Search Results (30)

A portable mass spectrometer (PMS) was combined with a mesoporous silica material (SBA-15) coated solid-phase microextraction (SPME) Arrow to develop a rapid, easy-to-operate and sensitive method for detecting five veterinary drugs—amantadine, thiabendazole, sulfamethazine, clenbuterol, and ractopamine—in milk and chicken samples. Equipped with a pulsed direct current electrospray ionization source and a hyperboloid linear ion trap, the PMS can simultaneously detect all five analytes in approximately 30 s using a one-microliter sample. Unlike traditional large-scale instruments, this method shows great potential for on-site detection with no need for chromatographic pre-separation and minimal sample preparation. The SBA-15-SPME Arrow, fabricated via electrospinning, demonstrated superior extraction efficiency compared to commercially available SPME Arrows. Optimization of the coating preparation conditions and SPME procedures was conducted to enhance the extraction efficiency of the SBA-15-SPME Arrow. The extraction and desorption processes were optimized to require only 15 and 30 min, respectively. The SBA-15-SPME Arrow–PMS method showed high precision and sensitivity, with detection limits and quantitation limits of 2.8–9.3 µg kg⁻¹ and 10–28 µg kg⁻¹, respectively, in milk. The LOD and LOQ ranged from 3.5 to 11.7 µg kg⁻¹ and 12 to 35 µg kg⁻¹, respectively, in chicken. The method sensitivity meets the requirements of domestic and international regulations. This method was successfully applied to detect the five analytes in milk and chicken samples, with recoveries ranging from 85% to 116%. This approach represents a significant advancement in food safety by facilitating rapid, in-field monitoring of veterinary drug residues. Full article

Stress-induced fetal programming diminishes β2 adrenergic tone, which coincides with intrauterine growth restriction (IUGR) and lifelong metabolic dysfunction. We determined if stimulating β2 adrenergic activity in IUGR-born lambs would improve metabolic outcomes. IUGR lambs that received daily injections of saline or the β2 agonist clenbuterol from birth to 60 days were compared with controls from pair-fed thermoneutral pregnancies. As juveniles, IUGR lambs exhibited systemic inflammation and robust metabolic dysfunction, including greater (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, increased (p < 0.05) intramuscular glycogen, reduced (p < 0.05) circulating IGF-1, hindlimb blood flow, glucose-stimulated insulin secretion, and muscle glucose oxidation. Daily clenbuterol fully recovered (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, hindlimb blood flow, muscle glucose oxidation, and intramuscular glycogen. Glucose-stimulated insulin secretion was partially recovered (p < 0.05) in clenbuterol-treated IUGR lambs, but circulating IGF-1 was not improved. Circulating triglycerides and HDL cholesterol were elevated (p < 0.05) in clenbuterol-treated IUGR lambs, despite being normal in untreated IUGR lambs. We conclude that deficient β2 adrenergic regulation is a primary mechanism for several components of metabolic dysfunction in IUGR-born offspring and thus represents a potential therapeutic target for improving metabolic outcomes. Moreover, benefits from the β2 agonist were likely complemented by its suppression of IUGR-associated inflammation. Full article

A simple, effective, and highly sensitive analytical approach was created and applied in this study for the accurate measurement of three β₂-agonist residues (clenbuterol, salbutamol, and ractopamine) in meat samples. In the course of the experiment, new adsorbent molecular sieves (ZMS)@nitrogen-doped carbon quantum dots (N-CQDs) composite materials were synthesized with the aid of hydrothermal synthesis. The composite adsorbent materials were prepared and characterized through scanning electron microscopy, transmission electron microscope, X-ray photoelectron spectroscopy, fluorescence, and zeta potential. Four determinants affecting the extraction and elution’s efficiency, such as the amount of adsorbent, the extraction time, desorption time, and the amount of extraction salt, were substantially optimized. The analytes were quantified by gas chromatography–mass spectrometry. Final results of the methodological validation reflected that the ZMS@N-CQDs composite materials were able to adsorb three β₂-agonist residues well and had good reproducibility. In the meantime, all analytes indicated good linearity with coefficient of determination R² ≥ 0.9908. The limit of detection was 0.7–2.0 ng·g⁻¹, the limit of quantification varied from 2.4 to 5.0 ng·g⁻¹, the precision was lower than 11.9%, and the spiked recoveries were in the range of 79.5–97.8%. To sum up, the proposed approach was quite effective, reliable, and convenient for the simultaneous analysis of multiple β₂-agonist residues. Consequently, this kind of approach was successfully applied for the analysis of such compounds in meat samples. Full article

