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A molecularly imprinted electrochemiluminescence sensor (MIECLS) is constructed to selectively detect clenbuterol (CLB) based on boron nitride quantum dots@gold nanoflowers/silver nanowires (BNQDs@AuNFs/AgNWs). The abundant amino and hydroxyl groups on the surface of the BNQDs generate an electrostatic self-assembly effect with the multi-tipped spatial structure of AuNFs, constituting a novel nanoscale co-reaction accelerator (NCRA) with high activity and large load capacity. An NCRA embedded in the network structure of the AgNW luminophores significantly promotes the reduction of peroxydisulfate (S 2 O 8 2- ) to sulfate anion radicals (SO 4 -• ) through the catalysis of amino groups and boron radicals (B • ) and the electron acceleration of AuNFs while also reducing the reaction distance between SO 4 -• and AgNWs -• , realizing the multiple synergistic amplification of the electrochemiluminescence (ECL) signal. Imprinted cavities in the molecularly imprinted polymers (MIPs) prepared by electropolymerization can generate a "blocking-effect" by recognizing CLB, realizing ECL signal quenching. Analytical results indicate that the established MIECLS detects CLB in a line concentration range of 0.5-50000 nM and detection limit of 0.00693 nM. The spiked recoveries are 85.90%-97.77%, with the relative standard deviations (RSD) under 5.1%, consistent with those of high-performance liquid chromatography (HPLC). This work demonstrates that an efficient NCRA can significantly enhance the output of the ECL signal in collaboration with the original luminophore, providing a new method to realize the ultra-detection of targeted substances by MIECLS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Clenbuterol (CLE) and ractopamine (RAC) are two kinds of typical β 2 -adrenergic agonists which pose a serious threat to the health of human beings. In this work, 10 kinds of metal-organic frameworks (MOFs) with high stability and various pore features are screened to assess adsorption performance for CLE and RAC. An Al(III)-MOF (BUT-19) with abundant ethyl groups exhibits exceptional performance in removing CLE and RAC from water. The maximum adsorption capacity for CLE and RAC are up to 294.1 and 366.3 mg/g under the optimum adsorption conditions, respectively. Meanwhile, the adsorption mechanism effects of pH, temperature, and coexisted ions are investigated systematically. It is found that the MOF pore size and weak hydrogen-bond interactions between CLE/RAC molecules and the MOF are the main causes leading to the extraordinary adsorption. This study provides a new idea for the purposeful design and synthesis of MOFs for removing environmental pollutants and sheds light on the depuration of contaminated water.

Clenbuterol hydrochloride (CLT), β 2 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., Competing Interests: The authors declare that they have not conflict of interest., (Thieme. All rights reserved.)

In this paper, we synthesized HNbMoO 6 /C composite through the calcination of octylamine-intercalated HNbMoO 6 precursor. The resulting HNbMoO 6 /C composite showed some new phases of MoO 2 , MoO 3 , NbO 2 , Nb 2 O 5 , and carbon, which was fully confirmed via powder X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and X-ray photoelectron spectroscopy (XPS) technologies. Besides, the HNbMoO 6 /C hybrid was coated on glass carbon electrode to construct an electrochemical sensor for sensitive determination of clenbuterol. The electrochemical behaviors of clenbuterol on the prepared electrode were tested by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) analysis. The results showed that the intercalated carbon can act as active sites to accelerate electron transfer. In addition, more exposed surface areas of the HNbMoO 6 /C composite will facilitate the electrolyte to permeate. The oxidation peak current of clenbuterol was linearly related to its concentration in the range of 1.04 × 10 -5 to 7.51 × 10 -4  mol L -1 , and the determination limit was calculated to be 3.03 × 10 -6  mol L -1 (S/N = 3). This sensor exhibits excellent stability, reproducibility, specificity, and good recoveries when applied to monitor clenbuterol in real samples.

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%., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A visualization strategy is described for the detection of clenbuterol (CLB). It is using of antibody against dsDNA and G-quadruplex/hemin labeled on a metal organic framework of type MIL-101(Fe) (G-quadruplex/hemin-anti-DNA/MIL-101) acting as a peroxidase mimetic, and magnetic beads modified with aptamer and complementary DNA (MB/Apt-cDNA) as capture probes. The detection reagent was prepared via the reactions between the double stranded DNA (Apt-cDNA) in capture probes and anti-DNA in peroxidase mimetic. In the presence of CLB, the aptamer on the magnetic beads preferentially binds CLB, and the peroxidase mimetic is released to the supernatant after magnetic separation. The released peroxidase mimetic can catalyze the TMB/H 2 O 2 chromogenic system under mild conditions. This leads to the development of a blue-green coloration whose absorbance is measured at 650 nm. The detection limit is as low as 34 fM of CLB. The method was applied to the determination of CLB in pork samples and gave results that were consistent with data obtained with an ELISA kit. Graphical abstract A visualization strategy is described for the detection of clenbuterol. The selectivity of detection system for clenbuterol is excellent compared with other interferents. The method was applied to the determination of CLB in pork samples.

