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Zilpaterol and clenbuterol are two β-adrenergic agonist drugs used in animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries. Clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used in unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proved yet of zilpaterol to produce reactive oxygen and nitrogen species. Regarding glutathione which is the main intracellular antioxidant plays detoxification functions on liver metabolism; in this work, it is our interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures (PCLS) exposed to clenbuterol and zilpaterol. A single drug assay was performed in first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both β-adrenergic agonists modify in a dose-dependent manner in oxide-reduction response through ROS generation. The activity or content of glutathione peroxidase activity, intracellular GSH, gamma glutamyl-transpeptidase, aspartate aminotrasnferase and alanine aminotrasnferase were modified. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

Clenbuterol is a β-adrenergic receptor agonist that can be used as a bronchodilator for the treatment of respiratory diseases or as an additive feed in livestock to improve conversion rate of lean meat. However, the residue of clenbuterol in food can be a threat to human health. Therefore, it is important to monitor clenbuterol in foods. In this paper, a fluorescence inner filtration immunoassay (FIFI) method for the detection of clenbuterol was developed based on the principle of immunoreactive competitive method and the theory of fluorescence quenching, which was constructed from a fluorescence-quenching detection system and a separation system of magnetic immunization. In the magnetic immunization separation system, the detection antigen competes with the biotin-labeled antigen for binding to colloidal gold-labeled clenbuterol antibody and the immunocomplex is enriched by streptavidin- and biotin-specific reactions on the surface of magnetic beads. The remaining complex as a fluorescence quencher in the detection system after centrifugation, and rhodamine 6G acts as a fluorescence agent. The quantitative detection of clenbuterol was accomplished by achieving fluorescence quenching under excitation light irradiation. The standard solutions of clenbuterol in the range of 0.03–5.0 ng/mL exhibited good correlation (r = 0.9996) with the fluorescence intensity, with limit of detection 0.01 ng/mL and recoveries ranging from 98.1 to 101.2%. Moreover, there was no significant difference between the results obtained after testing valid samples by FIFI and ELISA. Therefore, our developed method is sensitive and efficient, simple to operate, and has great potential for on-site detection of clenbuterol.

Context Exercise blunts the effect of beta2-agonists on peripheral glucose uptake and energy expenditure. Whether such attenuation extends into recovery is unknown. Objective To examine the effect of a beta2-agonist on leg glucose uptake and metabolic rate in recovery from exercise. Methods Using leg arteriovenous balance technique and analyses of thigh muscle biopsies, we investigated the effect of a beta2-agonist (24 mg of oral salbutamol) vs placebo on leg glucose, lactate, and oxygen exchange before and during quadriceps exercise, and 0.5 to 5 hours in recovery from quadriceps exercise, as well as on muscle glycogen resynthesis and activity in recovery. Twelve healthy, lean, young men participated. Results Before exercise, leg glucose uptake was 0.42 ± 0.12 and 0.20 ± 0.02 mmol × min–1 (mean ± SD) for salbutamol and placebo (P = .06), respectively, while leg oxygen consumption was around 2-fold higher (P Conclusion Exercise blunts the effect of beta2-agonist salbutamol on leg glucose uptake, but this attenuation diminishes in recovery. Salbutamol increases leg lactate release in recovery, which may relate to glycolytic trafficking due to excessive myocellular glucose uptake.

This work developed a fast and simple method for the quantification of two important β-agonists, ractopamine (RAC) and clenbuterol (CLEN) in animal urines. The method was based on micro-extraction by a mixed mode monolithic material and analysis by HPLC-UV and UHPLC-Q ExactiveTM Plus Orbitrap mass spectrometer. A mixed functional monolith with nonpolar ester-based structure containing polar carboxylic groups was thermally synthesized in situ in the pipette tips. The material combines both hydrophobic and ionic interactions. Parameters including conditions and composition of reagents for the in-pipette tip monolith synthesis, as well as the extraction process, were investigated and optimized. The procedure for extraction is 800 μL washing solvent of 10:90 (v/v) ACN:water and 150 μL eluting solvent of 30:70 (v/v) ACN:200 mM acetate buffer pH 4.0. Extraction efficiencies of 92% and 100% for RAC and CLEN, respectively, were achieved within 5 min with total organic solvent consumption of 395 μL. The extracts of spiked cattle and swine urines were analyzed by HPLC-UV and UHPLC-Q ExactiveTM Plus Orbitrap mass spectrometer. Good recovery with acceptable precision was observed. The mass spectrometry data confirmed efficient matrix removal by the synthesized extraction sorbent allowing routine analysis by the HPLC-UV method.

Metabolites of β2-agonists of 2-(4-amino-3,5-dichlorophenyl)-2-(alkylamino)ethanols, derivatives of hippuric and mandelic acids, have been synthesized. In experiments on laboratory animals, the excretion profile of target compounds and metabolites with urine was studied. After single oral administration of drugs in doses of 270 and 540 mg/kg, 9.7% of 2-(4-amino-3,5-dichlorophenyl)-2-(isopropylamino)ethanol and 0,7 % of 2-(4-amino-3,5-dichlorophenyl)-2-( tert -amylamino)ethanol from the administered dose were isolated in unchanged form. The times for determination of target components and metabolites in the urine of laboratory animals at the method sensitivity of 0.1 ng/mL has been established: 2-(4-amino-3,5-dichlorophenyl)-2-(isopropylamino)ethanol is identified for more than 120 h after drug taking and 2-(4-amino-3,5-dichlorophenyl)-2-( tert -amylamino)ethanol is identified for 96 h. Metabolite of 2-(4-amino-3,5-dichlorophenyl)-2-( tert -amylamino)ethanol (derivative of hippuric acid) is detected in urine longer than the initial compound – for more than 120 h.

