Single-dose administration of clenbuterol is detectable in dried blood spots.

Clenbuterol is a ß2 -agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time-consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 µg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post-ingestion, with additional urine collections on days 7 and 10. Using LC-MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post-ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7-10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 µg.

Comparison of three sample addition methods in competitive and sandwich colloidal gold immunochromatographic assay.

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.

The use of hair as a long-term indicator of low-dose ß2 agonist treatments in cattle: Implications for growth-promoting purposes monitoring.

The objective of this study was to assess the feasibility of using hair as a long-term indicator of cocktail (low-dose ß2 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 ß2 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 ß2 agonists in cattle, which could have implications for growth-promoting purposes monitoring.

A highly stable black phosphorene nanocomposite for voltammetric detection of clenbuterol.

A nanocomposite was prepared from graphene-like two-dimensional black phosphorene (BP, an allotrope of phosphorus) and nafion (Nf) treated with isopropanol (IP). A glassy carbon electrode (GCE) modified with this nanocomposite was found to be a viable sensor for voltammetric determination of clenbuterol (CLB). Unlike previously reported pure BP, the BP nanocomposite was stable towards water and oxygen. Its morphology, structure, electrochemically active surface area and electrochemical stability were investigated. The BP-Nf (IP) modified GCE displayed good electrochemical stability and electrocatalytic capacity with a low working potential of 0.94 V (vs. SCE), excellent peak current response for CLB in a linear concentration range of 0.06-24 µM with a detection limit of 3.7 nM (3σ/m) and a sensitivity of 0.14 µA·µM-1·cm-2 under optimal conditions. A sensing mechanism for the electro-oxidation of CLB was suggested and verified by density functional theory calculations under imitation of aqueous solution conditions. The sensor was successfully applied to the determination of CLB in bovine meat and bovine serum samples. Graphical abstract Highly-stable black phosphorene (BP) nanocomposite based on Nafion (Nf) was used to modify a glassy carbon electrode (GCE). It is shonw to be a viable electrochemical platform for sensitive voltammetric determination of trace clenbuterol (CLB) in bovine beef and bovine serum.

The Potential of Various Living Tissues for Monitoring Clenbuterol Abuse in Food-Producing Chinese Simmental Beef Cattle.

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

Quantification of trantinterol, its two metabolites and their primary conjugated metabolites in human plasma by ultra-high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

A highly rapid, selective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to simultaneously determine trantinterol, its major phase-I metabolites and their primary conjugated metabolites in human plasma. Waters Oasis HLB C18 solid phase extraction cartridges were used in the 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 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in selective reaction monitoring (SRM) mode with the use of an electrospray ionization (ESI) source. The linear calibration curves for trantinterol, tert-butyl hydroxylated trantinterol (tert-OH-trantinterol) and 1-carbonyl trantinterol (trantinterol-COOH) were obtained in the concentration ranges of 0.200-250, 0.108-4.00 and 0.0840-5.02 ng/mL, respectively (r(2)≥0.99). The intra- and inter-day precision (relative standard deviation, RSD) values were less than 13%, and the accuracy (relative error, RE) was within ±9.9%, as determined from quality control (QC) samples for the analytes. The concentrations of conjugated forms of trantinterol and tert-OH- trantinterol in plasma were determined using selective enzyme hydrolysis. The method described herein was fully validated and successfully applied for the pharmacokinetic study of trantinterol in healthy volunteers after oral administration.

An LC-MS/MS method for simultaneous determination of trantinterol and its major metabolite in rat plasma and its application to a comparative pharmacokinetic study.

Trantinterol is a novel ß2-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.

Clenbuterol Distribution and Residues in Goat Tissues After the Repeated Administration of a Growth-Promoting Dose.