A simple, efficient and sensitive molecularly imprinted electrochemiluminescence sensor (MIECLS) based on reduced graphene oxide (rGO) and upconversion nanoparticles (UCNPs) was developed for determination of clenbuterol (CLB). In this study, rGO generated by electrodeposition of graphene oxide not only acted as carrier for immobilizing UCNPs, but also had a significant impact in boosting electrochemiluminescence (ECL) response of UCNPs thanks to its high conductivity, superior electron transport rate and large specific surface area. UCNPs as an advanced ECL emitter possessed wonderful ECL performance. Furthermore, the introduction of molecularly imprinted polymers (MIP) endowed the ECL sensor a new character of specifically identifying analyte CLB. Under the optimal experimental conditions, the ECL signal was proportional to the logarithm of CLB concentration in the range of 10nM to 100µM with a low detection limit of 6.3nM. The proposed MIECLS combining the advantages of UCNPs-ECL and MIP exhibited good sensitivity, desirable selectivity and favorable stability, indicating enormous potential in the future of food safety detection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Several banned substances are illegally used by athletes in racemic mixtures for performance enhancement. These include clenbuterol, methyl hexaneamine, methamphetamines, and amphetamines. Clenbuterol is present in a large number of doping samples from Olympic and non-Olympic athletes that have adverse analytical findings (AAFs). In some cases, the presence of these substances could be the result of consumption of meat contaminated with clenbuterol. In other cases, the origin is not clear. In this study, 27 products with racemic clenbuterol were evaluated using a new analytical methodology for the resolution of R-(-) and S-(+)-enantiomers of clenbuterol by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a chiral column in 15 min with good separation. The method developed can also be used for the analysis of other biological matrices such as urine, serum, and meat. The resolution between two peaks' (R s ) value obtained using chromatographic data was 1.03. Both clenbuterol enantiomers were present in all products analyzed and the ratio was nearly 1. The origin of the product was not important for determining the presence of one or both enantiomers. All products displayed a 50:50 ratio of clenbuterol enantiomers. To the best of our knowledge, clenbuterol ratio determination of a large number of pharmaceutical preparations and black-market products has not been reported previously. The information shown could be used by national anti-doping organizations and the athletes with AAFs attributed to clenbuterol. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Mitochondrial dysfunction is associated with numerous acute and chronic degenerative diseases. The beta-2 adrenergic receptor (β 2 AR) agonist formoterol induces mitochondrial biogenesis (MB), but other β 2 AR agonists, such as clenbuterol, do not. We sought to identify the MB signaling pathway of formoterol and the differences in signaling between these two ligands that result in the differential induction of MB. While formoterol and clenbuterol increased cAMP, only formoterol increased the phosphorylation of Akt and its downstream target eNOS. The increase in Akt phosphorylation was Gβγ- and PI3K-dependent, and the increase in eNOS phosphorylation was Gβγ- and Akt-dependent. Only formoterol increased cGMP. Formoterol induced MB as measured by increases in uncoupled cellular respiration and PGC-1α and NDUFS1 mRNA expression and was blocked by inhibitors of Gβγ, Akt, NOS, and soluble guanylate cyclase. To identify distinct receptor-ligand interactions leading to these differences in signaling, we docked formoterol and clenbuterol to six structures of the β 2 AR. Compared to clenbuterol, the methoxyphenyl group of formoterol interacted more frequently with V114 and F193, while its formamide group interacted more frequently with C191. These data indicate that the unique structural features of formoterol allow it to interact with the β 2 AR to activate the Gβγ-Akt-eNOS-sGC pathway to induce MB.

A fluffy porous ethylenediamine-connected graphene/carbon nanotube composite (EGC), prepared by a simple and time-saving one-pot synthesis, was successfully applied as an adsorbent in pipette-tip solid-phase extraction (PT-SPE) for the rapid extraction and determination of clenbuterol (CLB) from pork. In the one-pot synthesis, carbon nanotubes were inserted into graphene sheets and then connected with ethylenediamine through chemical modification to form a three-dimensional framework structure to prevent agglomeration of the graphene sheets. Under the optimum conditions for extraction and determination, good linearity was achieved for CLB in the range of 15.0-1000.0ngg -1 (r=0.9998) and the recoveries at three spiked levels were in the range of 92.2-96.2% with relative standard deviation ≤9.2% (n=3). In comparison with other adsorbents, including silica, NH 2 , C 18 , and Al 2 O 3 , EGC showed higher extraction and purification efficiency for CLB from pork samples. This analytical method combines excellent adsorption performance of EGC and high extraction efficiency of PT-SPE., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As an ideal biomimetic antibody, molecularly imprinted polymer (MIP) has shown great promise in immunoassays. Here, we developed a covalent imprinting method to prepare MIP artificial antibody towards clenbuterol on the well surface of a microtiter plate. Template molecule (clenbuterol) was splited by hydrolysis with functional monomer and removed by methanol/acetic acid solution, and then three-dimensional cavities were fabricated in the MIP, which can rebind template molecule via hydrogen bond interaction. Using this artificial antibody, we developed a novel biomimetic enzyme-linked immunosorbent assay (ELISA) with excellent sensitivity and specificity. The linear range for clenbuterol was 10 -5 to 1000μgL -1 , with a detection limit of 10 -7 μgL -1 . Cross reactivity of this MIP artificial antibody to other four structural analogs was less than 0.93%. This method was applied to determine clenbuterol in real samples with satisfactory result, suggesting a promising application of the biomimetic ELISA in food and environmental analysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β 2 adrenergic receptor (β 2 AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the G s heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β 2 AR-G s complexes are formed and the efficiency of nucleotide exchange leading to G s activation. These data also reveal transient nucleotide-bound β 2 AR-G s species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes' claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed.

Clenbuterol (Broncodil and trade) is a direct-acting sympathomimetic agent with mainly beta-adrenergic activity and a selective action on β2 receptors (a β2 agonist). It has properties similar to those of salbutamol. It is used as a bronchodilator in the management of reversible airways obstruction, as in asthma and in certain patients with chronic obstructive pulmonary disease. The uses, applications, and the synthetic pathways of this drug are outlined. Physical characteristics including: ionization constant, solubility, X-ray powder diffraction pattern, thermal methods of analysis, UV spectrum, IR spectrum, mass spectrum are all produced. This profile also includes the monograph of British Pharmacopoeia, together with several reported analytical methods including spectrophotometric, electrochemical, chromatographic, immunochemical methods, and capillary electrophoretic methods. The stability, the pharmacokinetic behavior, and the pharmacology of the drug are also provided., (© 2017 Elsevier Inc. All rights reserved.)