β₂-agonists are a class of synthetic sympathomimetic drugs with acute poisoning effects if consumed as residues in foods. To improve the efficiency of sample preparation and to overcome matrix-dependent signal suppression in the quantitative analysis of four β₂-agonists (clenbuterol, ractopamine, salbutamol, and terbutaline) residues in fermented ham, an enzyme digestion coupled cation exchange purification method for sample preparation was established using ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). Enzymatic digests were subject to cleanup treatment on three different solid phase extraction (SPE) columns and a polymer-based strong cation resin (SCR) cartridge containing sulfonic resin was found to be optimal compared with silica-based sulfonic acid and polymer sulfonic acid resins based SPEs. The analytes were investigated over the linear range of 0.5 to 10.0 μg/kg with recovery rates of 76.0–102.0%, and a relative standard deviation of 1.8–13.3% (n = 6). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 μg/kg and 0.3 μg/kg, respectively. This newly developed method was applied to the detection of β₂-agonist residues in 50 commercial ham products and only one sample was found to contain β₂-agonist residues (clenbuterol at 15.2 µg/kg). Full article

The regularities of chromatographic retention and separation enantioselectivity of the selected β-blockers (propranolol, pindolol, alprenolol, atenolol, oxprenalol, metoprolol, clenbuterol, sotalol, pronethalol, salbutamol, and labetalol) were studied with eight chiral stationary phases (CSPs) in polar ionic mode (PIM) elution system. A range of novel CSPs was prepared by immobilisation of macrocyclic glycopeptide antibiotic eremomycin (E-CSP); structurally related antibiotics chloreremomycin (Chloro-E-CSP) and semi-synthetic oritavancin (O-CSP); and five eremomycin derivatives including amide- (Amide-E-CSP), adamantyl-2-amide- (Adamantylamide-E-CSP), eremomycin aglycon (EAg-CSP), eremosaminyl eremomycin aglycon (EEA-CSP), and des-eremosamynyl eremomycin (DEE-CSP) onto microspherical silica (Kromasil, particle size 5 micron, pore size 11 nm). The effect of different functional groups in eremomycin structure on chiral recognition of β-blockers was studied. The original E-CSP revealed moderate enantioseparation for all studied β-blockers. The presence of a free carboxylic group in a chiral selector molecule is found to be critical for the general retention of enantiomers as no separation enantioselectivity was recorded for Amide-E-CSP and Adamantyl-E-CSP. Modification of the aromatic system of eremomycin by the introduction of a chloro- substituent in the aromatic ring (Chloro-E-CSP) or a hydrophobic 4’-chlorobiphenylmethyl substituent to the disaccharide sugar residue (O-CSP) resulted in decreased enantioselectivity. The best enantioseparation of β-blockers was obtained for CSPs with eremosaminyl eremomycin aglycon and des-eremosamynyl eremomycin as chiral selectors. Full article

Clenbuterol is one of the most misused anabolic agents in professional sports. Therefore, the monitoring of clenbuterol in body fluids such as human urine is related to the development of rapid, selective and sensitive analytical methods that produce reliable results. In this work, these requirements were met by a two-dimensional separation method based on online solid-phase extraction coupled with ultra-high performance liquid chromatography–tandem mass spectrometry (SPE–UHPLC–MS/MS). The developed method provides favorable performance parameters, and it is characterized by minimum manual steps (only dilution and the addition of an internal standard) in the sample preparation. A limit of quantification (LOQ) of 0.1 ng/mL, excellent linearity (0.9999), remarkable precision (1.26% to 8.99%) and high accuracy (93.1% to 98.7%) were achieved. From a practical point of view, the analytical performance of the validated SPE–UHPLC–MS/MS method was demonstrated on blinded spiked urine samples from ten healthy volunteers. The estimated concentrations of clenbuterol were in accordance with their corresponding nominal values, as supported by the precision and accuracy data (relative standard deviation ≤5.4%, relative error ≤11%). The fulfillment of the World Anti-Doping Agency’s screening and confirmation criteria indicates that the proposed method is suitable for implementation in routine use in toxicologic and antidoping laboratories. Due to its high orthogonality and separation efficiency, the SPE–UHPLC–MS/MS method should also be easily adapted to the separation of structurally related compounds (such as clenbuterol metabolites). Thus, future antidoping applications could also include monitoring of clenbuterol metabolites, providing a longer detection widow. Full article

