Distribution and depletion of flubendazole and its metabolites in edible tissues of guinea fowl.

1. We measured the distribution and depletion of residues of flubendazole and its major metabolites in breast muscle, thigh muscle and liver of guinea fowls during and after oral administration of the veterinary medicine Flubenol 5% at two doses. 2. The guinea fowls were treated orally with normal feed, medicated at doses of 56 and 86 mg per kg feed for 7 successive days. Afterwards, depletion was observed for 8 d. Just before slaughter, body weights were measured. Thigh muscle, breast muscle and liver of three female and three male birds were sampled. The concentrations of the flubendazole-derived residues were determined by a liquid chromatographic-mass spectrometric method. 3. The highest residue concentrations were obtained for the reduced metabolite. With the therapeutic dose, the maximum mean residue concentrations obtained for this compound in thigh muscle, breast muscle and liver were 312, 288 and 1043 microg/kg, respectively. The values for flubendazole, the parent molecule, were 114, 108 and 108 microg/kg, respectively. The residues of the hydrolysed metabolite were negligible in the sampled muscle tissues. After 24 h of depletion, the sum of the residues of parent and metabolites in muscle tissue still exceeded 50 microg/kg. After 8 d of depletion, flubendazole-derived residues at low concentrations could still be measured in both muscle tissues and liver. Generally, the disposition of residues in breast and thigh muscle was comparable. 4. The European Union has not established a maximum residue limit (MRL) for flubendazole in edible tissues of guinea fowl. In contrast, the existing MRLs for other bird species are expressed as the sum of parent flubendazole and its hydrolysed metabolites. An estimated withdrawal period of three days will assure residue safety in the edible tissues of guinea fowl treated with flubendazole at therapeutic dose. After this withdrawal period following treatment of the guinea fowl, the residues were approximately constant, very low and far below the established safe MRL level for other bird species.

Determination of flubendazole and its metabolites in eggs and poultry muscle with liquid chromatography-tandem mass spectrometry.

The optimization of a quantitative and sensitive LC-MS/MS method to determine flubendazole and its hydrolyzed and reduced metabolites in eggs and poultry muscle is described. The benzimidazole components were extracted from the two matrices with ethyl acetate after the sample mixtures had been made alkaline. The HPLC separation was performed on an RP C-18 column with gradient elution, using ammonium acetate and acetonitrile as mobile phase. The analytes were detected after atmospheric pressure electrospray ionization on a tandem quadrupole mass spectrometer in MS/MS mode. The components were measured by the MS/MS transition of the molecular ion to the most abundant daughter ion. The overall extraction recovery values for flubendazole, the hydrolyzed metabolite, and the reduced metabolite in eggs (fortification levels of 200, 400, and 800 microg kg(-1)) and muscle (fortification levels of 25, 50, and 100 microg kg(-1)) were, respectively, 77, 78, and 80% and 92, 95, and 90%. The trueness (fortification levels of 400 and 50 microg kg(-1), respectively, for eggs and muscle), expressed as a percentage of the added values for these analytes, was, respectively, 89, 100, and 86 and 110, 110, and 98%. The proposed MS detection method operating in the MS/MS mode is very selective and very sensitive. The limits of detection for flubendazole and its hydrolyzed and reduced metabolites in egg and muscle were, respectively, 0.19, 0.29, and 1.14 microg kg(-1) and 0.14, 0.75, and 0.31 microg kg(-1). The limits of quantification were, respectively, 1, 1, and 2 microg kg(-1) and 1, 1, and 1 microg kg(-1). The discussed method was applied to a pharmacokinetic study with turkeys. Residue concentrations in breast and thigh muscle of turkeys orally treated with flubendazole were quantified. Medicated feed containing 19.9 and 29.6 mg kg(-1) flubendazole was provided to the turkeys for seven consecutive days. For the trial with the recommended dose of 19.9 mg kg(-1), one day after the end of the treatment, the mean sum of the flubendazole plus hydrolyzed metabolite residue values in thigh and breast muscle declined to below the maximum residue limit (50 microg kg(-1)) and were, respectively, 36.6 and 54.1 microg kg(-1). The corresponding values with the higher dose of 29.6 mg kg(-1) were, respectively, 101.7 and 119.7 microg kg(-1).

Confirmatory assay for the simultaneous detection of penicillins and cephalosporins in milk using liquid chromatography/tandem mass spectrometry.