Three stable and simple synthetic routes of labeled D 9 -Mabuterol, D 9 -Bambuterol, and D 9 -Cimbuterol were described with 98.5%, 99.7%, and 98.4% isotopic abundance and good purity. These structures and isotope-abundance were confirmed according to 1 H NMR and liquid chromatography-tandem mass spectrometry., (Copyright © 2016 John Wiley & Sons, Ltd.)

This report presents an efficient synthesis of D 6 -clenproperol and D 6 -cimaterol with 99.5% and 99.7% isotopic abundance in acceptable yields and excellent chemical purities with deuterium isopropylamine as labelled precursor. Their structures and the isotope-abundance were confirmed by proton nuclear magnetic resonance and liquid chromatography-mass spectrometry., (Copyright © 2016 John Wiley & Sons, Ltd.)

A pre-treatment methodology for clenbuterol hydrochloride (CLEN) isolation and enrichment in a complex matrix environment was developed through exploiting molecularly imprinted polymers (MIPs). CLEN-imprinted polymers were synthesized by the combined use of ally-β-cyclodextrin (ally-β-CD) and methacrylic acid (MAA), allyl-β-CD and acrylonitrile (AN), and allyl-β-CD and methyl methacrylate (MMA) as the binary functional monomers. MAA-linked allyl-β-CD MIPs (M-MAA) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and a scanning electron microscope (SEM). Based upon the results, M-MAA polymers generally proved to be an excellent selective extraction compared to its references: AN-linked allyl-β-CD MIPs (M-AN) and MMA-linked allyl-β-CD MIPs (M-MMA). M-MAA polymers were eventually chosen to run through a molecularly imprinted solid-phase extraction (MISPE) micro-column to enrich CLEN residues spiked in pig livers. A high recovery was achieved, ranging from 91.03% to 96.76% with relative standard deviation (RSD) ≤4.45%.

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 (vLOD) of UCP-ICA for CL was 0.1 ng/mL. Calculated LOD (cLOD) 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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A highly selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of trantinterol and one of its major metabolites, 1-carbonyl trantinterol, in human plasma. An Oasis MCX 96-well solid-phase extraction cartridge and a SeQuantTM ZIC(®)-HILIC LC column were used for sample preparation and chromatographic separation, respectively. The analytes were monitored by a QTrap 5500 mass spectrometer with positive electrospray ionization. Multiple reaction monitoring was used for quantification using the precursor to product ion pairs of m/z 311.1 → 237.9 (trantinterol), m/z 325.1 → 251.9 (1-carbonyl trantinterol) and m/z 368.4 → 294.0 (bambuterol as internal standard). The assay had a calibration range from 0.2 to 50 pg/mL and a lower limit of quantification of 0.2 pg/mL for both trantinterol and 1-carbonyl trantinterol. The inter-day and intra-day precisions were <12.0% and the accuracies were within the range of 87.1-111%. The mean recovery ranged from 82.0 to 97.7% and internal standard normalized matrix effect from 0.813 to 0.899. The analytes were stable under all tested conditions. This validated method was successfully applied to a pilot pharmacokinetic study in healthy subjects administered a single 50 μg oral dose., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Trantinterol is a novel β2-adrenoceptor agonist, currently undergoing clinical trials for the treatment of asthma. We developed and validated an liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trantinterol and its major metabolite, 1-carbonyl trantinterol (SPFFCOOH), in rat plasma. Aliquots (100μL) of heparinized plasma samples were processed by protein precipitation with acetonitrile. Chromatographic separation used an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7μm) and acetonitrile-0.1% formic acid (20:80, v/v) as mobile phase, at a flow rate of 0.25mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple-reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The precursor-to-product ion transitions m/z 310.9→m/z 237.9 for trantinterol, m/z 324.9→m/z 251.9 for SPFFCOOH and m/z 368.0→m/z 294.0 for bambuterol (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentration of 0.25-100ng/mL for both trantinterol and SPFFCOOH. The intra- and inter-day precision (relative standard deviations, RSD) values were below 15% and accuracy (relative error, RE) was from -4.3% to 6.6% at all quality control (QC) levels. The method was successfully applied to compare the pharmacokinetics of trantinterol and SPFFCOOH in male and female Wistar rats after a single oral administration of trantinterol., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A rapid, selective and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously determine trantinterol and its major metabolites in human urine. Waters Oasis HLB C18 solid phase extraction cartridges were used in the urine sample preparation. The separation was carried out on an ACQUITY UPLC™ BEH C18 column with methanol-0.2% formic acid (30:70, v/v) as the mobile phase at a flow rate of 0.25mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves for trantinterol, arylhydroxylamine trantinterol (N-OH-trantinterol), the tert-butyl hydroxylated trantinterol (tert-OH-trantinterol) and the 1-carbonyl trantinterol (trantinterol-COOH) were obtained in the concentration range of 0.414-207, 0.578-385, 0.168-84.0, and 0.954-477ng/mL, respectively. The linear correlation coefficients were greater than 0.990. The intra and inter-day precision (relative standard deviation, RSD) values were less than 12% and the accuracy (relative error, RE) was 6.7-11%. The method herein described was superior to previous methods in sample throughput and sensitivity and successfully applied to the human excretion study., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Zonal elution and nonlinear chromatography are two mainstream models for the determination of drug-protein interaction in affinity chromatography. This work intended to compare the results by zonal elution with that by nonlinear chromatography when it comes to the analysis of the interaction between seven drugs and immobilised β2-adrenoceptor (β2-AR). The results of the zonal elution showed that clorprenaline, clenbuterol, methoxyphenamine, salbutamol, terbutaline, tulobuterol and bambuterol have only one type of binding site on immobilised β2-AR, while nonlinear chromatography confirmed the existence of at least two types of binding sites between β2-AR and clorprenaline, clenbuterol and bambuterol. On these sites, both zonal elution and nonlinear chromatography presented the same rank order for the association constants of the seven drugs. Compared with the data from zonal elution, the association constants calculated using nonlinear chromatography gave a good linear response to the corresponding values by radio-ligand binding assay. The sampling efficiencies of nonlinear chromatography were clearly higher than zonal elution. Nonlinear chromatography will probably become a powerful alternative for the high throughput determination of drug-protein interaction., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A sensitive and enantioselective vancomycin chiral stationary phase high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile-methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from -6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human., (© 2015 Wiley Periodicals, Inc.)

