Development of extremely stable dual functionalized gold nanoparticles for effective colorimetric detection of clenbuterol and ractopamine in human urine samples.

New glutamic acid (Glu) and polyethylenimine (PE) functionalized ultra-stable gold nanoparticles (PE-Glu-AuNPs) were developed via a simple NaBH4 reduction method. The low toxicity and biocompatibility of PE-Glu-AuNPs were confirmed via an MTT assay in Raw 264.7 cells. Excitingly, PE-Glu-AuNPs were found to be extremely stable at room temperature up to six months and were utilized in an effective colorimetric naked eye assay of clenbuterol (CLB) and ractopamine (RCT) at pH 5. It was found that the selective assay of CLB and RCT is not affected by any other interferences (such as alanine, phenylalanine, NaCl, CaCl2, threonine, cysteine, glycine, glucose, urea and salbutamol). Furthermore, the detection of these ß-agonists can be visually accomplished through change color from wine red to purple blue. Notably, the aggregation induced detection of CLB and RCT was well confirmed through transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies. DLS investigations, clearly showed, that in the presence of CLB and RCT, the initial size of PE-Glu-AuNPs (12.8 ±â€¯8.6 nm) was changed to 84.8 ±â€¯52.3 and 79.5 ±â€¯47.8 nm, respectively, via aggregation. Furthermore, the colorimetric assays of CLB and RCT with PE-Glu-AuNPs were effective starting from CLB and RCT concentrations of 200 nM and 400 nM, respectively, and could be visualized using the naked eyes. Remarkably, UV-vis titrations of PE-Glu-AuNPs with CLB and RCT could be used to well estimate their sub nanomolar detection limits (LODs) via standard deviation and linear fittings. The contribution of surface functional groups that support the analyte recognition was confirmed by fourier-transform infrared spectroscopy (FTIR) analysis. Moreover, the CLB and RCT assays with PE-Glu-AuNPs were supported by examination of human urine samples.

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.

Protein expression changes in skeletal muscle in response to growth promoter abuse in beef cattle.

The fraudulent treatment of cattle with growth promoting agents (GPAs) is a matter of great concern for the European Union (EU) authorities and consumers. It has been estimated that 10% of animals are being illegally treated in the EU. In contrast, only a much lower percentage of animals (<0.5%) are actually found as being noncompliant by conventional analytical methods. Thus, it has been proposed that methods should be developed that can detect the use of the substances via the biological effects of these substances on target organs, such as the alteration of protein expression profiles. Here we present a study aimed at evaluating if a correlation exists between the treatment with GPAs and alterations in the two-dimensional electrophoresis (2DE) protein pattern obtained from the biceps brachii skeletal muscle from mixed-bred cattle. After image analysis and statistical evaluation, protein spots that differentiate between treated and control groups were selected for analysis by mass spectrometry. A set of proteins could be defined that accurately detect the use of glucocorticoids and ß(2)-agonists as growth promoters through the changes caused in muscle differentiation. As a further validation, we repeated the analysis using an independent set of samples from a strain of pure-bred cattle and verified these proteins by Western blot analysis.

Analysis of beta-agonists and beta-blockers in urine using hollow fibre-protected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry.

A method using hollow fibre-protected liquid-phase microextraction (HF-LPME) with in situ derivatization followed by gas chromatography/mass spectrometry (GC/MS) was established for the analysis of beta-agonists and beta-blockers in urine. Because it can simultaneously extract and derivatize compounds of interest by methylbenzol and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) in HF-LPME, the approach overcomes the drawbacks of considerable time-consuming and tedious operation, meanwhile improves enrichment multiple. The optimized conditions were extraction for 20 min at 35 degrees C with 5.0 microL of mixed extraction solvent (methylbenzol/MSTFA=1:1, v/v) with stirring speed of 925 rpm in 5.0 mL sample under pH 12.0 and 14% (w/v) NaCl. The method provided very wide linear ranges (0.25-400 ng mL(-1)) and low detection limits in the range of 0.08-0.10 ng mL(-1) for clenbuterol, metoprolol and propranolol while enrichment factors reached up to 256. The analytes could be determined in spiked urine by the method with high extraction efficacy (93.79-109.04% recoveries) and precision (<9.70% RSD). It has a satisfactory result for metoprolol in practical human urine samples for a single-dose administration of 50 mg after 36 h. The proposed method only needs few microliters of organic solvent and derivatizing agent; the operation is simple, convenient and rapid for the trace analysis of beta-agonists and beta-blockers in biological fluids; it can be readily generalized for high sample throughput. So, it is hopeful that the study will facilitate the monitoring of beta-agonists and beta-blockers in the competition sports.