Phosphatidylinositol 3-kinase (PI3K) plays an important role in protein metabolism and cell growth. We here show that mice (M-PDK1KO mice) with skeletal muscle–specific deficiency of 3′-phosphoinositide–dependent kinase 1 (PDK1), a key component of PI3K signaling pathway, manifest a reduced skeletal muscle mass under the static condition as well as impairment of mechanical load–induced muscle hypertrophy. Whereas mechanical load-induced changes in gene expression were not affected, the phosphorylation of ribosomal protein S6 kinase (S6K) and S6 induced by mechanical load was attenuated in skeletal muscle of M-PDK1KO mice, suggesting that PDK1 regulates muscle hypertrophy not through changes in gene expression but through stimulation of kinase cascades such as the S6K-S6 axis, which plays a key role in protein synthesis. Administration of the β2-adrenergic receptor (AR) agonist clenbuterol activated the S6K-S6 axis in skeletal muscle and induced muscle hypertrophy in mice. These effects of clenbuterol were attenuated in M-PDK1KO mice, and mechanical load–induced activation of the S6K-S6 axis and muscle hypertrophy were inhibited in mice with skeletal muscle–specific deficiency of β2-AR. Our results suggest that PDK1 regulates skeletal muscle mass under the static condition and that it contributes to mechanical load–induced muscle hypertrophy, at least in part by mediating signaling from β2-AR.

In the present study, an antibody against phenylethanolamine A (PEA) was produced, confirmed, and used in a surface plasmon resonance (SPR)-based measurement. Bovine serum albumin (BSA)-conjugated PEA was linked to nano-gold particles bound to l-cysteine modified on the surface of a Au-NP sensor chip. The concentrations of antigen and antibody were optimized, and the designed biosensor chip was investigated to examine the stability and accuracy of the proposed method. The recovery of PEA ranged from 80.4-93.4% in swine urine samples with spike levels of 5, 10 and 20 ng mL-1, and the relative standard deviations of PEA were less than 2%. PEA analogues, such as clenbuterol, ractopamine, and salbutamol, did not influence the PEA measurement. The developed method could be used to measure PEA in swine urine samples.

Clenbuterol hydrochloride (CLT), β2 adrenergic agonist is used as a bronchodilator in the therapeutic treatment of asthma. It is important to know the stability behaviour of the drug in different degradation conditions as per ICH Q1A (R2) guidelines for safety and efficacy purpose. The main objective of the study is to develop and validate stability indicating LC-MS/MS method for the determination of Clenbuterol HCl. The separation was achieved using Phenomenex Gemini NX C18 (250*4.6 mm, 5 μ) column and the mobile phase consisting of ammonium acetate buffer (5 mM), 0.15% triethylamine (TEA), pH 7.5 with acetic acid: methanol (70:30, v/v) at flow rate 1 ml/min. The detection was done using PDA detector at 245 nm. The validation was performed as per ICH Q2 (R1) guideline. The drug was subjected to stress degradation conditions as per ICH Q1A (R2) guidelines. The significant degradation was observed in acidic (8.78%) and sunlight (liquid) (9%) condition while no degradation was observed in neutral, basic, oxidation and thermal condition. The drug and its degradation products were characterized using LC-MS/MS and the proposed degradation mechanism was communicated. The developed method was found to be stability-indicating, simple, specific, selective, sensitive, linear, accurate, robust and precise and used as a routine analysis in quality control laboratory.

Aims/hypothesis Chronic stimulation of β2-adrenoceptors, opposite to acute treatment, was reported to reduce blood glucose levels, as well as to improve glucose and insulin tolerance in rodent models of diabetes by essentially unknown mechanisms. We recently described a novel pathway that mediates glucose uptake in skeletal muscle cells via stimulation of β2-adrenoceptors. In the current study we further explored the potential therapeutic relevance of β2-adrenoceptor stimulation to improve glucose homeostasis and the mechanisms responsible for the effect. Methods C57Bl/6N mice with diet-induced obesity were treated both acutely and for up to 42 days with a wide range of clenbuterol dosages and treatment durations. Glucose homeostasis was assessed by glucose tolerance test. We also measured in vivo glucose uptake in skeletal muscle, insulin sensitivity by insulin tolerance test, plasma insulin levels, hepatic lipids and glycogen. Results Consistent with previous findings, acute clenbuterol administration increased blood glucose and insulin levels. However, already after 4 days of treatment, beneficial effects of clenbuterol were manifested in glucose homeostasis (32% improvement of glucose tolerance after 4 days of treatment, p −1 day−1 (40 times lower than previously studied). Mechanistically, these effects were not due to increased insulin levels, but clenbuterol enhanced glucose uptake in skeletal muscle in vivo both acutely in lean mice (by 64%, p p p p p Conclusions/interpretation Clenbuterol improved glucose tolerance after 4 days of treatment and these effects were maintained for up to 42 days. Effects were achieved with doses in a clinically relevant microgram range. Mechanistically, prolonged treatment with a low dose of clenbuterol improved glucose homeostasis in insulin resistant mice, most likely by stimulating glucose uptake in skeletal muscle and improving whole-body insulin sensitivity as well as by reducing hepatic lipids and glycogen. We conclude that selective β2-adrenergic agonists might be an attractive potential treatment for type 2 diabetes. This remains to be confirmed in humans.

Both clenbuterol (CLB) and ractopamine (RAC) are β-adrenergic agonists. After long-term excessive intake, there will be adverse reactions such as headache, chest tightness, limb numbness, and serious life-threatening. Simultaneous detection of CLB and RAC in related samples is of great importance for human health. In this work, we outline a microfluidics-based indirect competitive immunoassay (MICI) system that can sensitively detect residual CLB and RAC in pork, swine blood and swine urine. The rapid detection of multiple samples can be achieved in one chip, which greatly improves the detection efficiency. This method has good stability and reproducibility and the microfluidic chips are easy to manufacture. The linear ranges for CLB and RAC detection by MICI are 0.1–2.5 ng/mL and 0.1–5 ng/mL, and the limits of detection (LODs) are 0.094 ng/mL and 0.091 ng/mL, respectively. This straightforward and portable immunoassay system provides a good platform for rapid detection of harmful substances in food samples.