P-glycoprotein (P-gp)-mediated drug-drug interactions are important factors causing adverse effects of drugs in clinical use. The aim of this study was to determine whether trantinterol (also known as SPFF), a novel β2-adrenoceptor agonist, was a P-gp inhibitor or substrate. The results showed that trantinterol was not a substrate of P-gp but increased rhodamine 123 (Rho 123) uptake by MDCK-MDR1 cells and decreased the efflux transport of both Rho 123 and cyclosporine A (CsA) in bidirectional transport studies across MDCK-MDR1 cell monolayers. This suggested that trantinterol was a P-gp inhibitor but not a P-gp substrate. The mechanism of inhibition was investigated in the P-gp-Glo assay system, where it was found that trantinterol inhibited P-gp ATPase activity in a dose-dependent manner. A subsequent study using the antibody binding assay with the conformation-sensitive P-gp-specific antibody UIC2 confirmed that trantinterol decreased UIC2 binding at 10 μM in contrast to the competitive inhibitor, verapamil. This suggested that trantinterol was a noncompetitive inhibitor of P-gp. Finally, a pharmacokinetic study in rat showed that trantinterol significantly increased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of digoxin and paclitaxel (PAC), and the Cmax of cyclosporine A (CsA). In summary, trantinterol is a potent noncompetitive P-gp inhibitor which may increase the bioavailability of other P-gp substrate drugs coadministered with it.

In this study, binding properties of clenbuterol hydrochloride (CL) with human serum albumin (HSA) and bovine serum albumin (BSA) were examined using constant protein concentrations and various CL contents under physiological conditions. The binding parameters were confirmed using fluorescence quenching spectroscopy at various temperatures. The experimental results confirmed that the quenching mechanisms of CL and HSA/BSA were both static quenching processes. The thermodynamic parameters, namely, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to the van't Hoff equation, which suggested that the electrostatic interactions were the predominant intermolecular forces in stabilizing the CL-HSA complex, and hydrogen bonds and van der Waals force were the predominant intermolecular forces in stabilizing the CL-BSA complex. Furthermore, the conformational changes of HSA/BSA in the presence of CL were determined using the data obtained from three-dimensional fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy., (Copyright © 2013 John Wiley & Sons, Ltd.)

Clenbuterol is a long-acting β2-adrenoceptor agonist and bronchodilator that is used for the treatment of asthma, but the desired activities reside almost exclusively in the (-)-R-enantiomer. This study examined enantioselectivity in the disposition of clenbuterol following administration of clenbuterol racemate to rats. Concentrations of clenbuterol enantiomers in plasma, urine and bile were determined by LC-MS/MS assay with a Chirobiotic T column. This method was confirmed to show high sensitivity, specificity and precision, and clenbuterol enantiomers in 0.1 ml volumes of plasma were precisely quantified at concentrations as low as 0.25 ng/ml. The pharmacokinetic profiles of clenbuterol enantiomers following intravenous and intraduodenal administration of clenbuterol racemate (2 mg/kg) in rats were significantly different. The distribution volume of (-)-R-clenbuterol (9.17 l/kg) was significantly higher than that of (+)-S-clenbuterol (4.14 l/kg). The total body clearance of (-)-R-clenbuterol (13.5 ml/min/kg) was significantly higher than that of the (+)-S-enantiomer (11.5 ml/min/kg). An in situ absorption study in jejunal loops showed no difference in the residual amount between the (-)-R- and (+)-S-enantiomers. Urinary clearance was the same for the two enantiomers, but biliary excretion of (-)-R-clenbuterol was higher than that of the (+)-S-enantiomer. The fractions of free (non-protein-bound) (-)-R- and (+)-S-clenbuterol in rat plasma were 48.8% and 33.1%, respectively. These results indicated that there are differences in the distribution and excretion of the clenbuterol enantiomers, and these may be predominantly due to enantioselective protein binding., (Copyright © 2013 John Wiley & Sons, Ltd.)

