Immunobiosensor analysis- of clenbuterol in bovine hair

Due to the potential speed and flexibility of surface plasmon resonance (SPR)-biosensor analysis, the technique is currently being exploited for detection of residues in food. In the present study an antibody-based inhibition assay for analysis of clenbuterol in cattle hair was developed using a Biacore 2000 instrument. Analysis of clenbuterol with and without enhancement with a secondary antibody was compared. A fast extraction method for hair, which enabled direct analysis in the biosensor with no clean up other than microscale ultrafiltration was developed. In contrast to most of the published methods in the area, no organic solvent or solid phase extraction step was needed. The biosensor method was validated by analysis of hair samples from animals treated with clenbuterol and the results were compared to results from GC-MS analysis. The correlation was good (r²=92.7%). The detection limit of the new method was 10 ng g^-1 hair.     

Screening of clenbuterol,salbutamol and terbutaline in animal feeds

A screening method was developed providing reliable detection of three chemically different β‐agonist model compounds, clenbuterol (50 ppb), salbutamol (500 ppb) and terbutaline (500 ppb). The method comprised an extraction with a mixture of acidic KH₂PO₄ buffer plus methanol followed by detection by enzyme immunoassay using an antibody raised against clenbuterol‐diazo‐bovine serum albumin. The background signals of 107 feed samples of different composition were measured, and 86% were ≤ 2.45 ng clenbuterol equivalents g^‐1. The highest values were 9–56 and 8–98 ng clenbuterol equivalents g^‐1, found in two samples of mineral supplements. Analysis of spiked samples showed mean recoveries of 106 (clenbuterol) to 110% (salbutamol) and coefficients of variation of 13–22% over the different materials. Selected groups of feed materials, such as soybean meal (n = 8), milk replacer (n = 15), dairy concentrate (n = 12), feed for poultry (n = 5), protein concentrate (n = 3) and ground grain meals (n = 5) provided blanks all ≤ 2.58 ng clenbuterol equivalents g^?1 and recoveries between 97 and 124%; the coefficients of variation ranged from 4 to 14%. Although mineral supplements gave slightly less reliable results using this method with varying recoveries (22–189%) a clear differentiation of positives and negatives was always possible at the levels of interest. It may be possible to detect other β‐agonists that show cross‐reactivity with the antibody used for the immunoassay.     

Dexamethasone and clenbuterol detection by enzyme immunoassay in Bovine Liver Tissue: A new multiresidue extraction procedure

A fast and simple extraction procedure was developed for simultaneous determination in bovine liver of two veterinary drugs, widely used as growth promoters in meat production: dexamethasone (a synthetic corticosteroid drug) and clenbuterol (a beta2‐adrenergic agonist drug). Liver samples were extracted by acetonitrile, without any clean‐up step. Two different ELISAs, specific for the two classes of drugs, were used to determine the residue concentration in the extracts. The intra‐ and inter‐extraction variability was determined at different concentrations: the intra‐extraction coefficients of variation (CVs) were between 2.5 and 17.7% for dexamethasone and between 0.9 and 9.8% for clenbuterol; the inter‐extraction CVs were between 2.0 and 16.8% for dexamethasone and between 0.5 and 10.8% for clenbuterol. Recovery ranged from 92 to 154% for dexamethasone and from 78 to 105% for clenbuterol. The limit of detection was 1.43 ng g^?1 and 0.43 ng g^?1, respectively. The limit of quantification for dexamethasone was 2.09 ng g^?1 and for clenbuterol was 0.72 ng g^?1. The combination of the new extraction procedure with an ELISA detection permitted the rapid semi‐quantitative determination of both dexamethasone at its maximum residue level (MRL: 2.5 ng g^?1 in liver tissue), and clenbuterol at low concentration level.     

Accumulation of the xenobiotic anabolic steroids ethinyloestradiol and methyltestosterone in calf hair

