Production and characterization of polyclonal antibodies to sulfamethazine and their potential use in immunoaffinity chromatography for urine sample pre-treatment

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.     

Screening and confirmation of thyreostatics in urine by gas chromatography with nitrogen-phosphorus detection and gas chromatography-mass spectrometry after sample clean-up with a mercurated affinity column

Methods are described for the screening and confirmation of residues of the thyreostatics thiouracil, methylthiouracil and propylthiouracil in urine samples of cattle at levels down to 25 micrograms/l. After a selective preconcentration of the thiol-containing thyreostatics on a mercurated affinity column, the analytes are derivatized by extractive alkylation and analysed by gas chromatography with nitrogen-phosphorus or mass spectrometric detection.     

Pour on application of growth promoters in veal calves: analytical and histological results.

To investigate the possibilities for screening and confirmation methods when the 'pour on' method of application is used for administration of growth promoters, an animal experiment was performed using a cocktail of a combination of growth promoters derived from (illegal) practice. Two cocktails were used, cocktail A consisting of stanozolol and estradiol benzoate and cocktail B consisting of stanozolol, estradiol benzoate and beclomethasone dipropionate. The intended dose per animal was 110 mg stanozolol, 25 mg estradiol and 10 mg beclomethasone. The experiment was performed on 20 male veal calves, 16 treated and 4 vehicle treated controls and 3 female veal calves, 2 treated and 1 vehicle treated control. Half of the animals were shaven prior to the application of the drugs. The cocktails were administered using two types of vehicles: vehicle A; Miglyol 840 with butylated hydroxytoluene and vehicle B; di(ethyleneglycol) monobutylether. During a 28 day treatment period, one group of animals was treated once a week, another group of animals was treated once every two weeks and slaughtered. Preliminary results showed that pour on application of anabolic steroids markedly increased growth performance of veal calves, the animals treated with cocktail A performed better than the animals treated with cocktail B. Macroscopically, the thymus was reduced in weight and size in the B animals. The bulbo-urethral glands were enlarged in all treated animals. Histologically all treated animals showed squamous metaplasia in the prostate, bulbo-urethral gland and Bartholins glands. Moreover, a changed secretion pattern was observed in both the prostate and the bulbo-urethral gland. Severe cortical atrophy was observed in the thymus and to a lesser extent the adrenals of the beclomethasone treated animals. The recently discovered 16 beta-hydroxy-metabolite of stanozolol was detected in urine, in relatively high concentrations. This is the first report of the excretion of this metabolite in urine after pour on administration showing the prospect for detection of dermal treatment. Estradiol levels were remarkably elevated (up to 200 micrograms l-1) exceeding the endogenous levels (< 1 microgram l-1).     

Prediction of anti and gauche vicinal proton-proton coupling constants in carbohydrates: A simple additivity rule for pyranose rings

The effect of the relative orientation and electronegativity of substituents on the magnitude of ³J(aa), ³J(ae) and ³J(ee) is well predicated by a simple set of additivity constants, valid for pyranose rings in carbohydrates. The proposed set of parameters is used to calculate 327 coupling constants [³J(HH)] in a variety of pyranosides and related compounds. A comparison with experimental values taken from the literature shows that couplings in molecules which are conformationally pure and underformed can be predicted with a surprising accuracy. An overall root-mean-square agreement of 0.29 Hz is attained for a selected group of 305 coupling values. A statistical breakdown of ΔJ(aa) and ΔJ(ae) [ΔJ=J(exp)-J(calc)] along each carbon-carbon bond in the pyranose systems reveals an unexpected degree of geometrical homogeneity.     

Rapid HPLC method for the determination of aflatoxin MI in milk at the ng/kg level

Summary A combination of HPLC with a quick extraction and clean-up procedure is described which is suitable for 100 ml of milk. Clean-up is performed on small silica gel columns. One quarter of the extract ( 20 ml milk) is used for reverse phase HPLC with a filter fluorescence detector. Every ten minutes a sample can be injected. Limit of determination is about 0.005 g/kg. At that level, with the remaining of the extract, a confirmation test can be performed with two dimensional thin-layer chromatography. Recovery of the procedure at the 0.05 g/kg level is about 75%.

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Schnelle Bestimmung von Aflatoxin M_I in Milch im ng/kg-Bereich durch HPLC

Zusammenfassung Eine Kombination von HPLC mit einem Extraktions- und Reinigungsverfahren wird beschrieben, die für Volumina von 100 ml Milch geeignet ist. Die Reinigung erfolgt an kleinen Silicagel-Säulen. Etwa ein Viertel des Extrakts wird für die reversed-phase-HPLC mit einem Filter-Fluorescenz-Detektor verwendet. Erfassungsgrenze ist etwa 0,005 g/kg. Alle 10 min kann eine Probe injiziert werden. Mit dem übrigen Extrakt können zur Bestätigung zweidimensionale Dünnschicht-Chromatogramme hergestellt werden. Im Bereich von 0,05 g/kg beträgt die Wiederfindung 75%.

     

Low-spin state structure of [Fe(chloroethyltetrazole)6](BF4)2 obtained from synchrotron powder diffraction data

The complex [Fe(teec)6](BF4)2 (teec = chloroethyltetrazole) shows a two-step complete spin-crossover transition in the temperature range 300-90 K. Time-resolved synchrotron powder diffraction experiments have been carried out in this temperature range, and crystal structure models have been obtained from the powder patterns by using the parallel tempering technique. Of these models, the low-spin state structure at 90 K has been refined completely with Rietveld refinement. Its structural characteristics are discussed in relation to the high-spin state model and other spin-crossover compounds. The complex shows a remarkable anisotropic unit-cell parameter contraction that is dependent on the applied cooling rate. In addition, the possible important implications for the interpretation of spin-crossover behavior in terms of structural changes are discussed.