Aim: Follow-up investigations are often required for clenbuterol-positive cases. A method to distinguish doping abuse from meat contamination was developed. Materials & methods: A total of 26 volunteers were recruited to ingest clenbuterol contaminated-pork and clenbuterol tablets. Results: For 20 volunteers, after ingestion of contaminated-pork, R-(-)/S-(+)-clenbuterol ratio was 1.0 after taking clenbuterol tablets. However, after taking clenbuterol tablets, some ratio points of the other six volunteers were between 0.9 and 1.0. A case of an abnormal cold and fever, which returned to normal after recovery, was also reported firstly. Conclusion: A change in R-(-)/S-(+)-clenbuterol was reported in the Chinese population initially. A ratio of 0.9 was recommended in doping related cases for the Chinese population.

Ractopamine hydrochloride often used as a bronchodilator, but its β-adrenergic agonist effects on un-striated muscle and its withdrawal time have not been assessed for Etawah goats and sheep. The aim of this study was to determine the safe time to slaughter goats and sheep post-treatment with ractopamine. Five clinically healthy adult goats and sheep (20 kg body weight) were treated with a single dose of ractopamine (1 mg, intravenously). Whole blood was sampled from the jugular vein at 120 min, 180 min and 300 min post-treatment. Ractopamine as a veterinary drug was analysed using HPLC at wavelength 225 nm. The concentrations at 120 min, 180 min and 300 min were 817.156 ± 13.460 µg.mL-1, 554.468 ± 50.157 µg.mL-1, and 294.588 ± 52.845 µg.mL-1 in goats and 706.266 ± 89.856 µg.mL-1, 579.194 ± 45.664 µg.mL-1, and 209.36 ± 54.797 µg.mL-1 in sheep, respectively. The withdrawal times in goats and sheep were 1141.710 ± 255.85 h and 989.741 ± 167.633 h, respectively, no drug residues detected. The safe time to slaughter goats after administration of 1 mg of ractopamine was approximately 3 months and 5 days post-treatment at a safety factor of 2, which was later than the sheep at 2 months and 22 days by a safety factor of two.

According to an indirect competitive immunoassay, six β-agonists (clenbuterol (CL), salbutamol (SAL), ractopamine (RAC), terbutaline (TER), mabuterol (MAB), and tulobuterol (TUL)) were detected by three novel multiresidue immunosensors on the basis of the successful preparation of bovine serum albumin (BSA)-RAC-CL-SAL multideterminant antigen and anti-BSA-RAC-CL-SAL antibody. A new strategy was reported to detect six β-agonists by combining nanotechnology, electrochemical detection, and specific immune technology. At the same concentration, the amperometric response for detection of six β-agonists was in a sequence of GCE/GNP/SAL > GCE/GNP/RAC > GCE/GNP/CL. Detection limits of six β-agonists show that the multiresidue detection performance of the GCE/GNP/RAC immunosensor is better than those of GCE/GNP/SAL and GCE/GNP/CL immunosensors. Three immunosensors manifest superior properties with a wide linear range, low detection limit, excellent reproducibility, and stability. The proposed GCE/GNP/RAC immunosensor displays high accuracy and can be effectively used for real sample detection.

A high-performance liquid chromatography method employing a diode-array detector and mass spectrometry detector was developed, validated and implemented for determining Synephrine, Caffeine, Clenbuterol, Nandrolone, Testosterone and Methylhexaneamine in Nutritional supplements. The use of Nutritional supplements is widespread. Hazards relating to concentration, composition, individual contaminants, supplements interactions as well as positive doping results among athletes present increasing concerns regarding nutritional supplement consuming. The proposed method was validated according to the International Conference on the Harmonization of the Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standards. The proposed method observed to be accurate, linear, precise, sensitive, required minimal sample preparation and uncomplicated mobile phase. The implementation of the proposed method on nine commercial supplements shows that inaccurate labeling for some supplements regarding the concentration of the ingredients.

Clenbuterol is a beta2 -adrenoceptor agonist marketed as an asthma reliever but is not approved for human use in most countries due to concerns of adverse cardiac effects. Given its demonstrated hypertrophic and lipolytic actions in rodents, clenbuterol is one of the most widely abused doping substances amongt athletes and recreational body-builders seeking leanness. Herein, we examined the effect of clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six young men. Before and 140 min after ingestion of 80 μg clenbuterol, resting metabolic rate and contractile function of the quadriceps muscle were measured, and blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39% (P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35% (P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9% (P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque did not change significantly with clenbuterol. Glycogen content of the vastus lateralis muscle did not change with clenbuterol. Clenbuterol increased circulating levels of glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is an efficient thermogenic substance that possibly also exerts muscle hypertrophic actions in humans. For these reasons, the restrictions imposed against clenbuterol in competitive sports seem warranted.

There is increasing demand for anti-doping drug monitoring in sports and food safety checks by developing sensitive and fast analytical methods. Here we report the development of hybrid Ir/SiNW as a new MALDI matrix for the detection of small molecules. This matrix is characterized by sufficient UV absorption, low-noise background, and high efficiency in ionization of small molecules. Sensitive detection of clenbuterol (LOD: 0.18 pmol) and a variety of other small molecules has been achieved using the Ir/SiNW matrix with a reproducible performance. Compared to the individual components separately, the matrix of hybrid Ir/SiNW synthesized via in situ growth can promote the MS signal intensity by up to 10 fold under identical experimental conditions. We provide a unique mechanism for the high performance of the hybrid Ir/SiNW matrix with the characteristic properties of hydrogen atom transfer and enhanced protonation at the interface of the hybrid nanostructures. Our approach of using a hybrid Ir/SiNW matrix enables detection of clenbuterol quantitatively in complicated biological samples and in vivo experiments, promising a useful tool for food security and anti-doping drug monitoring in sports.

