A potentiometric biosensor assay based on a commercially available polyclonal antibody was developed to detect tylosin residues in animal feed. The method can be used as a rapid (less than 45 min) laboratory-based procedure or as a portable field-test for the simultaneous measurement of up to 12 different samples. For both procedures the qualitative detection capability (CC beta) for tylosin was determined as 0.2 mg kg(-1) in a range of animal feeds with a measurement repeatability at concentrations between 0.2 and 4 mg kg(-1) of <= 13% coefficient of variation (%CV). The field-test format was capable of detecting tylosin residues at operating (external air) temperatures ranging between +4 and 37 degrees C, although some reduction in signal was observed at the lower temperatures. The laboratory-based tylosin assay was evaluated using 16 medicated and 22 non-medicated feeds and was found to give comparable data with a confirmatory method based upon liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential to develop a multi-probe format assay for the simultaneous detection of tylosin, spiramycin and virginiamycin was also demonstrated. Cross-validation in a second laboratory showed the assay to be transferable, reliable and robust.

A post-screening classification assay for tetracycline compounds has been developed and integrated into the previously reported optimized Premi®Test methodology. The new post-antimicrobial screening assay is based on a metal ion chelation using calcium and sodium chloride and has been shown to be specific towards the tetracycline class. The assay is both cost-effective and complementary to the post-screening procedures that have previously been developed for the β-lactam and sulfonamide compounds. A validation study was conducted in accordance with 2002/657/EC (Commission Decision). The method is rugged and applicable to a range of tetracyclines of differing antimicrobial potencies over a wide concentration range. A blind trial was undertaken in which all antimicrobial residues in the unknown samples were successfully identified by the analyst following the integrated Premi®Test procedure for the classification of antimicrobial compounds.

Two new microbial inhibition assays for the screening of antibacterial substances in milk have been validated in accordance with the ISO/IDF 183 international standard. The detection limits for the 10 antimicrobial substances (penicillin, ampicillin, amoxicillin, cephapirin, ceftiofur, cloxacillin, sulphadiazine, oxytetracycline, neomycin and erythromycin) tested by the Delvotest® SP-NT (sulphur-penicillin test no tablet) (ampoule and multi-plate formats) were found to be comparable with the European (EU) maximum residue limits. Sample measurement end-points were determined visually and by the Delvo®Scan system. The Delvo®Scan was effective at discriminating between matrix effects and a residue of an antimicrobial substance when present in milk at concentrations close to the lowest detected screening level. Both test formats were found to be robust and unaffected by a variety of procedural changes (e.g., sample incubation time, test volume). They were also found to be applicable for screening of milk with a range of fat contents and milk with raised somatic cell count.

Biacore Q biosensor and liquid chromatography–tandem mass spectrometry (LC–MS/MS) based methods for the screening and confirmation of trace levels of chloramphenicol (CAP) and the mammalian metabolite chloramphenicol glucuronide (CAP-Glu) is reported. Both methods employ solvent extraction and clean-up by solid phase extraction (SPE) prior to analysis. The biosensor screening method utilises surface plasmon resonance (SPR) to determine chloramphenicol concentration in a range of matrices including honey and prawns. As the antibody used in the biosensor has a high cross-reactivity with CAP-Glu, direct detection of this metabolite is possible in matrices such as porcine kidney. LC–MS/MS is used in negative ion electrospray mode for the confirmatory procedure. Parent CAP is determined via the use of an internal standard. In the case of porcine kidney parent CAP is released from CAP-Glu following a short digestion with a β-glucuronidase. All methods have been validated to the latest EU requirements (Commission Decision 2002/657/EC). The calculated decision limits (CCα) and detection capabilities (CCβ) are less than 0.1 and 0.2μgkg−1 respectively for the screening and confirmatory techniques. Incurred tissues were used to study and confirm the long-term reproducibility and agreement between methods. In addition, the stability of CAP and CAP-Glu has been investigated. The analytes are stable under nearly all storage conditions for at least 20 weeks.

In recent years there has been an increase in the use of tylosin in apiculture as bacterial brood diseases become resistant to oxytetracycline. Confirmatory mass spectrometry based methods have been developed but up until now there has been no complementary screening method available capable of sub 10 microg kg(-1) detection limits. In this paper the development and validation of a screening method using optical biosensor technology is presented. The honey was first dissolved in a phosphate buffer and following solid-phase extraction (SPE) cleanup was analyzed using a Biacore Q instrument. Using the criteria specified in European Commission Decision 2002/657/EC for qualitative screening methods, the detection capability (CCbeta) of the method was determined to be 2.5 microg kg(-)(1). Honey samples containing trace residue levels of tylosin were analyzed by both the biosensor screening method and a LC-MS/MS confirmatory procedure; the results were in good agreement.