An analytical method to detect the illegal application of anabolic steroids in cattle by hair analysis was developed. The time course of incorporation of the orally active xenobiotic steroids ethinyl‐oestradiol (EE₂) and methyltestosterone (MT) into growing hair, the duration of their detection by hair analysis and the influence of hair pigmentation on steroid concentrations were investigated in detail. Female veal calves of different coat colour were fed with 3.5 μg of EE2 kg^‐1 of body weight and 35 μg of MT kg^‐1 of body weight, twice daily, for 10 days. Before, during and after treatment, hair samples were obtained and analyzed for EE₂ and MT residues. An appropriate method was developed to extract EE₂ and MT from hair. The extraction procedure consisted of liquid‐liquid and solid‐phase extraction and was followed by an essential high‐pressure liquid chromatography step for further purification of the extracts. The final quantification was carried out with specific enzyme immunoassays. EE₂ and MT residues could be detected in hair from day 4 (approximately 1 ng of EE₂ g^‐1 of hair and 5 ng of MT g^‐1) of the experiment up to day 38 (approximately 0.3 ng of EE₂ g^‐1) and day 94 (approximately 0.5 ng of MT g^‐1) respectively. The plasma concentrations of MT during the treatment were about 10‐fold higher than the plasma concentrations of EE2. Steroid accumulation in hair seemed to depend on the plasma levels of the steroids applied because the accumulation of MT in hair was higher (approximately 7 ng g^‐1) than the accumulation of EE₂ (approximately 1 ng g^‐1). In addition, the tracking of MT by hair analysis was possible for a much longer duration (up to day 94 of the experiment) than the detection of EE₂ (day 38). There was no influence of pigmentation on the steroid concentrations in hair. These results suggest that hair analysis is a powerful means with which to detect and track the illegal use of anabolic steroids in animal production, horse‐racing and sports.     

Determination of clenbuterol residues in bovine hair by using diphasic dialysis and gas chromatography-mass spectrometry.

A method for the determination of clenbuterol (4-amino-3,5-dichloro-alpha[(tert.-butylamino)methyl]-benzyl alcohol hydrochloride) in hair of living cows has been developed. Hair samples were digested in an alkaline medium. The diphasic dialysis technique is a semi-permeable membrane technology developed for the direct extraction of relatively low-molecular-mass analytes such as clenbuterol. In this case, we used sodium citrate buffer to homogenize the digested hair, dichloromethane was used as the extraction solvent at 37 degrees C, and stirring was applied at 150 rpm for 4 h. The analysis was carried out using gas chromatography-mass spectrometry. The calibration curve for clenbuterol in hair was linear in the range from 12.5 to 400 ng g(-1). The detection limit of clenbuterol was 5 ng g(-1) and the quantification limit was 12.5 ng g(-1), in hair. A good inter-day reproducibility was obtained (R.S.D. = 7.08%). The repeatability and intra-day reproducibility (50 ng g(-1) of hair, n = 10) show R.S.D.s of 7.1 and 9.5%, respectively.     

Long-term detection of clenbuterol in human scalp hair by gas chromatography-high-resolution mass spectrometry

A method for the detection of clenbuterol in human scalp hair by gas chromatography-high-resolution mass spectrometry (GC-HRMS) is described. The sample preparation involved chemical digestion of the protein structure, which was achieved by incubating the hair with 1 M KOH at 70 degrees C. A single extraction step with tert.-butyl methyl ether provided approximately 90% of the analyte, which was dried and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) to yield clenbuterol N,O-bis-trimethylsilyl (TMS). Hair was collected from four pregnant women who were therapeutically treated with Spiropent (clenbuterol-HCl) and from the infant of one female patient. Hair samples were taken during the application time and two to six months after completion of clenbuterol administration. The detection limit of the method was approximately 4 ng clenbuterol/g hair when 25 mg hair material were processed and 2 ng/g for 50 mg hair samples (corresponds to 4 pg per injection). The method allows clenbuterol to be measured retrospectively for up to at least six months. The levels of clenbuterol determined in hair ranged from 2 to 236 ng/g. No clenbuterol was found in the hair of the infant, which was taken five and a half months after delivery. To improve sample preparation, an additional purification step via immuno affinity chromatography (IAC) was integrated. The IAC purified extracts showed reduced biological background interference and an improved limit of detection (0.8 ng/g).     

An enzyme‐linked immunosorbent assay for the direct analysis of beta‐agonist drugs in urine and sera

A sensitive competitive solid‐phase enzyme immunoassay for the detection of tert‐butylic beta‐agonist drugs was developed. An antiserum was raised in rabbits against the diazo‐conjugate of clenbuterol and the human serum albumin. In the assay, the anticlenbuterol antibody was incubated with the salbutamol‐enzyme conjugate and unlabelled standard or samples in microtitre wells precoated with affinity‐purified antirabbit IgG. The absolute detection limit for clenbuterol and mabuterol was 0.3 ng/ml (15 pg/well) and 50% relative binding at 1.0 ng/ml (50 pg/well). The absolute detection limits for salbutamol and terbutaline were 0.9 and 1.3 ng/ml, respectively. The cross‐reactivity of iso‐propylic parent compounds was quite low (<1%). Urine and sera were tested without any extraction step. Sample blanks (96 urine and 76 sera from five different sources) showed low matrix effect, suggesting a limit of decision for bovine urine of 0.6 ng/ml clenbuterol equivalent. Mean recoveries of clenbuterol in spiked urine and serum samples ranged from 90 to 120% (mean recovery 96%). Comparative analysis by gas chromatography/mass spectrometry showed that false negative results do not occur. The assay is simple, rapid, sensitive, multiresidual, reliable and cost‐effective. The test is adaptable to high sample throughput laboratories as well as field test for rapid screening of a few samples in slaughterhouses.     