In this study, a selective sample pretreatment procedure combing surface molecularly imprinted polymers and microextraction in packed syringe (SMIPs-MEPS) was developed for the analysis of clenbuterol (CLB) from pork samples. SMIPs for CLB were synthesized on silica gel particles through a sol-gel process. A series of characterization and adsorption experiments revealed that the SMIPs exhibited porous structures, good thermal stability, high adsorption capacity and a fast mass transfer rate. The obtained SMIPs were employed as selective sorbents of SMIPs-MEPS for extraction of CLB from pork samples. Several parameters affecting the extraction efficiency were investigated, including the pH of sample solution, number of draw-eject cycles, volume of sample, type and volume of washing solution, and the type and volume of elution solution. Under the optimized conditions, a simple and rapid method for the determination of CLB from pork samples was established by coupling with high performance liquid chromatography (HPLC). The whole pretreatment process was rapid and it can be accomplished with 2min. The limit of quantitation and the limit of detection for CLB were 0.02 and 0.009μgkg(-1), respectively. The average recoveries of CLB at three spiked levels ranged from 86.5% to 91.2% with the relative standard deviations (RSD) ≤6.3%., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Clenbuterol interacting with bovine serum albumin (BSA) or lysozyme (LYS) in physiological buffer (pH 7.4) was investigated by the fluorescence spectroscopy and UV-vis absorption spectroscopy. The results indicated that clenbuterol quenched the intrinsic fluorescence of BSA and LYS via a static quenching procedure. The binding constants of clenbuterol with BSA and LYS were 1.16×10(3) and 1.49×10(3) L mol(-1) at 291 K. The values of ΔH and ΔS implied that hydrophobic and electrostatic interaction played a major role in stabilizing the complex (clenbuterol-BSA or clenbuterol-LYS). In the presence of Fe2+, Fe3+, Cu2+, Mg2+, Ca2+, or Zn2+, the binding constants of clenbuterol to BSA or LYS had no significant differences. The distances between the donor (BSA or LYS) and acceptor (clenbuterol) were 2.61 and 2.19 nm for clenbuterol-BSA and clenbuterol-LYS respectively. Furthermore, synchronous fluorescence spectrometry was used to analyze the conformational changes of BSA and LYS., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A simple and sensitive method based on the combination of solid-phase microextraction (SPME) and high-performance liquid chromatography with ultroviolet detection was developed for the simultaneous determination of clenbuterol, salbutamol and ractopamine in pig samples. Parameters of the SPME procedure affecting extraction efficiency, such as the type of fiber, extraction time, extraction temperature, ion strength, pH of sample and stirring rate, were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.5-50 µg/L for clenbuterol and ractopamine, and 0.2-20 µg/L for salbutamol. The limits of detection were 0.1 µg/L for clenbuterol, 0.05 µg/L for salbutamol and 0.1 μg/L for ractopamine, respectively. The averages of intra- and inter-day accuracy ranged from 79.8 to 92.4%. The intra-day and inter-day precision were below 9.6% for the three analytes. This method exhibited the advantages of simplicity, rapidity and low solvent consumption, and was suitable for the monitoring of β2 -agonists residue in pig samples., (Copyright © 2013 John Wiley & Sons, Ltd.)

The chiral inversion and pharmacokinetics of two enantiomers of trantinterol, a new β2 agonist, were studied in rats dosed (+)- or (-)-trantinterol separately. Plasma concentrations of (+)- and (-)-trantinterol were measured by chiral stationary phase liquid chromatography tandem mass spectroscopy (LC-MS/MS). The apparent inversion ratio was calculated as the ratio of AUC0-t of (-)-trantinterol or (+)-trantinterol inverted from their antipodes to the sum of the AUC0-t of (-)- and (+)-trantinterol. Following single intravenous administration, both given enantiomers declined in similar plasma concentrations, suggesting that the two enantiomers have approximately the same disposition kinetics by the route of intravenous administration. However, after single oral administration, plasma concentrations of uninverted (-)-trantinterol at many timepoints were significantly higher than those of uninverted (+)-trantinterol, suggesting that the two enantiomers undergo apparently different absorption or metabolism after oral administration. Significant bidirectional chiral inversion occurred after intravenous and oral administration of (+)- or (-)-trantinterol. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated in vivo. The AUC0-36 of (+)-trantinterol after intravenous and oral dosing of (-)-trantinterol were 16.6 ± 5.2 and 33.3 ± 16%, respectively of those of total [(+) + (-)] trantinterol. The AUC0-36 of (-)-trantinterol after intravenous and oral dosing of (+)-trantinterol were 19.6 ± 8.8 and 37.9 ± 4.5%, respectively, of those of total [(-) + (+)] trantinterol. After intravenous administration of (+)- and (-)-trantinterol the chiral inversion ratios of the two enantiomers were not significantly different and similar results were found for oral administration. The extent of chiral inversion after intravenous administration was apparently lower, indicating that the bidirectional chiral inversion was not only systemic but also presystemic., (© 2013 Wiley Periodicals, Inc.)

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of trantinterol (SPFF) and its major metabolites for the first time. The analytes were extracted from rat urine and feces samples by liquid-liquid extraction (LLE) and determined in multiple reaction monitoring (MRM) mode with clenbuterol as the internal standard. Chromatographic separation was achieved on a Venusil ASB C8 column (2.1mm×100mm, 3μm), with the mobile phase consisted of methanol-0.2% formic acid (30:70, v/v) at the flow rate of 0.2mL/min. Each sample was chromatographed within 5min. This method has a lower limit of quantification (LLOQ) of 0.450, 1.05, 1.35, 0.904 and 1.36ng/mL for trantinterol (SPFF), arylhydroxylamine trantinterol (N-OH-SPFF), tert-butyl hydroxylated trantinterol (Tert-OH-SPFF), 1-carbonyl trantinterol (SPFF-COOH) and 3-methyl sulfone-dechloro-trantinterol (SPFF-SO2CH3) in rat urine, and 0.450, 1.35 and 0.904ng/mL for SPFF, Tert-OH-SPFF and SPFF-COOH in rat feces, respectively. The linear correlation coefficients were greater than 0.990. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was -9.9% to 11% at three quality control levels. The method has been successfully applied to the excretion study following an oral administration of 1mg/kg trantinterol to rats., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A simple and selective molecularly imprinted matrix solid-phase dispersion (MI-MSPD) method coupled with high performance liquid chromatography (HPLC) ultraviolet detection was developed for rapid screening of clenbuterol hydrochloride (CH) in chicken samples. The new molecularly imprinted microspheres (MIM) were synthesized by using butylamine and chloroaniline as dummy template with aqueous suspension polymerization and revealed good affinity to CH in aqueous solution. The application of the obtained MIM as sorbent of matrix solid-phase dispersion (MSPD) improved the selectivity of extraction procedure and avoided the effect of template leakage on quantitative analysis. Under the optimized conditions, good linearity of CH was obtained in a range of 0.059 18.30 μg mL(-1) with the correlation coefficient (R) of 0.9996. The recoveries of CH at three spiked levels were ranged from 92.0 to 99.1% with the relative standard deviation less than 4.0% (n = 3). The presented MI-MSPD-HPLC method combined the superiority of MIM and MSPD, and therefore could be potentially applied for the determination of CH in complicated biological samples.