Immunochromatographic assays (ICAs) are mainstream point-of-care diagnostic tools in disease control, food safety, and environmental monitoring. However, the important issue pertaining to the influence of sample addition methods on the detection performance of ICAs has not been addressed, and related information is still lacking. Herein, we selected the well-accepted gold nanoparticles (AuNPs) as visual labels. AuNP-based ICA was then used to explore the effects of three sample addition methods (i.e., dry, wet, and insert) on the analytical performance of ICAs by using competitive and sandwich models. Under optimized conditions, the competitive ICA with clenbuterol as an analyte showed a negligible difference (p 0.05) in the detection performance of the three methods in ideal phosphate buffered saline solution. However, the wet method demonstrated the worst performance in pork samples (p 0.05). The sandwich ICA strip with human chorionic gonadotropin as an analyte revealed the significantly different analytical performances of the three approaches in phosphate buffer (PB) solution and spiked serum (p 0.05). Two independent linear correlations were observed with the increase in target concentration. However, for the wet method in the PB solution and serum, the first linear correlation was at a relatively narrow target concentration range, and the second linear correlation was at a wider concentration range compared with those for the dry and insert methods. Our findings demonstrated that sample addition methods slightly influence competitive ICAs (p 0.05) but remarkably affect sandwich ICAs (p 0.05). We believe that this study can further explain the differences in detection results for the same target analyte in actual ICA detection. The results may serve as a reference in the rational selection of the appropriate sample addition method for succeeding ICA works.

Clenbuterol enantiomers differ greatly in their bioactivities. By optimizing the conditions for chromatographic separation and method validation, ultra-performance convergence chromatography (UPC2) was adopted to separate the enantiomers of clenbuterol. Standard solutions of (+)-clenbuterol and (-)-clenbuterol were stored at -18 ℃ for 1, 3, 5, 7, 14, 30, and 60 d, and then, their stability was monitored. The impacts of different chromatographic columns, cosolvents, system backpressure, and chromatographic column temperature on the separation of the two enantiomers were investigated. Acquity Trefoil AMY1 (150 mm×3.0 mm, 2.5 μm) was used for separation, and CO2-0.5% (v/v) ammonium acetate was used as the mobile phase. Gradient elution at a flow rate of 2.0 mL/min was adopted. The detection wavelength was set to 241 nm, and the injection volume was set to 10 μL. The backpressure was set to 13.8 MPa, and the column temperature was maintained at 40 ℃. The two enantiomers showed good linear relationships in the range of 1.0 to 20.0 mg/L with correlation coefficients greater than 0.9997. The limits of detection (LODs, S/N=3) of (+)-clenbuterol and (-)-clenbuterol were both 0.5 mg/L. The relative standard deviation (RSD, n=6) for the peak area of the 10.0 mg/L mixed standard working solution with six replicate injections ranged from 0.65% to 0.76%. The effectiveness and practicability of this method were demonstrated by using it to detect standard clenbuterol racemate. The (+)-clenbuterol and (-)-clenbuterol contents were 5.6 mg/L and 5.5 mg/L, respectively, in the standard clenbuterol racemates, as determined by the external standard method of quantification. The detection results suggested that the content ratio of (+)-clenbuterol and (-)-clenbuterol was close to 1.02∶1.00, which is consistent with the literature data. The established method has the advantages of rapid analysis, good separation effect, and low consumption of organic solvents, and it is suitable for the separation of clenbuterol enantiomers. This method can also provide technical support for the separation of other chiral drugs, analysis of the effects of chiral drugs, and assessment of product quality.

Exercise has beneficial effects on human health and is affected by two different pathways, motoneuron and endocrine. For the advancement of exercise research, in vitro exercise models are essential. We established two in vitro exercise models using C2C12 myotubes, EPS (electrical pulse stimulation) for a motoneuron model and clenbuterol, a specific β2 adrenergic receptor agonist, treatment for an endocrine model. For clenbuterol treatment, we found that Ppargc1a was induced only in low glucose media (1 mg/mL) using a 1-h treatment of 30 ng/mL clenbuterol. Global transcriptional changes of clenbuterol treatment were analyzed by RNA-seq and gene ontology analyses and indicated that mitogenesis and the PI3K-Akt pathway were enhanced, which is consistent with the effects of exercise. Cxcl1 and Cxcl5 were identified as candidate myokines induced by adrenaline. As for the EPS model, we compared 1 Hz of 1-pulse EPS and 1 Hz of 10-pulse EPS for 24 h and determined Myh gene expressions. Ten-pulse EPS induced higher Myh2 and Myh7 expression. Global transcriptional changes of 10-pulse EPS were also analyzed using RNA-seq, and gene ontology analyses indicated that CaMK signaling and hypertrophy pathways were enhanced, which is also consistent with the effects of exercise. In this paper, we provided two transcriptome results of in vitro exercise models and these databases will contribute to advances in exercise research.

A nitrogen-doped Fe-MOF shows a high specific surface area and excellent electrical conductivity after high temperature carbonization. A novel electrochemical sensor based on a N@Fe-MOF@C loaded dual-template molecularly imprinted polymer (DTMIP) modified glassy carbon electrode (GCE) was proposed for the rapid and ultra-sensitive simultaneous detection of clenbuterol hydrochloride (CLB) and ractopamine (RAC). N@Fe-MOF@C combined with a MIP significantly enhanced the electrical signal. Cyclic voltammetry (CV) was used to detect CLB and RAC. The electrochemical polymerization was conducted with O-phenylenediamine as the functional monomer and CLB and RAC as template molecules. The factors affecting the sensor response were optimized. Under the optimal experimental conditions, the CV current response showed a linear range of 10-12-8 × 10-9 M for both CLB and RAC, and the detection limit (LOD) for both CLB and RAC was 3.03 × 10-13 M (S/N = 3). This electrochemical sensing system has high sensitivity, selectivity, excellent reproducibility, repeatability and stability. The recoveries of the actual samples (97.4%-101.2%) and reasonable relative standard deviations (RSDs) (1.06%-3.17%) indicate the practicability of the sensor system. The system has high application value in the rapid detection of CLB and RAC in clenbuterol hydrochloride tablets, human urine and raw pork.

