.06 ng/mL) and CL residue in urine was lower than LOD (0.16 ng/mL) 42 days after treatment. CL significantly accumulated in the white and red hair and maintained more than 7.19 ' 2.19 pg/mg within the early withdrawal period of 70 days. A large number of CL were determined in all tested biological tissues, in which residues were higher than the maximum residue limits (MRLs) after dietary administration of CL for 21 days and pre-slaughter withdrawal period of ~6 h. A particular concern is the slow depletion of residues of CL in some tissues like gluteus and liver still exceeding theirs MRLs, respectively, on Days 14 or 28 days after withdrawal. Our study indicated that plasma and urine could be available for monitoring CL abuse only within a short period of time. However, hair (including light-pigmented) as a target matrix can be selected to perform the long-period monitor of CL. [ABSTRACT FROM AUTHOR]

A sensitive and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for determination of tulobuterol in rat plasma for the first time. Plasma samples were extracted by liquid–liquid extraction method with methyl tert -butyl ether and the analyte and clenbuterol (IS) were separated on a Venusil MP C 18 column (100 mm × 2.1 mm, 3 μm) using 0.1% formic acid–water–methanol as mobile phase, with a runtime of 5 min. The analyte was detected in multiple reaction monitoring (MRM) mode with positive electrospray ionization. Transitions of m / z 228.2 → 154.0 for tulobuterol and m / z 277.1 → 203.0 for the clenbuterol were monitored. The linear range was 0.5–100 ng/ml ( r = 0.9967) for tulobuterol with the lower limit of quantitation of 0.5 ng/ml. The intra-day and inter-day precisions were less than 10.3% for the analyte and the accuracy was less than −8.6%. The RSD of matrix effect and recovery yield were within ±15% of nominal concentrations and tulobuterol was stable during stability studies. The validated method has been successfully applied to a pharmacokinetic study of three doses of tulobuterol patch in rats for the first time. [ABSTRACT FROM AUTHOR]

A novel molecularly imprinted polymer (MIP) for efficient separation and concentration of clenbuterol (CLB) was synthesized by covalent imprinting approach using CLB derivative as functional monomer. The MIPs synthesized were characterized by scanning electron microscope, nitrogen adsorption analysis, Fourier transform infrared spectrometer, and thermo-gravimetric analysis. The binding experimental results showed that the MIPs synthesized had fast adsorption kinetic (20 min at 25 mg L −1 ), high adsorption capacity and specific recognition ability for the analyte. In addition, the MIPs synthesized were successfully used as solid-phase sorbent for CLB sample preparation to be analyzed by high performance liquid chromatography with ultraviolet detector. Under optimized experimental conditions, the linear range of the calibration curve was 5–80 μg L −1 ( R 2 = 0.9938). The proposed method was also applied to the analysis of CLB in pork and potable water samples. [ABSTRACT FROM AUTHOR]