Multi‐residue enzyme immunoassays for screening for β‐agonists in faeces and feeds

Multi‐analyte screening methods for β‐agonists in faeces and feeds based on enzyme immunoassay (EIA) were developed. Commercially available EIA kits were used, and the results presented. The extraction for both matrices was performed under acid conditions. The addition of an organic solvent to the extradant, resulting in higher recoveries, was only retained for feeds as the background absorption for faeces increased substantially. Clean‐up for faecal extracts was achieved in a one‐step extraction with isobutyl alcohol. Detection limits in faeces were slightly different for the two types of kits used. They ranged from 0.8ng g^?1 for clenbuterol to 10ng g^?1 for terbutaline. In feeds, corresponding detection limits for both compounds were 5 ng g^?1 and 110 ng g^?1. Confirmation of EIA positives was established by liquid chromatography with post‐column diazotation and gas chromatography‐mass spectrometry. In the screening of faeces over 1 year about 70% of the EIA positive samples were confirmed as positive.     

Preparation and characterization of a monoclonal antibody specific for the β‐agonist clenbuterol

A monoclonal antibody was obtained from a Balb/c mouse immunized with a conjugate prepared by the direct coupling of diazotized clenbuterol to bovine serum albumin. The antibody, characterized by γ₁,κ isotype and an affinity constant of 3.5 × 10⁸ l mol^‐1, is highly specific as its cross‐reactivity to structurally related molecules is less than 1%. A competitive ELISA for the detection of clenbuterol in horse and human urine was developed using this antibody.     

A Mediated Glucose Biosensor Incorporated with Reverse Iontophoresis Function for Noninvasive Glucose Monitoring

The objective of this study was to develop a ferrocene mediated glucose biosensor for reverse iontophoresis. An amperometric ferrocene mediated glucose biosensor based on a three electrodes planar configuration was constructed using screen printing technique. Different combinations of glucose oxidase and ferrocene loading were drop coated onto the surface of the amperometric transducer. The amperometric transducer was characterized electrochemically using cyclic voltammetry and its electrochemical characteristics (ΔE _p = 70 mV, I _pa/I _pc = 0.89) were found close to an ideal amperometric transducer. The biosensor on the detection of glucose at 200 mV (vs. Ag/AgCl) showed a linear response range (0–4 mM). The response time of the biosensor was about 10 s. Finally, the biosensor was used together with reverse iontophoresis technique. By the use of an actual model for evaluation, an excellent linear relationship (r ² = 0.99) was found between the glucose concentration of the actual model and the biosensor current response. In conclusion, a ferrocene mediated glucose biosensor incorporated with reverse iontophoresis function was developed.     

Preparation of anti-melamine antibody and development of an indirect chemiluminescent competitive ELISA for melamine detection in milk

An indirect chemiluminescent competitive ELISA (CL-ELISA) method was developed to detect residues of melamine in milk. A high-quality polyclonal antibody towards melamine cyanurate (MC) was prepared based on synthesis of a novel immunogen. The method is applicable over the range of 0.5–7.0 µg.mL^?1 of MC, with an IC₅₀ value of 1.7 µg.mL^?1. The developed method was used in the detection of melamine residue in milk with the detection limit of 1 ng.mL^?1. There was no cross-reactivity with commonly used veterinary drugs. The CL-ELISA method developed provides an alternative to chromatography spectrometry for regulatory analysis of melamine in milk and could be promisingly used to improve the sensitivity of the available ELISA test kits.     

Detection of urine and blood clenbuterol following short-term oral administration in the horse

Background and aim: The pharmacokinetics of clenbuterol in equine urine and blood was investigated.

Material and methods: Urine and blood samples were collected following 3-day multiple oral administrations. The samples were examined using enzyme-linked immunosorbent assay and further confirmed by solid phase extraction and capillary electrophoresis.