Background and objective Sympathetic activity plays an important role in the proliferation and differentiation of stem cells, and it changes over time, thereby exerting differential effects on various stem cell types. Aging causes sympathetic hyperactivity in aged tissues and blunts sympathetic nerves regulation, and sympathetic abnormalities play a role in aging-related diseases. Currently, the effect of sympathetic activity on skeletal muscle stem cells, namely satellite cells (SCs), is unclear. This study aimed to investigate the effects of skeletal muscle sympathetic activity on SC aging and skeletal muscle repair. Materials and methods To evaluate skeletal muscle and fibrotic areas, numbers of SCs and myonuclei per muscle fiber, β2-adrenoceptor (β2-ADR) expression, muscle repair, and sympathetic innervation in skeletal muscle, aged mice, young mice that underwent chemical sympathectomy (CS) were utilized. Mice with a tibialis anterior muscle injury were treated by barium chloride (BaCl2) and clenbuterol (CLB) in vivo. SCs or C2C12 cells were differentiated into myotubes and treated with or without CLB. Immunofluorescence, western blot, sirius red, and hematoxylin–eosin were used to evaluate SCs, myogenic repair and differentiation. Results The number of SCs, sympathetic activity, and reparability of muscle injury in aged mice were significantly decreased, compared with those in young mice. The above characteristics of young mice that underwent CS were similar to those of aged mice. While CLB promoted the repair of muscle injury in aged and CS mice and ameliorated the reduction in the SC number and sympathetic activity, the effects of CLB on the SCs and sympathetic nerves in young mice were not significant. CLB inhibited the myogenic differentiation of C2C12 cells in vitro. We further found that NF-κB and ERK1/2 signaling pathways were activated during myogenic differentiation, and this process could be inhibited by CLB. Conclusion Normal sympathetic activity promoted the stemness of SCs to thereby maintain a steady state. It also could maintain total and self-renewing number of SCs and maintain a quiescent state, which was correlated with skeletal SCs via β2-ADR. Normal sympathetic activity was also beneficial for the myogenic repair of injured skeletal muscle.

The present study aimed to assess the in ovo administration of clenbuterol on chick fertility, growth performance, muscle growth, myogenic gene expression, fatty acid, amino acid profile, intestinal morphology, and hepatic lipid-related gene expressions. In this study, 750 healthy fertile eggs from the local chicken breed Dokki-4 strain were analyzed. Fertile eggs were randomly divided into five experimental groups (150 eggs/3 replicates for each group). On day 14 of incubation, in addition to the control group, four other groups were established where 0.5 mL of worm saline (30 °C) was injected into the second group of eggs. In the third, fourth, and fifth groups, 0.5 mL of worm saline (30 °C), 0.9% of NaCl, and 10, 15, and 20 ppm of clenbuterol were injected into the eggs. Results suggested that clenbuterol increased growth efficiency up to 12 weeks of age, especially at 15 ppm, followed by 10 ppm, decreased abdominal body fat mass, and improved hatchability (p &lt, 0.01). Clenbuterol also modulated saturated fatty acid levels in the breast muscles and improved essential amino acids when administered at 10 and 15 ppm. Additionally, clenbuterol at 15 ppm significantly decreased myostatin gene expression (p &lt, 0.01) and considerably increased IGF1r and IGF-binding protein (IGFBP) expression. Clenbuterol administration led to a significant upregulation of hepatic PPARα, growth hormone receptor, and Lipoprotein lipase (LPL) mRNA expression with a marked decrease in fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP-1c) expression. In conclusion, the current study revealed that in ovo injection of clenbuterol showed positive effects on the growth of hatched chicks through reduced abdominal fat deposition, improved intestinal morphology, and modulation of hepatic gene expressions in myogenesis, lipogenesis, and lipolysis.

Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzylalcohol) is a β2-adrenergic agonist. The consumption of meat contaminated with clenbuterol can lead to increased heart rate, blood pressure, anxiety, palpitations and skeletal muscle tremors. Several analytical methods have been developed to identify and quantify clenbuterol in different biological matrices. In this report, we have developed a specific and sensitive analytical method for quantifying clenbuterol and performed an in-depth enantiomeric analysis in bovine urine. The method was evaluated in accordance with international guidelines, and we used an isotopically labeled analog as an internal standard. The extraction efficiency for clenbuterol in bovine urine was > 98%, the limit of detection was 0.05 ng/mL and the limit of quantification was 0.10 ng/mL. Our assay showed high specificity, no carryover was observed and the assay was linear in the range 0.10–8.0 ng/mL. Fifteen bovine urine samples were analyzed (containing clenbuterol), and an enantiomeric analysis was performed. The clenbuterol concentration range was 0.10–10.56 ng/mL across these samples. The levorotatory enantiomer was detected at greater concentrations than the dextrorotatory enantiomer, the ratio being 1.7 ± 0.6 (n = 15), and a statistical difference was observed (P

Agonists of β2 adrenergic receptors (β2AR) and glucocorticoid receptors (GR) are prescribed to treat pulmonary diseases. Since ozone effects are mediated through the activation of AR and GR, we hypothesized that the treatment of rats with relevant therapeutic doses of long acting β2AR agonist (LABA; clenbuterol; CLEN) and/or GR agonist (dexamethasone; DEX) would exacerbate ozone-induced pulmonary and systemic changes. In the first study, male 12-week-old Wistar-Kyoto rats were injected intraperitoneally with vehicle (saline), CLEN (0.004 or 0.02 mg/kg), or DEX (0.02 or 0.1 mg/kg). Since dual therapy is commonly used, in the second study, rats received either saline or combined CLEN + DEX (each at 0.005 or 0.02 mg/kg) one day prior to and on both days of exposure (air or 0.8ppm ozone, 4 hr/day x 2-days). In air-exposed rats CLEN, DEX or CLEN + DEX did not induce lung injury or inflammation, however DEX and CLEN + DEX decreased circulating lymphocytes, spleen and thymus weights, increased free fatty acids (FFA) and produced hyperglycemia and glucose intolerance. Ozone exposure of vehicle-treated rats increased bronchoalveolar lavage fluid protein, albumin, neutrophils, IL-6 and TNF-α. Ozone decreased circulating lymphocytes, increased FFA, and induced hypeerglycemia and glucose intolerance. Drug treatment did not reverse ozone-induced ventillatory changes, however, lung effects (protein and albumin leakage, inflammation, and IL-6 increase) were exacerbated by CLEN and CLEN + DEX pre-treatment in a dose-dependent manner (CLEN > CLEN + DEX). Systemic effects induced by DEX and CLEN + DEX but not CLEN in air-exposed rats were analogous to and more pronounced than those induced by ozone. These data suggest that adverse air pollution effects might be exacerbated in people receiving LABA or LABA plus glucocorticoids.