A novel neuromuscular syndrome associated with clenbuterol-tainted heroin.

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.

Detection of clenbuterol in heroin users in twelve postmortem cases at the Philadelphia medical examiner's office.

The presence of clenbuterol, a beta2-adrenergic agonist banned for human use in the United States because of its serious side effects, is reported in a series of 12 postmortem cases in which the cause of death was attributed to illicit drug use. During the first three months of 2007, postmortem specimens from cases previously screening positive for opiates or fentanyl were screened specifically for clenbuterol using enzyme-linked immunosorbent assay. Confirmation of clenbuterol was performed using solid-phase extraction, derivatization with trimethylboroxine, and analysis utilizing a gas chromatograph-mass spectrometer (GC-MS) operated in the full-scan mode. The limits of detection and quantitation in blood were 2.5 and 5 ng/mL, respectively. Linearity was from 5 to 100 ng/mL. Clenbuterol was positive in 12/106 (11%) drug-related cases and in 12/575 (2.1%) of the total cases tested. In each of the 12 cases positive for clenbuterol, heroin use was either confirmed by the presence of 6-acetylmorphine or strongly suspected by the presence of morphine with a history of heroin abuse. Because the use of clenbuterol in the United States is restricted to veterinary medicine, its detection is an unexpected finding. Its presence in these cases serves as a caution to emergency room physicians and toxicologists to consider and test for clenbuterol when treating a suspected heroin user who presents atypically. This is the first known series of clenbuterol-positive cases of illicit drug users to be reported from a medical examiner's toxicology laboratory.

A descriptive study of an outbreak of clenbuterol-containing heroin.

STUDY OBJECTIVE: Illicit drugs may be adulterated with substances other than the sought-after substance of abuse. Although the true incidence and clinical effects of this practice are unknown, geographically disparate outbreaks of clinically significant adulteration continue to occur. We report on a recent outbreak of clenbuterol-adulterated heroin occurring along the East Coast of the United States. METHODS: After identification of index cases, 5 US poison centers collaborated with state and territorial health departments to alert the public of clenbuterol-tainted heroin. A case definition of clenbuterol-tainted heroin toxicity was promulgated, and emergency departments (EDs) were asked to contact poison centers when cases were identified. RESULTS: We identified 34 probable or confirmed ED presentations in 5 states during a 6-month period. Thirteen of the 34 patients met the criteria for "confirmed" exposures. Clenbuterol was identified in the blood and or urine of 12 of these 13 patients. Clenbuterol concentrations ranged from 2.4 to 26 ng/mL in the blood and 9.4 to 12,526 ng/mL in the urine. Symptoms included nausea, chest pain, palpitations, dyspnea, and tremor. Physical findings included significant tachycardia, hypotension, and laboratory evidence of hyperglycemia, hypokalemia, and increased lactate levels. Six patients demonstrated biochemical evidence of myocardial injury. Ten patients received beta-adrenergic antagonists without adverse effect. CONCLUSION: The adulteration of heroin by clenbuterol was associated with sympathomimetic effects, metabolic acidosis, and myocardial injury. The report also highlights how collaborative efforts among poison centers using the Centers for Disease Control and Prevention's Epi-X system rapidly identified a disease outbreak.

Extraction of clenbuterol from urine using hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith microextraction followed by high-performance liquid chromatography determination.

A hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic capillary was prepared for polymer monolith microextraction (PMME). Coupled to HPLC with UV detection, this extraction medium was successfully applied to establish a simple and fast method for the analysis of clenbuterol (CLB) in urine. To obtain optimum extraction performance, the effects of pH value and ionic strength of the sample matrix on extraction efficiency were investigated. The linearity of the method was evaluated over a concentration range of 10-2000 ng/mL and the correlation coefficient (R2 value) was 0.9985. The detection limit and quantification limit were 2.3 and 7.7 ng/mL, respectively. Good reproducibility of the method was obtained, yielding the intra- and interday RSDs less than 5.1 and 9.1%, respectively. Moreover, the hydroxylated poly(GMA-co-EDMA) monolithic capillary exhibited good preparation reproducibility and long-term extraction life. When applied to the determination of CLB in urine samples, an effective removal of interfering compounds was achieved and recoveries were in the range of 87.6-106%. The determination of CLB from one real sample including pretreatment, extraction, and analysis could be finished within 30 min.