Trantinterol is a novel β(2)-adrenoceptor agonist used for the treatment of asthma. The aim of this study is to identify the metabolites of trantinterol using liquid chromatography tandem mass spectrometry (LC-MS/MS), to isolate the main metabolites, and confirm their structures by nuclear magnetic resonance (NMR). Urine, feces, bile, and blood samples of rats were obtained and analyzed. Reference standards of six metabolites were achieved with the combination of chemical synthesis, microbial transformation, and the model systems of rats. Moreover, in order to investigate the phase I metabolism of trantinterol in humans and to study the species differences between rats and humans, incubations with liver microsomes were performed. The biotransformation by a microbial model Cunninghamella blakesleana AS 3.970 was also studied. A total of 18 metabolites were identified in vivo and in vitro together, 13 of which were newly detected. Three phase I metabolites were detected in vivo and in vitro as well as in the microbial model, including the arylhydroxylamine (M1), the tert-butyl hydroxylated trantinterol (M2) and the 1-carbonyltrantinterol (M3). Another important pathway in rats is glutathione conjugation and further catabolism and oxidation to form consecutive derivatives (M4 through M10). Other metabolites include glucuronide, glucoside, and sulfate conjugates. The results of in vitro experiments indicate no species difference exists among rats, humans, and C. blakesleana AS 3.970 on the phase I metabolism of trantinterol. Our study provided the most comprehensive picture for trantinterol in vivo and in vitro metabolism to this day, and may predict its metabolism in humans.

A capillary electrophoretic method has been established for the separation of the enantiomers of clenbuterol. The effects of pH value, composition of the background electrolyte, concentration of carboxymethyl-β-cyclodextrin (CM-β-CD), capillary temperature and running voltage have been investigated. The two enantiomers were separated in an uncoated capillary with phosphate buffer (50 mmol/L, pH 3.5) containing 10 mmol/L CM-β-CD. The capillary temperature was at 15°C and applied voltage was at 20 kV. The inclusion complex of CM-β-CD and clenbuterol was synthesized and characterized by two-dimensional rotating frame spectroscopy (2D ROESY). Based on the 2D ROESY analysis, an inclusion structure of the clenbuterol/CM-β-CD complex was proposed, in which clenbuterol penetrated CM-β-CD in a tilted manner due to the interaction of intermolecular hydrogen bonds between clenbuterol and CM-β-CD.

Rationale: Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzyl alcohol) is approved for human and veterinary use primarily for the treatment of pulmonary afflictions. Despite the authorized administration in cases of medical indications, the misuse of clenbuterol in animal husbandry as well as elite and amateur sport has frequently been reported, arguably due to growth-promoting properties. Due to various recent incidences of doping control specimens containing clenbuterol, strategies towards the discrimination of a surreptitious application from unintended intake via animal-derived edibles or dietary supplements were required., Methods: The enantiomeric compositions of clenbuterol in human urine samples derived from administration studies with therapeutic amounts of the β(2)-agonist and authentic doping control specimens were determined. Due to the facts that therapeutic clenbuterol consists of a racemic mixture of (+)- and (-)-stereoisomers and that the first mentioned (dextrorotatory) stereoisomer is retained to a greater extent in edible animal tissue, the differentiation of a recent administration of therapeutic (and thus racemic) clenbuterol from food contamination (stereoisomerically depleted clenbuterol) was considered. Employing deuterated clenbuterol as internal standard, the target analytes were extracted from human urine by means of concerted liquid-liquid and solid-phase extractions and subjected to chiral liquid chromatography hyphenated to high resolution/high accuracy mass spectrometry with electrospray ionization., Results: Both enantiomers of clenbuterol were baseline separated and relative abundances of corresponding labeled and unlabeled stereoisomers were determined, demonstrating that the therapeutic use of clenbuterol results in racemic mixtures in urine for at least 24 h while adverse analytical findings presumably originating from food contaminations can yield (-)-clenbuterol-depleted pairs of analytes., Conclusions: The determination of relative abundances of clenbuterol enantiomers can indicate the ingestion of clenbuterol via contaminated food; however, depletion of (-)-clenbuterol in edible animal tissue is time-dependent and thus results can still be inconclusive as to the inadvertent ingestion of clenbuterol when clenbuterol administration to animals was conducted until slaughter., (Copyright © 2013 John Wiley & Sons, Ltd.)

The temperature dependent vibrational spectra of,three biological and pharmaceutical sets, genistein and biochanin A, clenbuterol hydrochloride and salbutamol, as well as ginseng R2 and R3, in the range of 0.2-4.5 THz (6.6-150.0 cm(-)) are presented over the temperature range from 295 to 77 K Although there are only some minor difference in their molecular structures, the spectra of two samples in every group are quite differences in both absorption band positions and their relative intensities, and display strong linewidth narrowing and frequency blue-shift with cooling. Au 77 K, 13 highly resolved spectral features for biochanin A were obtained and the sensitivity of the experiment allows detection of amounts as small as 1.9 x 10(-5) mol of ginseng R3. Such high-resolution THz fingerprint spectra provide a rapid, nondestructive and reliable method for the identification of these pharmaceutical settings molecules.