β2-adrenergic receptor (β2-AR) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of β-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and 59.57 μg/l, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was 3.2 μg/l and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified β2-AR would have great application potential in detection of β-agonist residues.

In this work, a novel sulfated-β-cyclodextrin (S-β-CD) coated stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). The capillary was developed by attaching polydopamine/sulfated-β-cyclodextrin (PDA/S-β-CD) onto the gold nanoparticles (AuNPs) coated capillary which was pretreated with polydopamine. The results of scanning electron microscopy (SEM) and energy dispersive X-ray analysis spectroscopy (EDS) indicated that polydopamine/sulfated-β-cyclodextrin was successfully fixed on the gold nanoparticles coated capillary. To evaluate the performance of the prepared open tubular (OT) column, the enantioseparation was carried out by using ten chiral drugs as model analytes. Under the optimal conditions, salbutamol, terbutaline, trantinterol, tulobuterol, clorprenaline, pheniramine, chlorpheniramine, brompheniramine, isoprenaline and tolterodine were baseline separated with the resolution (Rs) values of 3.25, 1.76, 2.51, 1.89, 3.17, 2.17, 1.99, 1.72, 2.01 and 3.20, respectively. Repeatability of the column was studied, with the relative standard deviations for run-to-run, day-to-day and column-to-column lower than 5.7%.

Background: Thyrotoxicosis increases bone turnover, resulting in net bone loss. Sympathetic nervous system (SNS) activation, via β2-adrenoceptor (β2-AR) signaling, also has osteopenic effects. Because thyroid hormones (TH) interact with the SNS to regulate several physiological processes, we hypothesized that this interaction also occurs to regulate bone mass. Previous studies support this hypothesis, as α2-AR knockout (KO) mice are less susceptible to thyrotoxicosis-induced osteopenia. Here, we evaluated whether TH-SNS interactions in bone involve β2-AR signaling. Methods: Thyrotoxicosis was induced in 120-day-old female and male mice with β2-AR gene inactivation (β2-AR-/-) by daily treatment with supraphysiological doses of triiodothyronine (T3) for 12 weeks. The impact of thyrotoxicosis on femoral bone microarchitecture, remodeling, fracture risk, and gene expression of the receptor activator of nuclear factor-kappa-B (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) pathway was evaluated. In addition, the effect of the β2-AR-specific agonist clenbuterol (CL) on cAMP accumulation was determined in osteoblastic (MC3T3-E1) cells treated with T3 and/or 17β-estradiol (E2). Results: Thyrotoxicosis negatively affected trabecular bone microarchitecture in wild-type (WT) females, but this effect was milder or nonexistent in β2-AR-/- animals, whereas the opposite was seen in males. T3 treatment increased the femoral RANKL/OPG mRNA ratio and the endosteal perimeter and medullary area of the diaphysis in WT females and males, but not in β2-AR-/- mice, suggesting that T3 promotes endosteal resorption in cortical bone, in a mechanism that involves β2-AR signaling. T3 treatment increased endocortical mineral apposition rate only in WT females but not in β2-AR-/- mice, suggesting that TH also induce bone formation in a β2-AR signaling-dependent mechanism. T3 treatment decreased femoral resistance to fracture only in WT females, but not in KO mice. E2 and CL similarly increased cAMP accumulation in MC3T3-E1 cells; whereas T3 alone had no effect, but it completely blocked E2-stimulated cAMP accumulation, suggesting that some T3 effects on bone may involve E2/cAMP signaling in osteoblasts. Conclusions: These findings sustain the hypothesis that T3 interacts with the SNS to regulate bone morphophysiology in a β2-AR signaling-dependent mechanism. The data also reveal sex as an important modifier of skeletal manifestations of thyrotoxicosis, as well as a modifier of the TH-SNS interactions to control bone microarchitecture, remodeling, and resistance to fracture.

In this study, colloidal gold (CG) and time-resolved fluorescent nanobead (TRFN) probes were used to establish an integrated immunochromatographic assay (ICA) to qualitatively and quantitatively detect clenbuterol (CLE). The best experimental conditions for the two probes in separate ICAs were obtained by optimizing the antibody labeling concentration, the amount of antigen, and the concentration of probe. When the CG and TRFN probes co-existed in the ICA, the latter had no effect on the sensitivity of qualitative detection of the CG probe-based ICA. However, the CG probe optimized the linear range of quantitative detection in the TRFN probe-based ICA. The integrated test strip can be used for qualitative and quantitative detection of CLE in one step. When the amount of antigen reached 0.4 mg/mL, the CG probe concentration reached 1.2 μg/mL, and the TRFN probe concentration reached 0.68 μg/mL. The qualitative sensitivity of the integrated ICA was 0.5 ng/mL and its quantitative limit of detection was 0.04 ng/mL with a detection range of 0.1–2.7 ng/mL. This developed method is of great significance for large-scale samples screening and positive monitoring in the field of food safety testing.

Clenbuterol is known to improve competition resistance and muscular growth in athletes. Although it is an illegal drug, its use by farmers is widely spread to induce growth of their cattle. Thus, when clenbuterol is found in the urine of an athlete, there is doubt whether it was consumed with doping purposes or if it is due to the consumption of meat from a clenbuterol-fed animal. Previous studies suggest that enantiomeric relationship of clenbuterol may be different according to the intake source. However, the enantiomeric relationship throughout a doping cycle or a continuous intake of contaminated meat has not yet been explored. In this first approximation, our aim was the development and validation of a sensitive and rapid method for the determination of S- (+) and R- (─) clenbuterol enantiomers to be used in a controlled study in rats fed for one week with contaminated meat or simulating a doping cycle. Enantiomers were measured using liquid chromatography coupled to mass spectrometry with a triple quadrupole analyzer (LC-TQ-MS) and were separated on an AGP Chiralpak column. The method was fully validated following the VICH (Veterinary International Conference on Harmonization guidelines) and was linear in the range of 12.5-800 pg/mL with a correlation coefficient of ≥0.98 for each enantiomer, and with a limit of quantitation and detection (LOQ and LOD) of 12.5 pg/mL and 6.5 pg/mL, respectively, for both enantiomers. The application of this method pointed out the shift of the enantiomeric relationship in urine from rats during the first five days of the doping cycle compared to those fed with contaminated meat. This finding can be of substantial importance in further doping studies.