Development of a lateral-flow assay for rapid screening of the performance-enhancing sympathomimetic drug clenbuterol used in animal production; food safety assessments.

A lateral-flow assay that could provide visual evidence of the presence of clenbuterol in swine urine was developed. Colloidal gold was prepared and conjugated with anti-clenbuterol monoclonal antibody. Immunochromatographic test strips were produced, and then, 210 samples were tested on these strips. Analysis was completed in 10 min. Detection limit was 3 ppb of clenbuterol. Parallel GC-MS data indicated that clenbuterol rapid detection strip had no false negative. The false positive rate was 4.4%. Immunochromatographic strip has great applied value in the food safety field because it possesses benefits of sensitivity, stability, reproducibility, ease of use and inexpensive.

Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for beta2-agonists in human or equine urine.

Electrospray ionization (ESI) mass spectra of 19 common beta(2)-agonists were investigated in terms of fragmentation pattern and dissociation behavior of the analytes, proving the origin of fragment ions and indicating mechanisms of charge-driven and charge-remote fragmentation. Based on these data, liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) screening and confirmation methods were developed for doping control purposes. These procedures employ established sample preparation steps including either acidic or enzymatic hydrolysis, alkaline extraction and, in the case of equine urine specimens, acidic re-extraction of the analytes. In addition, a degradation product of formoterol caused by acidic hydrolysis during sample preparation could be identified and utilized as target compound in screening and also confirmation methods. The screening procedures cover 18 or 19beta(2)-agonists, the estimated limits of detection of which for equine and human urine samples vary between 2 and 100 ng ml(-1) and between 2 and 50 ng ml(-1), respectively. A single LC/MS/MS analysis can be performed in 9 min.

Positive chemical ionization and tandem mass spectrometric fragmentation for the gas chromatographic analysis of beta-agonists using the ion trap technique.

A procedure is described for the determination of three characteristic beta-agonists (clenbuterol, terbutalin and salbutamol) based on the formation of the corresponding protonated molecules and related collisional experiments. Quantification was carried out on selected collisional fragments and the reproducibility of the relative abundances of these fragments was estimated. The performance of the method was tested on bovine urine samples spiked at the lowest level of 0.2 ng ml(-1) in each of the chosen compounds.

Determination of clenbuterol in bovine urine using gas chromatography-mass spectrometry following clean-up on an ion-exchange resin.

A gas chromatographic-mass spectrometric method was developed for the determination of residues of clenbuterol in bovine urine. The method involves a simple cation-exchange clean-up and concentration of clenbuterol in the acidified urine, followed by ethyl acetate extraction. The analyte is determined as the di-trimethylsilyl derivative and quantitated against an internal standard of penbutolol. Using a 5-ml sample of urine, a detection limit of 0.07 ng/ml can be achieved with recoveries close to 100% for fortification levels of 0.2 and 1 ng/ml. By increasing the sample volume to 50 ml, a detection limit below 0.01 ng/ml was achievable with recovery averaging 70%. The coefficient of variation of the assay ranged from 15% at 0.01 ng/ml (50-ml sample) to 6% at 1 ng/ml (5-ml sample). It was demonstrated that the method can detect the presence of clenbuterol in bovine urine at sub-ppb (ng/ml) levels using low resolution GC-MS with electron impact (EI) ionization.

Beta2-adrenergic agonist residues: simultaneous methyl- and butylboronic derivatization for confirmatory analysis by gas chromatography-mass spectrometry.

A derivatization procedure for confirmatory residue analysis of beta2-agonists is described. Methyl (MBA) and butyl (BBA) boronic acids are simultaneously used for the derivatization of tulobuterol, mabuterol, mapenterol, salbutamol, clenproperol, clenbuterol, clenpenterol and bromobuterol by GC-MS determination. A temperature of 55 degrees C during 60 min was selected as optimal temperature-time condition for simultaneous MBA and BBA beta2-agonists derivatization. It was also observed that stability of boronic derivatives was maintained at -20 degrees C over a period of four days. The proposed methodology was tested in urine and it could be applied for confirmatory residue analysis of clenbuterol-like compounds.

Probenecid markedly reduces urinary excretion of ethinylestradiol and trimethoprim slightly reduces urinary excretion of clenbuterol.