Results: Urinary clenbuterol was detectable until day 14 after the last dose. The urinary excretion of clenbuterol was characterized by a biphasic pattern. The half-lives of the bi-exponential elimination (t_1/2α and t_1/2β) for urinary clenbuterol were about 12.1 and 48?hours. After a single oral administration (4 μg/kg) of clenbuterol, the half-life of serum clenbuterol was approximately 11.4?hours.     

Development of enzyme-linked immunosorbent assay for rapid determination of sildenafil in adulterated functional foods

To determine the sildenafil in adulterated functional foods, a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) procedure employing a polyclonal antibody generated from sildenafil-KLH was established. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC₅₀ of 6±0.1 ng/mL and the limit of detection was 0.6±0.02 ng/mL. The matrix effect of the functional food samples was easily eliminated by using methanol and distilled water for extraction and dilution with PBS, without any clean-up procedure such as solid phase extraction or liquid–liquid extraction, much more simple and rapid than the other reports. For the validation of the established SIL–ELISA, the functional food sample spiked at three different concentrations, analysed by HPLC. The results showed good correlation between the data of ELISA and HPLC (r ²=0.9832). It approved that HPLC methods show the same results with ELISA, and the developed immunoassay method is considered reliable for detection of sildenafil.     

New 16-plex PCR method for rapid detection of diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> directly from stool samples

A rapid 16-plex polymerase chain reaction (PCR) suitable for routine diagnostics of diarrheagenic Escherichia coli (EHEC, EIEC, EAEC, ETEC, and EPEC) was developed, validated with control strains, and tested with 250 diarrhoeal stool samples. The specificity was 100% when tested with 289 control bacterial strains, and the analytical sensitivity of automated DNA extraction directly from stool samples was made by boiling the bacterial culture (10⁴–10⁵ colony forming units/ml). The assay design starting directly from extraction of stool DNA allowed same day analysis without compromising sensitivity and specificity, which makes it superior compared to PCR after culturing the bacteria. The 16-plex PCR method demonstrated high prevalence of diarrheagenic E. coli in stool samples of patients returning from abroad (39.0%) in contrast to the patients with no travel history (8.7%; p < 0.001). The high prevalence of diarrheagenic E. coli suggests that their screening should be part of normal diarrhoea diagnostics, at least in the leading diagnostic laboratories.     

Optical biosensors with an integrated Mach-Zehnder Interferometer for detection of Listeria monocytogenes

In this work, we have demonstrated an efficient optical immunoassay technique for the detection of a food-borne pathogen, Listeria monocytogenes, using a Mach-Zehnder Interferometer (MZI) configuration. We have investigated ten different MZI configurations with angular and Sbend Y-junction geometries. An efficient Hydrofluoric acid (HF) based technique was used for rapid and specific binding of L. monocytogenes to the sensor arm of the MZI biosensor. The MZI biosensor was able to detect L. monocytogenes at concentrations of the order of 10⁵ CFU/ml, which is lower than the infection dose for healthy human beings. SEM analysis and light intensity measurements showed the biosensor is highly selective to L. monocytogenes over other microbial species (such as Escherichia coli). Finally, a novel calibration scheme of the MZI biosensor was developed from experimental data that can be used for determining unknown concentrations of L. monocytogenes.     

A competitive direct enzyme-linked immunosorbent assay for the rapid detection of deoxynivalenol: development and application in agricultural products and feedstuff

A competitive direct enzyme-linked immunosorbent assay (cd-ELISA) was developed for the rapid detection of deoxynivalenol (DON) in food and feedstuff. Polyclonal antibodies against DON were generated by immunizing rabbits with 3-HS-DON-BSA conjugates. In this assay, the sensitivity (measured as IC₅₀) and limit of detection (measured as IC₁₅) were 0.03 and 0.003?mg?kg^?1, respectively. A total of eight sample types (barley, wheat, oat, maize, rice, flour, milk and feedstuff) were chosen to evaluate the cd-ELISA assay performance. The sample could be directly detected after extraction and dilution with double-distilled water. The limit of detection in the samples was in the range of 0.15–0.48?mg?kg^?1. In the end, the spike and recovery results in both cd-ELISA and high-performance liquid chromatography (HPLC) were compared for assay validation. The recovery results ranged between 70% and 100%. A good correlation (R²?=?0.9613) between ELISA and HPLC was obtained.     