This study was conducted to investigate the deposition and depletion process of clenbuterol (CL) in goat tissues, plasma and urine after the repeated administration of a growth-promoting dose. The experiment was conducted in 24 goats (21 treated and 3 controls). Treated animals were administered orally in a dose of 16 µg/kg body mass once daily for 21 consecutive days and randomly sacrificed on days 0.25, 1, 3, 7, 14, 21 and 28 of the withdrawal period. CL in goat tissues was extracted with organic solvents and determined using liquid chromatography tandem mass spectrometry. The depletion rates of tissue differed significantly. The highest concentrations of CL in all tissues are detected on day 0.25 of treatment discontinuation. After administration had been discontinued for 28 days, CL still residues in all tissues, especially, in whole eye, where the concentrations reach 363.29 ± 31.60 µg/kg. These findings confirmed that the whole eye, which are rich in pigment, showed a much higher concentration than any other studied tissue during the withdrawal period.

Determination of trantinterol enantiomers in human plasma by high-performance liquid chromatography - tandem mass spectrometry using vancomycin chiral stationary phase and solid phase extraction and stereoselective pharmacokinetic application.

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.

Simultaneous Determination of Trantinterol and One of Its Major Metabolites, 1-Carbonyl Trantinterol, in Human Plasma by LC-MS-MS.

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.

Enantioselective disposition of clenbuterol in rats.

Clenbuterol is a long-acting ß2-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.

Detection, pharmacokinetics and cardiac effects following administration of clenbuterol to exercised horses.

REASONS FOR PERFORMING STUDY: The use of clenbuterol in performance horses necessitates the establishment of appropriate withdrawal times. OBJECTIVES: To describe plasma and urine concentrations of clenbuterol following administration of 2 commonly used dosing regimens to racing fit Thoroughbreds. STUDY DESIGN: Experimental. METHODS: Twenty-two horses received an oral dose of 0.8 µg/kg bwt of clenbuterol twice daily for 30 days. A second group of 6 horses received clenbuterol according to the escalating dose protocol on the manufacturer's label. Blood and urine samples were collected prior to, throughout and at various times up to 35 days post administration of the final dose. Drug concentrations were measured using liquid chromatography-mass spectrometry, and plasma data were analysed using noncompartmental analysis. Behavioural and physiological effects were monitored and heart rate was recorded throughout the course of the study. RESULTS: Clenbuterol plasma concentrations were below the limit of quantification (10 pg/ml) of the assay by Day 4 in all horses receiving the chronic low-dose regimen and by Day 7 in 5 of 6 horses receiving the escalating dosing protocol. Urine clenbuterol concentrations fell below the limit of quantification of the assay between Days 21 and 28 in all 22 horses in the low-dose group and in 5 of 6 of the horses in the escalating dose group. Muscle fasciculations, sweating and transient increases in heart rate were noted in a small number of horses following clenbuterol administration, but tolerance to these effects occurred rapidly. CONCLUSIONS AND POTENTIAL RELEVANCE: Establishment of appropriate withdrawal times for specific racing jurisdictions depends upon the threshold adopted by that specific jurisdiction. This study extends previous studies describing the pharmacokinetics of clenbuterol and describes plasma and urine concentrations following administration of 2 commonly used dosing regimens to racing fit Thoroughbreds, which will allow jurisdictions to establish withdrawal times in order to prevent inadvertent positive regulatory findings.

Bidirectional chiral inversion of trantinterol enantiomers after separate doses to rats.

The chiral inversion and pharmacokinetics of two enantiomers of trantinterol, a new ß2 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.

Preparation of clenbuterol imprinted monolithic polymer with hydrophilic outer layers by reversible addition-fragmentation chain transfer radical polymerization and its application in the clenbuterol determination from human serum by on-line solid-phase extraction/HPLC analysis.