This study investigated whether the illegal application of ethinylestradiol or clenbuterol in cattle as growth promotors may be concealed by co-treatment with drugs that affect urinary excretion. Therefore, six male veal calves were fed with ethinylestradiol and six different male veal calves were fed with clenbuterol for 13 days. Both groups received the growth promotors twice daily (days -2 to 11) with milk replacer. The calves receiving ethinylestradiol were additionally fed with probenecid on days 7-11, and the calves receiving clenbuterol were additionally fed with trimethoprim (days 7-11). During days 1-11 of the experiment, 24-h urine and blood samples (once daily) were collected and analyses for ethinylestradiol and clenbuterol by specific enzyme immunoassay. In four calves the average urinary excretion of ethinylestradiol during days 7-11 (co-treatment with probenecid) was only about 25% of their average urinary excretion of ethinylestradiol on days 1-6. In the other two calves of this group, the excretion of ethinylestradiol was reduced to 4% on days 7-11 compared with days 1-6. In these two calves several urine samples provided concentrations of ethinylestradiol around the limit of detection. As a consequence, there may be a chance of concealing ethinylestradiol application by co-treatment with probenecid. Co-treatment with trimethoprim led only to a slight reduction of urinary excretion of clenbuterol. The detection of clenbuterol in urine samples from calves which were co-treated with trimethoprim can thus not be prevented.

Gas chromatography-mass spectrometric analysis of clenbuterol from urine.

Clenbuterol which is mostly used as an anabolic agent. It is also used for treatment of asthma. Clenbuterol was analysed from urine by using gas chromatography-mass spectrometry. GC-MS parameters were determined. Timolol was used as an internal standard. Extraction and derivatisation procedure of clenbuterol from urine were developed. Clenbuterol was extracted by using diethylether/ter-butanol (4:1; v:v) and pH 12 K2CO3/KHCO3 (3:2; w:w) buffer. MSTFA/NH4I (1 ml/10 mg) mixture was used for derivatization of clenbuterol. Selected ions of clenbuterol-bis-TMS were m/z: 405, 337, 336, 335, 300, and 227. Extraction yield and minimum detection limit of clenbuterol from urine were identified. Extraction yield was 94.30% and minimum detection was found 0.02 ng ml(-1) urine. It has been concluded that the GC-MS method is sensitive, accurate, precise, and reproducible for analysing of clenbuterol from urine.

Effects of drugs which influence renal transport systems on the urinary excretion of the beta 2-adrenoceptor agonist clenbuterol and the anabolic steroids ethinylestradiol and methyltestosterone.

The aim of this study was to determine whether the illegal application of clenbuterol, ethinylestradiol and methyltestosterone in cattle as growth promoters can be concealed by co-treatment with drugs that affect urinary excretion. Six male veal calves were fed with 0.8 micrograms clenbuterol kg-1 of body weight (BW), 3.5 micrograms ethinylestradiol kg-1 BW and 35 micrograms methyltestosterone kg-1 BW together twice daily for 28 days. At the eighth day of clenbuterol, ethinylestradiol and methyltestosterone treatment each calf was additionally fed either with probenecid, para-aminohippuric acid, trimethoprim, famotidine or cimetidine at three different doses which were increased in weekly intervals. During the treatment 24 h-urine and blood samples (once daily) were obtained and analysed for clenbuterol, ethinylestradiol and methyltestosterone by specific enzyme immunoassay. By high performance liquid chromatography/enzyme immunoassay it was determined whether these drugs or their metabolites interfered with the immunological detection of the growth promoters. Clenbuterol, ethinylestradiol and methyltestosterone could be detected in plasma and urine throughout the whole experiment. Co-treatment with probenecid led to a five-fold reduction in urinary excretion of ethinylestradiol and co-treatment with trimethoprim led to a three-fold reduction in urinary excretion of clenbuterol. None of the drugs reduced urinary excretion of the growth promoters to concentrations below the limit of detection. The detection of these three growth promoters in urine samples from calves which were co-treated with the drugs tested in this study can thus not be prevented.