Clenbuterol (CB) is a synthetic β-receptor agonist which can be used to improve carcass leanness in swine, but its residues in pork also pose health risks. In this report, surface-enhanced Raman scattering (SERS) technology was used to achieve rapid detection and identification of clenbuterol hydrochloride (CB) residues. First, the effects of several different organic solvents on the extraction efficiency were compared, and it was found that clenbuterol in pork had a better enhancement effect using ethyl acetate as an extraction agent. Then, SERS signals of clenbuterol in different solvents were compared, and it was found that clenbuterol had a better enhancement effect in an aqueous solution. Therefore, water was chosen as the solvent for clenbuterol detection. Next, enhancement effect was compared using different concentration of sodium chloride solution as the aggregating compound. Finally, pork samples with different clenbuterol content (1, 3, 5, 7, 9, and 10 µg/g) were prepared for quantitative analysis. The SERS spectra of samples were collected with 0.5 mol/L of NaCl solution as aggregating compound and gold colloid as an enhanced substrate. Multiple scattering correction (MSC) and automatic Whittaker filter (AWF) were used for preprocessing, and the fluorescence background contained in the original Raman spectra was removed. A unary linear regression model was established between SERS intensity at 1472 cm^-1 and clenbuterol content in pork samples. The model had a better linear relationship with a correlation coefficient R² of 0.99 and a root mean square error of 0.263 µg/g. This method can be used for rapid screening of pork containing clenbuterol in the market. Full article

Direct and sensitive detection of multiple illegal additives in complex food samples is still a challenge in on-site detection. In this study, an ultrasensitive immunochromatographic assay (ICA) using magnetic Fe₃O₄@Au nanotags as a capture/detection difunctional tool was developed for the direct detection of β2-adrenoceptor agonists in real samples. The Fe₃O₄@Au tag is composed of a large magnetic core (~160 nm), a rough Au nanoshell, dense surface-modified Raman molecules, and antibodies, which cannot only effectively enrich targets from complex solutions to reduce the matrix effects of food samples and improve detection sensitivity, but also provide strong colorimetric/surface-enhanced Raman scattering (SERS) dual signals for ICA testing. The dual readout signals of the proposed ICA can meet the detection requirements in different environments. Specifically, the colorimetric signal allows for rapid visual detection of the analyte, and the SERS signal is used for the sensitive and quantitative detection modes. The proposed dual-signal ICA can achieve the simultaneous determination of two illegal additives, namely, clenbuterol hydrochloride and ractopamine. The detection limits for the two targets via colorimetric and SERS signals were down to ng mL⁻¹ and pg mL⁻¹ levels, respectively. Moreover, the proposed assay has demonstrated high accuracy and stability in real food samples. Full article

This paper developed a universal detection method by high-performance liquid chromatography-tandem mass spectrometry to detect three typical clenbuterols, CLB, SAL, and RAC, and to investigate the metabolism of β-agonists in vivo. The parent ions and daughter ions of the three β-receptor agonist standards and the residues in the muscle, liver, and blood samples of rats were obtained by Total Ions Scan mode. The metabolites produced in different tissues at a specific time were qualitatively and quantitatively analyzed, and the corresponding metabolic pathways were inferred. The results showed that the three β-receptor agonists mainly existed in the form of prototype drugs in rats, with a small amount of clenbuterol methyl compound and albuterol methyl compound. There were significant differences in residual metabolism between different tissues of the same species. In addition, different β-receptor agonists have different absorption and utilization rates in rats. Full article

