Sensitive spectrofluorimetric methods for determination of ethopabate and amprolium hydrochloride in chicken plasma and their residues in food samples.

Two sensitive and selective spectrofluorimetric methods are proposed to determine ethopabate (ETH) and amprolium hydrochloride (AMP). First derivative synchronous spectrofluorimetry determines the natively fluorescent ethopabate at 288 nm in presence of amprolium hydrochloride which is a non fluorescent quaternary compound with average recovery 100.54±0.721 over a concentration range of 0.01-0.8 µg/mL. Limits of detection (LOD) and quantification (LOQ) are 0.002 and 0.007 µg/mL, respectively. The second method is direct synchronous spectrofluorimetry for determining amprolium hydrochloride at 362 nm after a reaction with 5% NaOH and 0.08% potassium ferricyanide that is optimized by a two-level factorial design. This method is linear over a concentration range of 0.01-0.65 µg/mL with average recovery 99.4±1.28. Limits of detection (LOD) and quantification (LOQ) are 0.002 and 0.006 µg/mL, respectively. The proposed methods are found to be valid and applicable for the analysis of ETH and AMP in their veterinary formulation. They are successfully applied to determine the studied drugs in chicken plasma and their residues in chicken muscle, liver, egg and chicken-based baby food product with recoveries in the ranges of 95.71-108.73% and 97.36-111.89% and for ETH and AMP, respectively.

Rapid and sensitive determination of amprolium in chicken plasma by high-performance liquid chromatography with post-column reaction.

A rapid, sensitive and reproducible reversed-phase HPLC assay was developed for the determination of amprolium (APL) in chicken plasma. Protein in plasma sample was precipitated with 0.33 M perchloric acid and supernatant solution was injected into the HPLC system. Following the chromatographic separation of APL and the beclotiamine (I.S.) on a C18 column, the derivatives of APL and I.S. were formed by post-column reaction and detected by fluorescence detection (excitation at 400 nm, emission at 460 nm). The method showed excellent precision, accuracy and speed with a detection limit of 2 ng/ml. The intra- and inter-assay variance of this method were less than 11.2%. This method has been successfully applied to plasma determinations after oral administration of APL to chicken.

Inhibition of thiamine transport across the blood-brain barrier in the rat by a chemical analogue of the vitamin.

The flux of thiamine from the blood into the brain has been measured using a specially devised technique by which a steady raised level of the vitamin, with or without radioactive labelling, can be achieved rapidly and maintained in the bloodstream. This is done by a continuous injection, given at a rate which is adjusted by a pre-determined programme so as to replace the tracer at the rate at which it has been found to leave the circulation in previous experiments. A further programme was worked out to maintain, in a similar manner by a separate injection, a steady raised level in the bloodstream of a chemical analogue of thiamine, 1-[(4-amino-2-propyl-5-pyrimidinyl)methyl]-2-picolinium chloride HCl (amprolium). In the presence of a high concentration of amprolium the flux of thiamine across the blood-brain barrier was greatly reduced and no longer saturable by raising the blood thiamine concentration up to at least 10 microM. It was concluded that this analogue of thiamine inhibited the saturable component of thiamine transport across the barrier but not the non-saturable component. In a further series of experiments, progressively higher levels of thiamine were maintained in the bloodstream and the influx of the vitamin across the blood-brain barrier was measured. From kinetic analysis of the results, it was clear that the affinity of amprolium for the transport carrier was of a similar magnitude to that of thiamine itself. That the inhibition was competitive was shown by the way in which it could be overcome if the level of thiamine in the blood plasma was raised sufficiently above the normal.