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection of residues of penicillins and cephalosporins in milk has been developed. After a simple extraction with acetonitrile, the extract was directly injected into the LC/MS/MS system on a C(18) column. A gradient consisting of acetonitrile and water, each containing 0.1% formic acid, was applied. The abundant parent ions [M + H](+) produced by positive electrospray ionisation were selected for fragmentation with argon. For each compound at least one fragment was recorded with multiple reaction monitoring. The limits of detection ranged from 1.5 to 25 microg/kg and the limits of quantification ranged from 4 to 50 microg/kg. Recoveries were examined at three levels (MRL, 0.5 x MRL, 2 x MRL) and ranged from 57 to 88%. The coefficients of variation obtained for the repeatability experiments were in agreement with those specified by the Horwitz equation. Linearity was checked by injecting extracts of samples spiked with increasing amounts of the different standards ranging from 0 to 150 microg/kg. The advantage of this method over existing methods is the very simple sample pre-treatment which makes the method very suitable for routine analysis.

Validation of HPLC method of analysis of tetracycline residues in eggs and broiler meat and its application to a feeding trial.

HPLC with ion-pairing chromatography and diodearray detection at 355 nm was used to determine tetracycline antibiotics in eggs and broiler meat. The analytical methods were optimized and validated. The mean recovery values for oxytetracycline for eggs and for tetracycline for breast meat were 76%. The within-day precision ranged from 8.0 to 11.8% for oxytetracycline in eggs and from 6.1 to 15.5% for tetracycline in breast meat. The between-day precision was 4.8% and 5.0% respectively for oxytetracycline in eggs and tetracycline in breast meat. The limit of detection and the limit of quantitation for oxytetracycline in eggs were 2.2 and 13.0 ng/g respectively. These limits for tetracycline in breast meat were 10.5 and 20.9 ng/g respectively. Residue values of tetracycline antibiotics in eggs and broiler meat were determined after oral administration of medicated feed. Medicated feed with 840 mg/kg oxytetracycline was provided to laying hens for seven successive days. Two days after the administration was stopped, the mean oxytetracycline residue value in the eggs was already lower than the Maximum Residue Limit (MRL)-level and reached 118 ng/g. Broilers were supplied with medicated feed containing 480 mg/kg tetracycline for seven successive days. Four days after the administration was stopped, the mean tetracycline residue value in breast meat decreased below the MRL and was 86 ng/g.

Selenium content of sheep's milk and its distribution in protein fractions.

The selenium (Se) distribution in sheep's milk is reported. Skim milk was found to contain the major part (98%) of total milk Se. The Se distribution over casein and whey protein fractions depends on the separation method used, but irrespective of these methods, skim milk Se is mainly associated with the casein fraction (> 68%). Approximately 11%, 4% and 17% Se is removed by dialysis (molecular mass cut-off 10-12 kDa) from skim milk, casein and whey respectively, indicating a major association of Se with milk proteins. This observation is confirmed through Se analysis of individual caseins and whey proteins isolated through ion-exchange chromatography and gel filtration. Se concentrations of the different isolated milk proteins show considerable variations (caseins, 304-750 ng Se/g; whey proteins, 241-806 ng Se/g).

Selenium content of goat milk and its distribution in protein fractions.

This study reports on selenium distribution in goat milk. Skim milk was found to contain the major part (94%) of total milk selenium. The selenium distribution over casein and whey protein fractions depends on the separation method used, but irrespective of these methods, skim milk selenium is mainly associated with the casein fraction (greater than 69%). Approximately 9%, 7% and 24% of selenium is removed by dialysis (molecular cutoff 10-12 kDa) from skim milk, casein and whey respectively, indicating a major association of selenium with milk proteins. This observation is confirmed by selenium analysis of individual caseins and whey proteins isolated through ion-exchange chromatography and gel filtration. Selenium concentrations of the different isolated milk proteins show considerable variation (caseins: 294-550 ng Se/g; whey proteins: 217-457 ng Se/g).

Deformation of the dog aortic valve ring during the cardiac cycle.