Many food safety hazard factors pose a great threat to human health. To mornitor these factors, development of a sensitive, reliable, and efficient detection platform is significantly important. Herein, a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed, in which bacteria was introduced as natural signal carriers to construct probes. Through the carriers, the ratio between signal and recognition molecules was flexibly adjusted. As is known, in the competitive immunoassay, the limited number of antibodies was key to significantly improved sensitivity. By this protocol, horseradish peroxidase (HRP) was enriched on the bacterial surface to realize signal amplification, extremely reduce the applied quantity of antibody, and finally trigger intense competition between free target analytes and immobilized antigen. Clenbuterol (CL) as a model target was analyzed by the proposed biosensor system, with a linear range of 0.02–1.0 ng mL−1 and a sensitivity (expressed as IC50) of 0.16 ng mL−1. This contribution has opened up a new route to improve analytical performance of ic-ELISA and provided an application prospect in the detection of other small molecule targets.

WOS: 000456760700070, 30606580, beta-Agonists are illegally consumed in various products such as food and animal and effect the nutrition distribution owing to change of body fat. In addition, they result in acute poisoning and several symptoms such as muscular tremors and nervousness. A new electrochemical approach based on two-dimensional hexagonal boron nitride (2D-hBN) nanosheets decorated functionalized multi-walled carbon nanotubes (f-MWCNTs) was presented for simultaneous determination of beta-agonists such as phenylethanolamine A (PEA), clenbuterol (CLE), ractopamine (RAC) and salbutamol (SAL) in urine samples. X-ray diffraction (XRD) method, Raman spectroscopy, scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used characterizations of nanomaterials. After that, 2D-hBN/f-MWCNTs nanocomposite modified glassy carbon electrode (GCE) was prepared for simultaneous determination of beta-agonists. 1.0 x 10(-12)-1.0 x 10(-8) M and 1.0 x 10(-13) M were founded as the linearity range and the detection limit (LOD) for PEA, CLE, RAC and SAL. Finally, the prepared electrochemical sensor was used for urine sample analysis in presence of ascorbic acid (AA) and uric acid (UA).

Rapid sample preparation is a key step in the field of food safety. A mechanical high-pressure method using a laboratory-made meat press machine was first introduced in this study to process the incurred muscle samples of chicken and rabbit. By applying high pressure to animal muscle, the meat juice was obtained. After extraction and purification, veterinary drug residues in the juice were qualitatively and quantitatively analyzed by using high-performance liquid chromatography-tandem mass spectrometry. The sample press conditions and extraction solvents were optimized. Under the optimal conditions, all veterinary drug residues, including tetracycline, enrofloxacin, clenbuterol, ampicillin, lincomycin, erythromycin, and sulfadiazine, in the incurred samples were detected. The residual concentration of drugs in samples obtained by using the mechanical high-pressure method can reach up to 94.0% of that obtained by using the common homogenization method, suggesting that drug residues exist in the tissue juice, which justifies the use of the mechanical high-pressure method. Moreover, with the mechanical high-pressure method, the sample preparation time was shortened by five times, and the consumption of the extraction solvent was reduced by 50%, relative to the homogenization method.

The objective of this study was to assess the feasibility of using hair as a long-term indicator of cocktail (low-dose β2 agonists) treatments in cattle. Six male Simmental cattle were treated with a mixture of low-dose clenbuterol, ractopamine, and salbutamol at dosages of 5.3, 223.3, and 50.0 μg/kg, respectively. The trial lasted for 112 days and included 28 days of treatment and 84 days of withdrawal. Plasma and urine samples taken during the treatment period contained the highest residues, with maximum concentrations of clenbuterol, ractopamine, and salbutamol in plasma of 1.49 ng/mL (Day 21), 43.78 (Day 14) ng/mL, and 8.07 ng/mL (Day 7), respectively, and in urine of 62.40 ng/mL (Day 28), 3995.77 ng/mL (Day 28), and 503.72 ng/mL (Day 1), respectively. On day 42 of withdrawal, the residues of all three β2 agonists in plasma were below the limit of quantification (LOQ; 0.3 ng/mL for clenbuterol, and 0.5 ng/mL for ractopamine and salbutamol), and in urine samples were below or near the LOQ (the highest being ractopamine at 1.10 ng/mL). The highest concentrations of clenbuterol, ractopamine, and salbutamol in hair were 88.36, 1351.92, and 100.58 ng/g, respectively, on day 14 of withdrawal; and the residues were long-lasting, with 7.64, 28.55, and 8.77 ng/g, respectively, on day 84 of withdrawal. The results of this study demonstrate that hair could be utilized as a long-term indicator of the use of a combination of low-dose β2 agonists in cattle, which could have implications for growth-promoting purposes monitoring.

Lateral flow immunoassays (LFIAs) have been applied widely in clinical diagnosis, food safety, drug abuse, environmental monitoring and other fields due to their characteristics of rapidity, sensitivity, stability and low cost. Choosing the proper labels is key to the preparation of LFIAs. An ideal probe requires a simple synthesis, has stable performance and is economical. This study aimed to establish a simple, rapid and convenient method for the preparation of selenium nanoparticles (SeNPs) that can be used as labels in LFIAs. Appropriate SeNPs were successfully prepared by reducing 2 mM H2SeO3 with 16 mM l -ascorbic acid (Vc), 5 mM sodium dodecyl sulfate (SDS) and 100 mM polyethylene glycol (PEG) as stabilizing agents. The spherically formed and uniformly dispersed nanoparticles were applied to the preparation of an LFIA. In this study, the preparation of the SeNPs was economical and was accomplished at room temperature. We used clenbuterol (CLE) as an analyte in an LFIA; the limit of detection (LOD) of CLE in pig urine was found to be 3 ng/mL, which meets the industry standard and has the same sensitivity as commercially available flow assays. In addition, the SeNP-based LFIA can be stored at 37 °C for 60 days.