A novel imprinted monolithic material with the ability of protein exclusion was developed for the selective extraction of clenbuterol (CLE) from biological samples by direct injection in the HPLC analysis. The material has an imprinted inner structure and hydrophilic outer layer. The reversible addition-fragmentation chain transfer (RAFT) polymerization was employed in the material preparation by a two-step procedure. In the first step, clenbuterol imprinted monolithic polymer was synthesized by combining the molecular imprinting and the RAFT polymerization techniques. The resulting monolithic polymer has a RAFT chain transfer agent (trithioester groups) in its structure, which was used to graft poly(glycerol mono-methacrylate) [pGMMA] in the second step by post-RAFT polymerization. The hydrophilic pGMMA layers grafted on the surface of the imprinted monolith created barriers for protein diffusion. More than 90% of bovine serum albumin can be excluded from the pGMMA coated monolithic column. Meanwhile the clenbuterol was retained selectively with a large retention factor. The result indicated that the column, denoted as RA-MIM, has both the merits of a molecularly imprinted polymer and restricted access material. By using RA-MIM as the solid-phase extraction pre-column, an on-line column-switching HPLC method for the determination of clenbuterol in human serum has been established and validated. The recoveries of clenbuterol from the serum were 87.3-96.9% in the spiked level 2-1000 ng mL(-1). Both good linearity (R = 0.999) and acceptable reproducibility (RSD < 7.0%) were obtained. The limit of detection and the limit of quantitation were 0.7 ng mL(-1) and 2.0 ng mL(-1) respectively, which is sensitive in terms of UV detection. The results have demonstrated that the RAFT polymerization can be used to synthesize bi-functional monolithic columns by using its living reaction property. The resulting RA-MIM in this research can be used for efficient clenbuterol determination by HPLC from biological samples.

Rapid immunoassay method for the determination of clenbuterol and salbutamol in blood.

The aim of the study was to evaluate the adequacy of enzyme-linked immunosorbent assay (ELISA) in the post-exposure determination of the ß-agonists clenbuterol and salbutamol in animal plasma and serum. Experimental guinea pigs (n = 20) were treated with two doses (0.25 and 2.5 mg/kg) of clenbuterol (n = 10) and salbutamol (n = 10) for seven days, whereas the control animal group (n = 10) was left untreated. Validation of the applied method yielded acceptable recovery (mean > 70%) and repeatability rates, showing ELISA to be applicable for the semi-quantitative determination of both analytes in both matrices, preferably in plasma. In both matrices, clenbuterol concentrations were proven to be significantly (14-fold) higher than those of salbutamol. Concentrations of both analytes were higher in plasma than in serum. The application of a 10-fold higher clenbuterol and salbutamol dose (2.5 mg/kg) resulted in concentrations 3- to 4-fold higher for clenbuterol and 2- to 3-fold higher for salbutamol, indicating a different release rate of these two ß-agonists.

Detection of clenbuterol at trace levels in doping analysis using different gas chromatographic-mass spectrometric techniques.

This study demonstrates the development of a gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS-MS) assay to detect clenbuterol in human urine and the comparison of this method with GC-MS techniques and gas chromatography-high resolution mass spectrometry (GC-HRMS) techniques. Urine samples were hydrolyzed with ß-glucuronidase, extracted with methyl tert-butyl ether and dried under nitrogen. The derivative reagent was N-methyl-N-(trimethylsilyl)-trifluoroacetamide with NH4I and was analyzed by GC-MS, GC-MS-MS and GC-HRMS. A validation study was conducted by GC-MS-MS. The analyses of clenbuterol using different mass spectrometric techniques were compared. The limit of detection (LOD) for clenbuterol in human urine was 2 ng/mL by GC-MS (selected ion monitoring mode: SIM mode), 0.06 ng/mL by GC-HRMS and 0.03 ng/mL by GC-MS-MS, respectively, while the LOD by GC-HRMS was 0.06. With GC-MS-MS, the intra-assay and inter-assay precisions were less than 15%, the recoveries were 86 to 112% and the linear range was 0.06 to 8.0 ng/mL. The GC-MS under SIM mode can be used as a screening tool to detect clenbuterol at trace levels in human urine. The GC-MS-MS and GC-HRMS methods can confirm clenbuterol when its concentration is below 2 ng/mL. The results demonstrate that the GC-MS-MS method is quite sensitive, specific and reliable for the detection of clenbuterol in doping analysis.