A high performance liquid chromatographic (HPLC) method using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase (Lux Cellulose-1) for the separation of trantinterol hydrochloride enantiomers was developed. The method was based on normal phase model with n-heptane as the basal solvent of mobile phase. The influences of organic modifiers, mobile phase additives and column temperature on the retention and separation of the enantiomers were examined and discussed. It was demonstrated that the mobile phase additives had a dominant effect on the enantiomeric separation. No separation was observed with the addition of diethylamine only in the mobile phase, while the retention was increased and the enatiomeric separation was observed with the addition of trifluoroacetic acid. When both trifluoroacetic acid and diethylamine were added into the mobile phase, the enantioseparation was significantly improved with a resolution up to 4.0. Ethanol and isopropanol were investigated as the organic modifiers, and ethanol offered a better enatiomeric resolution for trantinterol hydrochloride. In the examined temperature range between 15 degrees C and 35 degrees C both separation factor and resolution were decreased with the increase of the column temperature. The optimized chiral HPLC method for the separation of trantinterol hydrochloride enantiomers involved a Lux Cellulose-1 column (250 mm x 4.6 mm, 5 microm), a mobile phase of n-heptane/ethanol/trifluoroacetic acid/diethylamine (88: 12: 0.3: 0.05, v/v/v/v) at a flow rate of 1.0 mL/min, a detection at 246 nm and a column temperature of 25 degrees C. The method is simple and rapid for the enantiomeric impurity determination of (-)-trantinterol hydrochloride bulk samples.

In this paper, a novel molecularly imprinted polymer (MIP) coated stir bar with ractopamine as template by glass capillary filling with magnetic core as substrate was prepared reproducibly. The ractopamine MIP coating was homogeneous and porous with the average thickness of 20.6 microm. The extraction apparatus for the stir bar was improved to avoid coating loss. The MIP-coated stir bar showed better extraction capacity and good selectivity than that of non-imprinted polymer (NIP) coated stir bar to ractopamine and its analogues. The extraction capacities of ractopamine, isoxsuprine, clenbuterol and fenoterol for MIP-coated stir bar were 3.3, 3.1, 2.8 and 2.4 times as much as that of the NIP coated stir bar, respectively. The MIP-coated stir bars could be used at least 40 times without apparent damage and kept in dried air for 8 months without reduce of extraction ability. A method for the determination of beta(2)-agonists in complex samples by MIP-coated stir bar sorptive extraction coupled with high-performance liquid chromatography (HPLC) was developed. The linear ranges were 0.5-40 microg/L for ractopamine and 1.0-40 microg/L for isoxsuprine and clenbuterol. The detection limits were within the range of 0.10-0.21 microg/L. The method was successfully applied to the analysis of beta(2)-agonists in spiked pork, liver and feed samples with the recoveries of 83.7-92.3%, 80.5-90.2% and 73.6-86.2%, respectively. The RSDs was within 2.9-8.1%. The method is very suitable for the determination of trace beta(2)-agonists in pork, liver and feed samples., (Copyright 2010 Elsevier B.V. All rights reserved.)

A liquid chromatography-tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel beta(2)-adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed-phase C(8) column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time-of-flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed., (2009 John Wiley & Sons, Ltd.)

Highly luminescent Eu(3+) and Tb(3+) complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu(3+) is a subset of 1 and Tb(3+) is a subset of 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lanthanide chelates are very stable and highly luminescent in aqueous solution and allowed to reach 10 microg L(-1) and 40 microg L(-1) sensitivities for CL and for HC, respectively. Application to the horse urine, that is a very complex matrix, has a considerable interest in the control of illegal use of these compounds.

Quantum dots (QDs) are semiconductor fluorescent nanoparticles, which can be used for food safety or environmental monitoring with high sensitivity. This work demonstrates the feasibility of detecting clenuterol residue in pig urine using CdSe/CdS quantum dots as fluorescent labels based magnetic core/shell Fe3O4/Au nanoparticles (MCFN) as solid carriers. The detection of clenbuterol is carried out by a fluoroimmunoassay-based biosensor using competitive binding between conjugated clenbuterol antigen-CdSe/CdS QDs and free clenbuterol with immobilized clenbuterol antibodies on MCFN. This assay method allows for clenbuterol determination in a linear working range of 0.5-20000 pg mL(-1). It would provide a simple, rapid, and ultra-sensitive detection method for clenbuterol or other biomolecular analysis.

Two clenbuterol O-glucuronide diastereomers were synthesized by the Koenigs-Knorr reaction. Structures and glucuronidation sites of the glucuronides were characterized by tandem mass spectrometry and nuclear magnetic resonance spectroscopy. The two diastereomers were used as standard compounds in studies of stereoselective glucuronidation of clenbuterol with liver microsomes from different species and with 15 human recombinant UDP-glucuronosyltransferases. In this study, chemical and enzymatic reactions produced only O-glucuronides of clenbuterol, although on the basis of the chemical structure of the aglycone, both O- and N-glucuronides of clenbuterol could be formed. Differences in the production of diastereomers of clenbuterol glucuronides were observed among liver microsomes from the various animals. Dog and bovine liver microsomes were significantly active, and also stereoselective, each producing only one but a different diastereomer. Liver microsomes from rabbit and rat were also rather actively glucuronidating clenbuterol, but human, pig, and moose liver microsomes produced only minor amounts of glucuronides. Human liver microsomes produced only one clenbuterol glucuronide diastereomer, and the same was true of the human UDP-glucuronosyltransferases that were active (formation of glucuronide: 1A9 > 1A10 >> 1A7). The marked differences in the stereoselective glucuronidation of clenbuterol show that UDP-glucuronosyltransferases in the livers of different animals do not have the same functions, activities, or distribution. This needs to be taken into account, particularly in toxicology testing.

Enantiomers of clenbuterol were separated by a new HPLC method on a chiral column. Enantiomeric resolution was achieved on a vancomycyin macrocyclic antibiotic chiral stationary phase known as chirobiotic V with UV detection at 247 nm. The polar ionic mobile phase consisting of methanol-triethylamine-glacial acetic acid (100 + 0.05 + 0.025, v/v/v), was used at a flow rate of 1.0 mL/min. The method was validated for linearity, accuracy, precision, and robustness. Standard linear calibration curves were established for the R-(-) and S-(+) enantiomers over the range of 0.2-20 microg/mL, and an average recovery of 98.0% and a mean relative standard deviation of 1.5% were obtained at 5.0 microg/mL. The lower limit of detection was 0.05 microg/mL for each enantiomer. The mean recovery for R-(-) and S-(+)-clenbuterol enantiomers from plasma was 91.0-97.0% at 0.20-20 microg/mL. The method was successfully used to identify and quantify the clenbuterol enantiomers in human plasma.

