Detection of streptomycin and dihydrostreptomycin residues in milk, honey and meat samples using an optical biosensor.

Immuno-biosensor inhibition assays for the detection of streptomycin and dihydrostreptomycin residues in whole cows' milk, honey, pig kidney and pig muscle are reported. The antibody showed high cross-reactivity with dihydrostreptomycin in various foodstuffs (buffer 103%, milk 96%, honey 84%, kidney extract 129% and muscle extract 98%). There was no significant cross-reaction with other aminoglycosides or commonly used antibiotics. A streptomycin derivative was used to prepare a stable, reusable sensor chip surface. The assay allowed the direct analysis of bovine whole milk (fat content approximately 3.5%). Honey samples required dilution with buffer, while kidney and muscle samples from pigs were homogenized in an aqueous extraction buffer and clarified by centrifugation. The limit of detection for each assay was determined from known streptomycin-free samples (n = 20; mean - (3 x standard deviation)) and the results were as follows: milk 30 microg kg(-1), honey 15 microg kg(-1), kidney 50 microg kg(-1) and muscle 70 microg kg(-1). Repeatability (or relative standard deviation) between runs were calculated (n = 3) at the respective Community maximum residue limits (MRL) and 0.5 x MRL with the exception of honey since no European MRL exists at present. Results were determined as 4.3% (200 microg kg(-1)) and 2.8% (100 microg kg(-1)) in milk, 13.3% (40 microg kg(-1)) and 9.5% (20 microg kg(-1)) in honey, 7.1% (1000 microg kg(-1)) and 7.6% (500 microg kg(-1)) in kidney and 7.1% (500 microg kg(-1)) and 11% (250 microg kg(-1)) in muscle.

Determination of clenbuterol residues in bovine urine by optical immunobiosensor assay.

Clenbuterol (CBL) is an orally active beta2-adrenoceptor agonist which has been used in veterinary medicine as a broncodilator and an agent of uterine relaxation. It has however become better known as a drug used illegally to promote growth in farm animals. A rapid and sensitive biosensor assay was developed to detect CBL residues in bovine urine. The method involved a simple extraction procedure using tert-butyl methyl ether followed by analysis on the biosensor with results obtained against a buffer calibration curve. The assay allowed up to 88 samples to be analyzed per working day, with each cycle on the biosensor taking approximately 7 min to complete. The limit of detection (LOD) was determined as 0.27 ng/mL using 20 EU reference blank urine samples. The intra-assay Sr ranged from 4.7-7.6% for 3 control samples while the interassay Sr ranged from 9.2-12.7%. The recovery was found to be approximately 95%. A series of incurred urine samples were assayed and the results compared by Enzyme immunoassay (EIA), radio-immunoassay (RIA), and gas chromatography/mass spectrometry (GC/MS) analysis. Urine samples taken from local abattoirs were also analyzed by the biosensor method and by EIA analysis. The antibody used in the biosensor test exhibited high cross reactivity with at least 7 other beta-agonists allowing detection of these compounds at less than 1 ng/mL in bovine urine.

Detection of streptomycin residues in whole milk using an optical immunobiosensor.

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples ( approximately 3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).

Biosensor assay of sulfadiazine and sulfamethazine residues in pork.

Biosensor-based immunochemical screening assays for the detection of sulfadiazine (SDZ) and sulfamethazine (SMT) in muscle extract from pigs were developed. Samples were extracted with aqueous buffer and then centrifuged. This simple and straightforward preparation allowed up to 40 samples to be processed and analysed in 1 d. The limits of detection for the assays were found to be 5.6 ng g-1 for SDZ and 7.4 ng g-1 for SMT. These figures were well below the European and US legal limits for sulfonamides (100 ng g-1). The precision (RSD) between runs was < 8% and the recovery was between 91 and 98%. The validation proved the assays to be accurate and the analysis of routine field samples showed good correlation with an established TLC screening procedure. No false negative or positive results were obtained with blank and spiked samples. The influence of cross-reacting metabolites on immunoassays was demonstrated by testing incurred tissue samples, collected from sulfonamide treated pigs after only a short withdrawal period. The quantitative results obtained by biosensor analysis were a combination of parent sulfonamide plus N4-acetyl metabolite while the HPLC method used for confirmatory analysis detected only the parent sulfonamide. This gave rise to some false positive results and highlighted the need to use real samples in evaluating any assay thoroughly. False negative results were not obtained.

Evaluation of an immunobiosensor for the on-site testing of veterinary drug residues at an abattoir. Screening for sulfamethazine in pigs.

A study was conducted to determine the feasibility of performing "on-site" screening for sulfamethazine (SMT), at an abattoir, using a rapid immunobiosensor method. This involved transfer of the biosensor technology and an assay developed in the laboratory, to the cold, humid conditions of a modern pig-processing factory. A pre-determined threshold limit of 0.4 microgram ml-1 SMT in bile was used to identify the likelihood that corresponding tissue samples contained SMT concentrations in excess of the European maximum permissible residue limit of 0.1 mg kg-1. Bile samples containing SMT concentrations above the threshold limit were deemed positive and the corresponding kidney and muscle samples were sent to the laboratory for HPLC analysis. The robustness of the biosensor instrumentation in the harsh operating conditions was monitored throughout the project. The performance of the assay, on-site, was assessed by the regular inclusion of QA samples and by the submission of control 'SMT-positive' pigs to the abattoir. Sampling procedures, identification and traceability were also under scrutiny. During the project, 337 (9.35%) of the total kill were tested for SMT residues, representing 75% of all producers submitting pigs for slaughter. Twelve animals, including the ten controls, gave positive bile results. HPLC analysis confirmed SMT residues in all 12 kidneys (11 in excess of the permissible level). Ten muscle samples also contained violative SMT levels. Throughout the project, the biosensor performed reliably, with no adverse reaction of any mechanical or electrical components. The SMT assay also performed reliably. This is the first report of a biosensor being used for 'on-site' drug screening.

Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist.

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists). This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples. The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELSA results. Four calves were orally treated with RCT (0.1 mg kg-1 body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli). High concentrations of RCT residues were found throughout the medication period (44-473 ng ml-1; LC-MS-MS data) and remained present for several days following removal of the drug from the diet. RCT residues were no longer detectable 2 weeks after withdrawal. Good agreement (r2 = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for sets of analyses. The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal. However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.

Use of biosensors for rapid drug residue analysis without sample deconjugation or clean-up: a possible way forward.

The drug salbutamol (SBL) is a beta-agonist that may be used illegally as an animal growth promoter. SBL is also a good example of a drug which is excreted in the form of glucuronides and sulfates. Such metabolites cause complexities in analysing for the presence of drug residues. In the majority of cases a process of deconjugation and sample clean-up is required prior to analysis. This is both time consuming and causes some loss of accuracy. In this study, the urine of calves treated with SBL orally for 3 d was collected during and after medication. Samples were assayed before and after hydrolysis by two different methods, radioimmunoassay (RIA) and a newly developed biosensor immunoassay (BIA). Some samples were also analysed by GC-MS. The results clearly showed that both screening assays (RIA and BIA) found high concentrations of SBL residues throughout the study. This was especially true in the BIA method. It was also demonstrated that urine sample analysis without the need for deconjugation or clean-up could be achieved. Results obtained by GC-MS tended to be an order of magnitude lower than the corresponding screening test results. This work showed that biosensor based veterinary drug residue testing procedures can be developed which can generate results in real time without the need for time consuming sample preparation.

Immunobiosensor--an alternative to enzyme immunoassay screening for residues of two sulfonamides in pigs.

A rapid immunoassay using an optical biosensor (BIAcore) for determining the presence of sulfamethazine (SMT) residues in pig bile was developed. The assay was used in a routine screening laboratory alongside a previously described biosensor method for sulfadiazine (SDZ). Sulfonamide bile concentrations, determined by enzyme immunoassay (EIA), have already been shown suitable for use in predicting the extent of sulfonamide accumulation in kidney. The ability of immunobiosensor based bile screening to predict violative tissue residues (greater than the maximum residue limit; MRL) was compared with results achieved using two conventional EIAs for two of these drug residues (SMT and SDZ). Analysis of 2081 samples for both sulphonamide residues, over an 8 month period, showed the false positive prediction rate of biosensor analysis to be 0.14% and 0.34% for SMT and SDZ, respectively, compared with false positive rates of 1.54% and 1.44% by EIA. Biosensor analysis showed no false negative predictions for either SMT or SDZ while EIA showed a false negative prediction rate of 0.14% for SMT and 0.24% for SDZ. The present study has clearly demonstrated that immunobiosensor assays can be developed for veterinary drug residue screening programmes. These methods have the potential for generating faster and more reliable results than conventional immunoassay methods.

A multicenter comparison of carvedilol with hydrochlorothiazide in the treatment of mild-to-moderate essential hypertension.

The efficacy and safety of carvedilol, a beta-blocker with vasodilatory properties, were compared with that of hydrochlorothiazide (HCTZ) both at a once-daily dose of 25-50 mg in a double-blind, randomized, parallel-group, multicenter study. Following a single-blind placebo phase of 3-6 weeks, 201 eligible patients (aged 27-88 years) were randomized to receive 8 weeks of treatment with either carvedilol or HCTZ, 25 mg doubling to 50 mg at week 4 if the respone was inadequate. Sitting and standing blood pressure and heart rate were recorded 2 h after the first dose and 24 h postdose at weeks 4 and 8. The analysis included 179 patients (11 having withdrawn, including 5 for adverse events, and 11 excluded for protocol violations). There were no statistically or clinically significant differences between treatment groups. Eighty-six percent of patients in the carvedilol group and 88% in the HCTZ group had an 8-week sitting diastolic blood pressure less than or equal to 90 mm Hg or decreased by greater than or equal to 10 mm Hg. Safety profiles were similar for both agents, with a tendency to lower uric acid and total cholesterol levels with carvedilol. Carvedilol and HCTZ at doses compared in this study have similar efficacy and tolerability, with laboratory evidence to suggest a more favorable metabolic profile for carvedilol.

Performance of children under a multiple random-ratio random-interval schedule of reinforcement.

Three children, aged 1.5, 2.5, and 4.5 years, pressed telegraph keys under a two-component multiple random-ratio random-interval schedule of reinforcement. In the first condition, responses on the left key were reinforced under a random-interval schedule and responses on the right key were reinforced under a random-ratio schedule. In the second condition, the schedule components were reversed. In the third condition, the original arrangement was reinstated. For all subjects, rates of responding were higher in the random-ratio component despite higher rates of reinforcement in the random-interval component. The average interreinforcement interval of the random-interval component was increased in the fourth condition, resulting in more similar rates of reinforcement for both schedule components, and then returned to its original value in the fifth condition. In both conditions, all subjects continued to exhibit higher rates of responding in the ratio component than in the interval component. Although these observations are consistent with results from studies with pigeons, it is argued that the response-rate differences between the interval and ratio schedule components are sufficient to demonstrate schedule sensitivity.