This study was designed to investigate clenbuterol, salbutamol, and ractopamine concentrations in fresh meat products. A total of 801 samples of ribs, loin, and internal organs of pigs, cattle, and lamb were collected from retail and wholesale markets in nine districts of Jilin Province, Northeast China. The three β-agonists were analyzed by LC-MS/MS. Results showed that 11.36% of the samples contained prohibited β-agonists. Detection rates of β-agonists decreased in the following order: Clenbuterol (6.37%) > Ractopamine (2.62%) > Salbutamol (2.37%). The β-agonists levels were higher for pork than for beef and lamb. The detection rate of meat products decreased in the following order: pork loin (28.72%) > pork ribs (24.72%) > pork internal organs (20.24%) > beef loin (6.67%) > beef internal organs (6.19%) > beef ribs (5.00%) > lamb internal organs (3.45%) > lamb ribs (3.41%) > lamb loin (3.26%). Only one β-agonists was detected per sample. On the surface of our research, the amount β-agonists of added to fresh meat products in Jilin Province of China is relatively safe.

A rapid, selective, and quantitative method to determine clenbuterol content in animal urine based on Surface-Enhanced Raman Spectroscopy (SERS) by aggregated re-oxidized graphene oxide/Au nanoparticle (roGO/AuNPs) hybrids and liquid-liquid extraction has been proposed in this study. To realize the quantitative calculation, melamine was optimized as the internal standard. The peak intensity of melamine observed at △v = 709 cm−1 was first set to 1000 as basis, and the peak at △v = 1474 cm−1 was selected as the characteristic peak of clenbuterol used for the quantitative calculation. Sufficient linearity was obtained in the range of 1–20 ng mL−1 (R2 = 0.997). The limit of detection and quantification in urine matrix were as low as 0.5 and 1 ng mL−1, respectively. The recovery rates were 78.6–89.4% with coefficients of variation less than 9.8%, and the storage time of the substrates is approximately 180 days under 4℃. The detection results of the developed SERS detection method agree well with those determined by LC–MS/MS, while the cost time significantly reduced to 8 min/sample. This new protocol could be easily developed for the routine monitoring of the illicit use of clenbuterol in animal farming.

Here, a gold nanoflowers (AuNFs) labelled lateral flow assay integrated with smartphone was developed. In the proposed assay, high sensitivity was achieved without additional excitation light source as AuNFs was used as label under natural light. Moreover, with smartphone, subtle changes of optical signals from the test line was collected and distinguished portably. Thus, portable quantitative detection of clenbuterol (Clen) with high sensitivity was achieved. The limit of detection (LOD) of the proposed assay for Clen was 12.5 pg/mL, which was 4.9 times lower than that of the traditional lateral flow assay based on gold nanospheres (61.4 pg/mL). The linear range was 0.1–5.0 ng/mL. The average recoveries in swine urine were 92.7–112.0%, with coefficients of variation of 1.8–7.4%. The detection results were highly correlated with those of LC-MS/MS. Therefore, the assay is promising for the extend analysis fields with the advantages of sensitive, portable, and accurate.

Zilpaterol and clenbuterol are two β-adrenergic agonist drugs used on animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries; clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used at unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proven yet of zilpaterol to produce reactive oxygen (ROS) and nitrogen species. Regarding glutathione which is the main intracellular antioxidant and plays detoxification functions on liver metabolism; case study is a primary interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures exposed to clenbuterol and zilpaterol. A single drug assay was performed in the first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both agonist β-adrenergic modify in a dose-dependent manner oxide-reduction response through ROS generation, glutathione peroxidase activity, and intracellular GSH content; and de release of liver enzymes associated with hepatocellular damage like gamma glutamyl-transpeptidase, aspartate aminotransferase, alanine aminotransferase. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

Lateral flow immunoassay (LFIA) has emerged as an effective technique in the field of food safety and environmental monitoring. However, sensitive and quantitative detection is still challenging for LFIAs in complex environments. In this work, a dual-model colorimetric/SERS lateral flow immunoassay for ultrasensitive determination of clenbuterol was constructed based on a metallic core-shell Au/Au nanostar acting as a multifunction tag. Raman reporter molecules are located between the core (AuNP) and shell (Au nanostar) to form a sandwich structure, which contributes to eliminate the environmental interference and improve the detection stability. In addition, the Au/Au nanostar provides a much higher Raman enhancement due to the presence of sharp tips and larger surface roughness in comparison with gold nanoparticles (AuNPs). Thus, on the basis of the antibody-antigen interaction, the dual-model immunoassay can produce strong colorimetric and surface-enhanced Raman spectroscopy (SERS) signals for highly sensitive detection of the target analyte, clenbuterol. Under optimal conditions, clenbuterol could be detected by the colorimetric model with a visual detection limit of 5 ng/mL. Meanwhile, the SERS signal of the Au/Au nanostar was accumulated on the test line for the SERS model detection with a quantitative detection limit as low as 0.05 ng/mL, which is at least 200-fold lower than that of the traditional AuNPs-based immunoassay. Furthermore, recovery rates of the proposed method in food samples were 86-110%. This dual-model immunoassay provides an effective tool for antibiotic residues analysis and demonstrates a broad potential for future applications in food safety monitoring.

A growing number of β-agonists are illegally using for reducing animal fat deposition in animals, but the development of analytical methods always lags behind the emergence of new illegal compounds. Therefore, class specificity antibody-based immunoassays that can detect a great many β-agonists are important for timely supervision. In this study, a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) based on a clenbuterol monoclonal antibody was developed to recognize 23 β-agonists and analogues. Holographic and three-dimensional quantitative structure–activity relationship (HQSAR and 3D QSAR) revealed that there are two critical binding epitopes on β-agonist hapten affecting antibody specificity, and these epitopes have been further validated using a ractopamine antibody with narrow specificity. Tert-butyl at C-2′ epitope is needed to generate class specific antibodies, and different characteristics of substituents at benzene ring epitope would adjust antibody specificity. This investigation could provide reference for future design of β-agonist haptens.