Determination of L-trantinterol in rat plasma by using chiral liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and rapid method for determination of L-trantinterol in rat plasma was developed for the first time by using LC coupled to MS/MS based on chiral stationary phase. A baseline separation of the enantiomers of trantinterol was achieved on a Chirobiotic V column, using a mixture of acetonitrile-methanol-ammonia-acetic acid (80:20:0.01:0.02, v/v/v/v) as the mobile phase. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via ESI. The calibration curve was linear in concentration range from 0.270 to 108 ng/mL in plasma with the lower limit of quantification of 0.270 ng/mL. The intra- and interday precision (relative standard deviation) values were within 10.9% and the accuracy (relative error) was from 2.6 to 9.2% at all quality control levels. The method has been successfully applied to a study of L-trantinterol pharmacokinetics in rats.

Effects of clenbuterol administration on serum biochemical, histologic, and echocardiographic measurements of muscle injury in exercising horses.

OBJECTIVE: To determine the effects of clenbuterol, at a dosage of up to 3.2 µg/kg for 14 days, PO, on skeletal and cardiac muscle in healthy horses undergoing treadmill exercise. ANIMALS: 12 healthy horses from 3 to 10 years old. PROCEDURES: Horses were randomly assigned to a control group (n = 6) or clenbuterol group (6) and received either saline (0.9% NaCl) solution or clenbuterol, PO, every 12 hours for 14 days. Horses were subjected to submaximal treadmill exercise daily during treatment. Muscle biopsy specimens were collected before and after treatment for determination of apoptosis. Echocardiographic measurements, serum clenbuterol and cardiac troponin I concentrations, and serum activities of creatine kinase and aspartate aminotransferase were measured before, during, and after treatment. Jugular venous blood samples were collected every 3 days during treatment. Echocardiography was repeated every 7 days after beginning treatment. Response variables were compared between treatment groups and across time periods. RESULTS: No significant effect of clenbuterol or exercise on response variables was found between treatment and control groups at any time point or within groups over time. CONCLUSIONS AND CLINICAL RELEVANCE: Results did not reveal any adverse effects of treatment with an approved dose of clenbuterol on equine cardiac or skeletal muscle in the small number of horses tested.

Separation and determination of clenbuterol by HPLC using a vancomycin chiral stationary phase.

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.

Distribution of blood-muscle for clenbuterol in rat using microdialysis.

Clenbuterol is clinically used as a bronchodilator, but it is also illegally used to increase lean meat in animal husbandry. To investigate the muscle distribution of protein-unbound clenbuterol, a microdialysis technique coupled to liquid chromatography system was applied to simultaneously monitor clenbuterol in rat blood and muscle. Two microdialysis probes were implanted into the jugular vein/right atrium and hind leg muscle of rat for sampling after clenbuterol administration (10 mg/kg) through the femoral vein. Dialysate samples of clenbuterol were separated by a reversed-phase column (250 mm x 4 mm I.D., particle size 5 microm). The results indicate that the maximum concentration of clenbuterol in muscle was found at 30-45 min after clenbuterol administration (10 mg/kg) and the area under concentration curve (AUC) of clenbuterol in blood and in muscle were 942.75+/-101.92 and 174.81+/-13.03 min microg/mL, respectively. The AUC(muscle)/AUC(blood) was 0.20+/-0.03 representing about 20% of the clenbuterol distributing into the muscle. The elimination half-life of clenbuterol in the blood and muscle were about 2 and 6h, respectively. These results suggest that the protein-unbound concentration of clenbuterol sustained a high level and prolonged elimination in the muscle. The accumulation of clenbuterol might result in some clinical effects when clenbuterol-contaminated meat was consumed.