Changes in strain in the line of aortic valve leaflet attachment (aortic ring) were measured during the cardiac cycle by means of an inductive technique. To that purpose coils were sutured to each commissure and base point of the aortic ring, when the animals were on a cardiopulmonary bypass. After bypass and stabilization of the hemodynamic variables changes in the aortic strain were measured at aortic pressures ranging from 4 to 20 kPa. Aortic pressure at the level of the commissure points and left ventricular pressure were measured to assess transvalvular pressure. Commissure strain appeared to depend on aortic and transvalvular pressure throughout the cardiac cycle. At an aortic pressure of 10 kPa (75 mm Hg), the derivative of commissure strain to aortic pressure was found to be 1.9 x 10(-5) +/- 1.2 x 10(-5) Pa-1 (mean +/- SD). During the ventricular ejection phase commissure strain was 0.04 +/- 0.03 higher than during ventricular filling. Maximum variations in basal strain during the cardiac cycle ranged from 0.03 to 0.15. During the ejection phase the basal segments adjoining the myocardium shortened whereas the segment close to the non-contracting anterior mitral valve leaflet lengthened. Strain between a base and a commissure point of the aortic ring were synchronous with the cardiac cycle, but no specific pattern could be found.

On-line measurement of aortic valve ring deformation during the cardiac cycle.

An electronic device is described for the measurement of relative changes in segment length within the aortic valve ring during the cardiac cycle. The technique is based on the principle of magnetic induction. A magnetic field generated in one coil induces a voltage in another coil. From the amplitude of this voltage the strain between both coils can be determined because the strength of the magnetic field decreases with distance. In vitro, over a range of 5-25 mm, strains less than or equal to 0.20 strain units can be measured with an accuracy of 0.008 strain units. The frequency response is 0-150 Hz (-3 dB). By varying the generator and receiver assignment at a frequency of 2 kHz and multiplexing the signals of six coils, six strains can be measured simultaneously. As an example, simultaneous recordings of commissure strains in the aortic valve and left ventricular and ascending aortic pressures, as obtained in open-chest dogs, are shown.

Determination of chloride concentration in cheese: collaborative study.

Three samples of ground Gouda cheese containing 1-2% chloride were analyzed by 7 laboratories by 3 methods: oxidation with KMnO4 and HNO3 followed by a Volhard titration; the same but with filtering off the precipitated AgC1 before back-titration; and the general potentiometric method without ashing or oxidation. The data were analyzed by ISO statistics (ISO-R 5725) and by AOAC statistics (Youden), the major differences being the rejection of different values as outliers and in the statement of the precision parameters. The within-laboratory variability (repeatability) is comparable for all 3 methods; the between-laboratory variability (reproducibility) is comparable for the Volhard method with filtration and the potentiometric methods, but the direct Volhard method is inferior. Because of its generality and simplicity, the potentiometric method has been adopted official first action; the Volhard method with filtration has been reinstated official final action as an alternative.

In vitro closing behaviour of Björk-Shiley, St Jude and Hancock heart valve prostheses in relation to the in vivo recorded aortic valve closure.

To compare prosthetic valve behaviour with that of the natural aortic valve, experiments were performed in vitro as well as in vivo. In a mock circulation system, cinematographic high-speed recordings of the valvular behaviour were made for Björk-Shiley, St Jude and Hancock heart valve prostheses. Simultaneously with the film recording, the aortic flow and the left ventricular and aortic pressures were measured. The closing behaviour of the natural aortic valve was recorded in in vivo experiments following the same measuring technique. Comparison of the film frames with the aortic flow signal revealed that the mechanical prostheses mainly close due to the back flow in the early phase of diastole; they close only for 5% of their cross-sectional area during systolic ejection. The Hancock bioprosthesis closes already for 45% during the flow deceleration phase of systole, which is however significantly less than the corresponding closure of the aortic valve as recorded in vivo (74%). The change in fluid viscosity from 3.10(-3) Ns/m2 to 1.10(-3) Ns/m2 does not affect the closing behaviour of the mechanical prostheses. Reducing the peak value of systolic aortic flow by a factor two decreases the cross-sectional area of the prosthetic valves at peak systole. When the natural aortic valve closing mechanism is applied to a disc-valve prosthesis, a partial (30%) systolic valve closure is found.

Identification of antibiotic residues in milk by thin-layer chromatography.

A scheme has been devised that makes it possible to separate and identify, by means of thin-layer chromatography, the 14 different antibiotic resudues in milk which are, besides penicillin, the most widely used in mastitis control: cloxacillin, dihydrostreptomycin, tetracycline, oxytetracycline, chlortetracycline, chloramphenicol, neomycin, novobiocin, bacitracin, erythromycin, oleandomycin, ampicillin, streptomycin and oxacillin. The limits of detectability of the antibiotics studied vary between 0.1 and 3 mug/ml, with the exception of neomycin the minimum detectable concentration of which is 15 mug/ml.