To detect two types of β₂-agonist residues at the same time, we coupled two haptens of clenbuterol (CLE) and ractopamine (RAC) to the same carrier protein through diazotization to prepare dimeric artificial antigen, and a fluorescent lateral flow immunoassay method based on europium nanoparticles (EuNP-FLFIA) was established by combining polyclonal antibodies with europium nanoparticles to form probes. Under optimized conditions, the EuNP-FLFIA could simultaneously detect eight aniline-type and one phenol-type β₂-agonists, and the limits of detection (LOD) were 0.11–0.19 ng/mL and 0.12 ng/mL, respectively. The recovery rate of this method was 84.00–114.00%. This method was verified by liquid chromatography–tandem mass spectrometry (LC-MS/MS), and the test results were consistent (R² > 0.98). Therefore, the method established in this study could be used as a high-throughput screening for the efficient and sensitive detection of β₂-agonists in food. Full article

The use of clenbuterol (CLB) in large quantities in feedstuffs worldwide is illegal and potentially dangerous for human health. In this study, we directly prepared nitrogen-doped graphene quantum dots (N-GQDs) by a one-step method using cationic-etherified starch as raw material without pollution, which has the advantages of simple, green, and rapid synthesis of N-GQDs and high doping efficiency of nitrogen elements, compared with the traditional nitrogen doping method of reacting nitrogen source raw material with quantum dots. The N-GQDs synthesized by cationic etherification starch with different substitution degrees (DSs) exhibit good blue-green photoluminescence, good fluorescence stability, and water solubility. By comparing the fluorescence emission intensity of the two methods, the N-GQDs prepared by this method have higher fluorescence emission intensity and good fluorescence stability. Based on the static quenching mechanism between CLB and N-GQDs, a fluorescent probe was designed to detect CLB, which exhibited a wide linear range in the concentration range of 5 × 10⁻¹⁰~5 × 10⁻⁷ M (R² = 0.9879) with a limit of detection (LOD) of 2.083 × 10⁻¹³ M. More excitingly, the N-GQDs fluorescent probe exhibited a satisfactory high selectivity. Meanwhile, it can be used for the detection of CLB in chicken and beef, and good recoveries were obtained. In summary, the strategic approach in this paper has potential applications in the detection of risky substances in the field of food safety. Full article

Recently, C-reactive protein (CRP) was shown to affect intracellular calcium signaling and blood pressure in vitro and in vivo, respectively. The aim of the present study was to further investigate if a direct effect on G-protein coupled receptor (GPCR) signaling by CRP can be observed by using CRP in combination with different GPCR agonists on spontaneously beating cultured neonatal rat cardiomyocytes. All used agonists (isoprenaline, clenbuterol, phenylephrine, angiotensin II and endothelin 1) affected the beat rate of cardiomyocytes significantly and after washing them out and re-stimulation the cells developed a pronounced desensitization of the corresponding receptors. CRP did not affect the basal beating-rate nor the initial increase/decrease in beat-rate triggered by different agonists. However, CRP co-incubated cells did not exhibit desensitization of the respective GPCRs after the stimulation with the different agonists. This lack of desensitization was independent of the GPCR type, but it was dependent on the CRP concentration. Therefore, CRP interferes with the desensitization of GPCRs and has to be considered as a novel regulator of adrenergic, angiotensin-1 and endothelin receptors. Full article

A method for the determination of β-agonists was developed by combining the separation of analytes through high-performance liquid chromatography, with a reversed-phase column, coupled to the pulsed amperometric detection at a glassy carbon electrode. Preliminary experiments, using cyclic voltammetry, allowed for an understanding of the electrochemical behavior of clenbuterol, fenoterol, and terbutaline. By analyzing the electrochemical response, the conditions for detecting the analytes and for cleaning the working electrode were identified. The proposed potential-time profile was designed to prevent contamination of the carbon electrode following consecutive analyses, so ensuring a reproducible and sensitive quantitative determination. The waveform electrochemical parameters, including detection and delay times, have been optimized in terms of sensitivity, detection limits, and long-term response stability. The chromatographic separation was carried out using a C8 column in isocratic mode, and a mixture of acetic acid and acetonitrile. The optimized experimental conditions were used for the analysis of standard solutions and real samples. Detection limits, lower than the maximum residue limit set for clenbuterol by European directives, were obtained for all β-agonists investigated. The method validation was performed by evaluating the linearity, selectivity, precision, and recovery. Calf urine samples were used to verify the applicability of the proposed method, analyzing both enriched and naturally contaminated urine samples. Full article

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. Full article

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 < 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 < 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. Full article