Clenbuterol hydrochloride (CLB) could catalyze NaIO(4) oxidation of eosine Y (R), which caused the room temperature phosphorescence (RTP) signal of R to quench sharply. The DeltaI(P) (= I(P2)-I(P1), I(P2) was RTP intensities of reagent blank and I(P1) was RTP intensities of test solution) of the system was directly proportional to the content of CLB. According to that academic thought, a new solid substrate-room temperature phosphorimetry (SS-RTP) for the determination of trace CLB has been established. This method has high sensitivity (detection limit (LD): 0.021 zg spot(-1), corresponding concentration: 5.2x10(-20) g mL(-1)) and good selectivity (Er = +/-5%, interfering species were of no interference). It has been applied to the determination of residual CLB in the practical samples. The results were verified using HPLC and GC/MS methods. The reaction mechanism of catalytic SS-RTP for the determination of residual CLB was also discussed.

Clenbuterol is a well-known growth promoter, illegally used in farm animals, especially in cattle. Samples collected for the screening of beta(2)-agonist residues in Portuguese Azores Islands must travel through all the nine islands until they reach Azores Central Laboratory. If any suspicious sample is detected, it must be further transported to the National Reference Laboratory in Lisbon for confirmation. As a consequence of these circumstances, samples are submitted to different transport and storage times, as well as different temperature conditions and in some cases successive freezing and thawing cycles. As clenbuterol is the most detected beta(2)-agonist growth promoter in the Portuguese Residue Monitoring Plan, studies were conducted on the stability of this compound in incurred samples (bovine liver and urine) at +4, -20 and -60 degrees C over time. Samples kept at -20 degrees C were also analyzed over time after successive freezing and thawing cycles. The analyses of clenbuterol over time were performed by gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). Clenbuterol in incurred urine and liver samples was significantly stable up to 20 weeks at -20 and -60 degrees C and after, at least, six consecutive freezings and thawings. At +4 degrees C, clenbuterol remained stable, at least until 12 weeks in urine and up to 20 weeks in liver.

Background: Clenbuterol is a potent, long-acting beta-adrenergic agonist that has been reported as an adulterant of heroin. We describe an atypical syndrome in five users of clenbuterol-tainted heroin., Methods: All cases were referred to a regional Poison Control Center. Urine and blood were analyzed using gas and liquid chromatography as well as mass spectrometry., Case Series: Five heroin users presented with a syndrome characterized by muscular spasm, tremor, hyperreflexia, and elevated serum creatine phosphokinase concentrations. All patients lacked findings of acute clenbuterol toxicity but tested positive for clenbuterol and negative for strychnine and a battery of common potential adulterants., Conclusions: We report five cases of a novel neuromuscular syndrome in users of clenbuterol-adulterated heroin. It is unclear whether these reactions represent an atypical response to clenbuterol or another unidentified contaminant.

The electrochemical modification of clenbuterol (CLB) was studied at paraffin-impregnated graphite electrode (WGE) in two potential ranges of 0.0-1.6V and -1.2 to 1.2V. Various methods including X-ray photoelectron spectroscopy (XPS), UV-spectroelectrochemistry, infrared (IR) spectra and electrochemical techniques have been used to characterizing the modification. Clenbuterol can be modified at the electrode surface by carbon-nitrogen linkage or carbon-carbon linkage in 0.0-1.6V or -1.2 to 1.2V, respectively. The electrochemical behaviors of dopamine (DA), norepinephrine (NE), adrenalin (EP), ascorbic acid (AA) and uric acid (UA) were studied at clenbuterol-modified paraffin-impregnated graphite electrode (CLB/WGE), and it was found that all these compounds could be detected successfully.

Capillary electrophoresis (CE) with UV detection for the simultaneous and short-time analysis of clenbuterol, salbutamol, procaterol, fenoterol is described and validated. Optimized conditions were found to be a 10 mmoll(-1) borate buffer (pH 10.0), an separation voltage of 19 kV, and a separation temperature of 32 degrees C. Detection was set at 205 nm. Under the optimized conditions, analyses of the four analytes in pharmaceutical and human urine samples were carried out in approximately 1 min. The interference of the sample matrix was not observed. The LOD (limits of detection) defined at S/N of 3:1 was found between 0.5 and 2.0 mgl(-1) for the analytes. The linearity of the detector response was within the range from 2.0 to 30 mgl(-1) with correlation coefficient >0.996.

Clenbuterol (CLB) is an antiasthmatic drug used also illegally as a lean muscle mass enhancer in both humans and animals. CLB and amine-related drugs in general are nitrosatable, thus raising concerns regarding possible genotoxic/carcinogenic activity. Oral administration of CLB raises the issue of its possible transformation by salivary nitrite at the acidic pH of gastric juice. In acidic human saliva CLB was rapidly transformed to the CLB arenediazonium ion. This suggests a reaction of CLB with salivary nitrite, as confirmed in aerobic HNO(2) solution by a drastic decrease in nitric oxide, nitrite, and nitrate. In human saliva, both glutathione and ascorbic acid were able to inhibit CLB arenediazonium formation and to react with preformed CLB arenediazonium. The effect of ascorbic acid is particularly pertinent because this vitamin is actively concentrated within the gastric juice. EPR spin trapping experiments showed that preformed CLB arenediazonium ion was reduced to the aryl radical by ascorbic acid, glutathione, and serum albumin, the major protein of saliva. As demonstrated by anti-CLB antibodies and MS, the CLB-albumin interaction leads to the formation of a covalent drug-protein adduct, with a preference for Tyr-rich regions. This study highlights the possible hazards associated with the use/abuse of this drug.