A rapid, high-throughput antimicrobial screening assay has been developed that combines either a physical fluid extraction or a solvent extraction technique with the commercially available PremioTest.In order to remove the subjectivity of the visual end-point measurement associated with this microbial inhibition assay, work has been conducted to couple the Premi(R)Test to scanner technology. The use of the solvent extraction provides an enhanced detection capability for a wide range of drugs at or below one-half the maximum residue limit (MRL) concentrations in a variety of matrices, as demonstrated by dose response curve data. Secondary class-specific assays, for the identification of beta-lactams and sulphonamides following the primary screen have been previously developed and recently validated using the scanner technology.Method validation using both fortified and incurred tissues has been undertaken to establish the ruggedness of the technique. The false-positive and -negative rates have been established at less than 5% for a range of drug/matrix combinations.The CCbeta values determined for this qualitative screening assay are at concentrations less than the MRL. This integrated screening strategy provides a reliable tool for antimicrobial residue monitoring in surveillance programmes. (C) 2004 Elsevier B.V. All rights reserved.

Immunochemical screening assays using surface plasmon resonance have been developed for chloramphenicol and chloramphenicol glucuronide residues in poultry muscle, honey, prawn and cows’ milk using a sensor chip coated with a chloramphenicol derivative and an antibody. The antibody cross-reacted with chloramphenicol glucuronide 73.8% (poultry), 69.2% (honey), 75.7% (prawn) and 84.8% (milk). There was no cross-reaction with similar drugs or other commonly used antibiotics. The assay allowed the direct analysis of bovine milk (fat content ∼3.5%). Poultry, honey and prawn samples were extracted with ethyl acetate followed by analysis on the biosensor. The decision limits (CCα) for each assay were determined as: poultry (0.005μgkg−1), honey (0.02μgkg−1), prawn (0.04μgkg−1) and milk (0.04μgkg−1) and the detection capabilities (CCβ) were 0.02, 0.02, 0.07 and 0.05μgkg−1, respectively. Poultry muscle, honey and milk were spiked at 0.1μgkg−1 and prawn at 0.15μgkg−1 and the intra-assay precision (n=10) calculated as 10.5, 5.0, 4.6 and 8.8%, respectively. Between run precision (n=3) performed at the same levels yielded the following results: 3.0% (poultry), 4.7% (honey), 7.6% (milk) and 5.5% (prawn).

A rapid, high-throughput antimicrobial screening assay was developed using either a physical fluid extraction or a solvent extraction technique coupled to the commercially available PremiTest. The solvent extraction approach was fully validated for a wide range of tissues and the fluid extraction approach partially validated for porcine muscle. Both procedures can detect a wide range of antimicrobial compounds at or below maximum residue limit concentrations. The use of a solvent extraction provides an enhanced test capable of detecting a wider range of drugs than the fluid extraction approach at or below half maximum residue limit levels in a variety of matrices. Biochemical methods for the class-specific identification of beta-lactams and sulphonamides following initial screening were developed and validated. The approach is a significant improvement on existing methodologies as a tool for residues monitoring in surveillance programmes.

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10−3 M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10−4 and 10−3 M than their respective controls. Acidified saline solution (pH 4.8, as control for 10−3 M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (≥10−4 M), should be re-considered.

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.

Apoptosis (programmed cell death) of smooth muscle cells (SMC) in blood vessels is an essential process involved in the control of vessel wall structure. Several antihypertensive drugs currently used in therapy may exert their pharmacological effects by promoting SMC apoptosis. The biochemical events which regulate SMC apoptosis in the vessel wall are complex, and not well understood. We therefore investigated whether treatment of cultured SMC from normotensive Wistar-Kyoto rats (WKY) and from spontaneously hypertensive rats (SHR) with selected antihypertensive drugs would induce SMC apoptosis. We treated aortic SMC from WKY and SHR in vitro with the L-type Ca2+ channel antagonist, nifedipine; with the nitric oxide donor, sodium nitroprusside (SNAP); with forskolin (an activator of adenylyl cyclase); or with thapsigargin (a selective inhibitor of the sarcoplasmic reticulum (SR), Ca2+-ATPase); and compared their apoptosis-promoting effects in SMC derived from the two strains of rats. SMC were derived from the thoracic aorta of 3-4-week-old WKY and SHR, and were used in passages 7–10. Apoptotic cells were detected by in-situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labeling (TUNEL) method, and by morphological examination. We found that: 1) Treatment of cultured aortic SMC with the L-type Ca2+ channel antagonist, nifedipine (5 × 10−5 M) for 24 hours induced a significantly higher level of apoptosis in SHR cells than in SMC from WKY. Cells from WKY, following exposure to nifedipine for 72 hours, exhibited a similar response to the cells from SHR treated for 24 hours. This was detectable by both morphological criteria as well as DNA labeling by the TUNEL technique. 2) Similar treatment of these cells with thapsigargin (1 × 10−7 M) led to morphological alterations characteristic of apoptotic cells in SMC from both WKY and SHR, and cells from SHR but not WKY were labeled by the TUNEL technique at 24 hours. The TUNEL method did however identify cells from both WKY and SHR as apoptotic after 48 and 72 hours of treatment. 3) The addition of SNAP, or forskolin to the cultured SMC induced significant, but low levels of apoptosis in WKY SMC only. This selective apoptosis-promoting effect of nifedipine in SHR SMC may result from differences in the control of intracellular Ca2+ between the two strains of cells, or it may indicate that the signaling pathways which regulate apoptosis are different in SMC from the normotensive and the hypertensive rats. Our findings imply that SMC apoptosis may be a selective target for pharmacological intervention in hypertension.