Gas and liquid chromatography coupled to tandem mass spectrometry for the multiresidue analysis of beta-agonists in biological matrices.

beta-Agonists are substances used in veterinary and human medicine for the treatment of pulmonary disorders. They have found a use as growth promoters to improve meat-to-fat ratios in cattle but they are not authorized for use in the European Union. Due to their presence in trace levels (usually less than 1 microgram/kg), to the diversity of the illegally used compounds and to the complexity of the biological matrices analysed, the detection of these residues requires a very sensitive and specific method of determination. This work describes the strategy of analysis we developed for five beta-agonists in urine and liver. The combination of improved solid- or liquid-phase extraction methods and LC or GC-MS-MS (in the multiple reaction monitoring mode) has shown to provide a system suitable for the control of these substances. The efficiency of extraction and the sensitivity and selectivity allow this multiresidue detection down to, and below, the UK regulatory level of 0.5 microgram/kg. Moreover, the use of LC removes the need for the derivatisation step (cyclic methylboronate derivatives) which is required prior to GC-MS-MS analysis.

Probenecid, sulfinpyrazone and pyrazinamide do not inhibit urinary excretion of the beta 2-adrenoceptor agonist clenbuterol in cattle.

The objective of this study was to develop an analytical approach to determine whether the illegal application of clenbuterol in cattle as an anabolic agent can be concealed by co-treatment with substances that affect urinary excretion. Female veal calves were dosed orally with 0.8 microgram clenbuterol per kg of body weight twice daily for 28 days, as licensed for the therapeutic use which is registered in most European countries. On the eighth day of clenbuterol treatment each calf was additionally dosed orally either with probenecid, sulfinpyrazone or pyrazinamide at three different doses that were increased in weekly intervals. During the treatment blood and urine samples were obtained and analysed for clenbuterol by enzyme immunoassay and by high performance liquid chromatography/ enzyme immunoassay to determine whether these drugs or their metabolites interfered with the immunological detection of clenbuterol. Clenbuterol could be in plasma (approximately 200 pg ml-1) and urine (1-40 ng ml-1) 5 h after the initial intake and throughout the whole treatment. None of the drugs reduced urinary excretion of clenbuterol to concentrations below the limit of detection. There was no prevention of clenbuterol detection in urine samples from calves that were co-treated with the drugs tested in this study. Our results demonstrate the uselessness of applying these drugs in order to conceal the illegal use of clenbuterol in meat production.

Determination of beta-sympathomimetics in liver and urine by immunoaffinity chromatography and gas chromatography-mass-selective detection.

A specific and sensitive method for the determination of several beta-agonistic drugs in liver and urine is described. Following clean-up by immunoaffinity chromatography and two different derivatizations, gas chromatography-mass spectrometry with electron-impact ionization is performed. The immunoaffinity chromatography columns were packed with Sepharose-immobilized polyclonal antibodies raised against the beta-agonist clenbuterol. Owing to the high clean-up efficiency of the immunoaffinity column large sample volumes can be used (up to 100 ml urine or 25 gram liver). The immunoaffinity sample pretreatment is highly specific and no further sample pretreatment was necessary. Due to the combination of two different derivatizations only GC-MS with electron-impact ionization is necessary to fulfil legal requirements. The first confirmation step consists of a derivatization reaction between the hydroxyl group of the parent compound and trimethylsilane. The second confirmation method is a derivatization to a cyclic derivative with the hydroxyl group and the aliphatic nitrogen group. Limits of determination in liver as well in urine are at the 10 ng/kg or ng/l (ppt) level with acceptable signal-to-noise ratio. The method is suitable for identification and quantification of trace amounts of several similar beta-agonistic drugs either used separately or in combination and can be used also for quantification of clenbuterol in liver with regard to levels exceeding the maximum residue limit (MRL) of 1 microgram/kg (ppb).

Determination of clenbuterol and salbutamol in urine by capillary gas chromatography with capillary columns of 100 microns.

A method to determine clenbuterol and salbutamol in calf urine is described. Two independent extraction procedures using Extrelut (clenbuterol) and octadecylsilica (salbutamol) were used; the extracts obtained were mixed and purified over a cyanopropyl minicolumn. Trimethylsilyl derivatives were prepared and analysed by GC-MS in selected-ion monitoring mode using a fused-silica open tubular capillary column, 10 m x 100 microns coated with 5% phenylmethylsilicone. Splitless injection was optimized to achieve low percentage residual standard deviation of absolute areas. The best conditions were: injection volume 0.5 microliter, column head pressure 22 p.s.i. (1 p.s.i. = 6894.76 Pa), inlet temperature 250 degrees C and glass liner volume 250 microliters. The recoveries of the complete procedure were in the range 50-60% for both compounds.