Localized surface plasmon resonance biosensor integrated with microfluidic chip

A sensitive and low-cost microfluidic integrated biosensor is developed based on the localized surface plasmon resonance (LSPR) properties of gold nanoparticles, which allows label-free monitoring of biomolecular interactions in real-time. A novel quadrant detection scheme is introduced which continuously measures the change of the light transmitted through the nanoparticle-coated sensor surface. Using a green light emitting diode (LED) as a light source in combination with the quadrant detection scheme, a resolution of 10⁻⁴ in refractive index units (RIU) is determined. This performance is comparable to conventional LSPR-based biosensors. The biological sensing is demonstrated using an antigen/antibody (biotin/anti-biotin) system with an optimized gold nanoparticle film. The immobilization of biotin on a thiol-based self-assembled monolayer (SAM) and the subsequent affinity binding of anti-biotin are quantitatively detected by the microfluidic integrated biosensor and a detection limit of 270 ng/mL of anti-biotin was achieved. The microfluidic chip is capable of transporting a precise amount of biological samples to the detection areas to achieve highly sensitive and specific biosensing with decreased reaction time and less reagent consumption. The obtained results are compared with those measured by a surface plasmon resonance (SPR)-based Biacore system for the same binding event. This study demonstrates the feasibility of the integration of LSPR-based biosensing with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments.     

Highly sensitive capacitive biosensor for detecting white spot syndrome virus in shrimp pond water

Water is one major pathways by which the white spot syndrome virus (WSSV) pathogen enters aquaculture facilities. This paper describes the production and use of a capacitive biosensor for the quantitative detection of as little as 1 copy/μl of WSSV in shrimp pond water. A glutathione-S-transferase tag for white spot binding protein (GST-WBP) was immobilized on a gold electrode through a self-assembled monolayer. Binding between WSSV and the immobilized GST-WBP was directly detected by a capacitance measurement. Under optimum conditions, the capacitive biosensor detected WSSV over a wide linear range of between 1 and 1 × 10⁵ copies/μl. The system was highly selective for WSSV. One analysis cycle required only 20-25 min of analysis time and 25 min of regeneration time. The capacitive biosensor was applied to analyze WSSV concentration in eight shrimp pond water samples and the results were in good agreement with those obtained by a real time quantitative polymerase chain reaction (real-time PCR) method (P > 0.05). The immobilized GST-WBP provided and could be reused for up to 39 analysis cycles for one electrode preparation with a relative standard deviation (RSD) of 2.4% and a good reproducibility of residual activity (95.8 ± 2.3%). The appealing performance of this biosensor indicated that it had great potential for an accurate very sensitive, quantitative, detection method for WSSV.     

Therapeutic clenbuterol treatment does not alter Ca2+ sensitivity of permeabilized fast muscle fibres from exercise trained or untrained horses

Clenbuterol is a β₂-adrenoceptor agonist primarily used for treating bronchospasm and alleviating the symptoms of chronic obstructive pulmonary disease (COPD) in the horse. In other species (rats, mice, sheep, and cattle), chronic high doses of clenbuterol (typically in the milligram per kilogram body weight range) has been shown to cause a muscle directed protein anabolic response. Clenbuterol can also modify muscle fibre composition and therefore potentially affect muscle function. This has implications for the performance of exercising horses being treated with therapeutic doses of clenbuterol (typically in the microgram per kilogram body weight range) for bronchospasm or COPD. It is not known whether clenbuterol treatment affects muscle fibre function in horses. The purpose of this study was to examine the effects of a therapeutic dose of clenbuterol, with and without exercise, on the contractile activation characteristics of single membrane permeabilized fibres prepared from muscle biopsies. We tested the hypothesis that therapeutic treatment with clenbuterol would not affect muscle fibre function. Unfit Standardbred mares were treated for 8 weeks with; clenbuterol (2.4 μg/kg twice/day, 5 days/week) plus exercise (20 min at 50% O_2max 3 d/wk; CLENEX), clenbuterol only (CLEN), or exercise only (EX). Muscle biopsies were taken from the gluteus medius muscle before and after treatment and stored in a glycerol-based solution to prepare permeabilized muscle fibres. The force–pCa relationship for fibres from CLEN horses was steeper (P < 0.05) indicative of greater cooperative interactions within the thin filament, however, fibre sensitivity to Ca²⁺ was unchanged. In contrast, the steepness of the force–pCa relationship was not changed in fibres from EX and CLENEX horses and Ca²⁺ sensitivity was also unaffected. Rigor force, activation in the absence of ATP, was not affected by any treatment indicating an approximately equivalent number of participating cross-bridges during activation. The results indicate that a therapeutic dose of clenbuterol to Standardbred horses does not affect the Ca²⁺-activated contractile characteristics of isolated muscle fibres. This revised version was published online in July 2006 with corrections to the Cover Date.