Comprehensive stability-indicating high-performance liquid chromatography coupled with diode array detection method for simultaneous determination of amprolium hydrochloride and ethopabate in powder dosage form for veterinary use.

This research deals with the development of a stability-indicating high-performance liquid chromatography method for simultaneous determination of amprolium hydrochloride and ethopabate. To the best of our knowledge, no comprehensive stability-indicating method has been reported for analysis of this mixture. Separation was achieved using Kromasil cyano column with gradient elution of the mobile phase composed of sodium hexane sulfonate solution and methanol. Quantification was based on measuring peak areas at 266 nm. Amprolium and ethopabate peaks eluted at retention times 10.42 and 18.53 min, respectively. The proposed procedure was validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Linearity ranges for amprolium and ethopabate were 1.5-240 and 1-160 µg/mL, respectively. Analytes were subjected to stress conditions of hydrolysis, oxidation and thermal degradation. The proposed method enabled resolution of drugs from their forced-degradation products and amprolium related substance (2-picoline). Moreover, specificity was verified by resolution of the analytes from about 22 drugs used in antimicrobial veterinary products. The validated method was successfully applied to assay of the combined veterinary powder dosage form, additionally it was implemented in the accelerated stability study of the dosage form when stored for six months at 40°C and 75% relative humidity.

Five different spectrophotometric methods for determination of Amprolium hydrochloride and Ethopabate binary mixture.

Five simple, specific, accurate and precise UV-spectrophotometric methods are adopted for the simultaneous determination of Amprolium hydrochloride (AMP) and Ethopabate (ETH), a binary mixture with overlapping spectra, without preliminary separation. The first method is first derivative of the ratio spectra ((1)DD) for determination of AMP and ETH at 234.7nm and 306.8nm respectively with mean percentage recoveries 99.76±0.907 and 100.29±0.842 respectively. The second method is the mean centering of the ratio spectra for determination of AMP and ETH at 238.8nm and 313nm respectively with mean percentage recoveries 100.26±1.018 and 99.94±1.286 respectively. The third method is based on dual wavelength selection for determination of AMP and ETH at 235.3nm & 308nm and 244nm & 268.4nm respectively with mean percentage recoveries 99.30±1.097 and 100.03±1.065 respectively. The fourth method is ratio difference method for determination of AMP and ETH at 239nm & 310nm and 239nm & 313nm respectively with mean percentage recoveries 99.27±0.892 and 100.40±1.814 respectively. The fifth one is area under the curve (AUC) method where the areas between 235.6-243nm and 268.3-275nm are selected for determination of AMP and ETH with mean percentage recoveries 100.35±1.031 and 100.39±0.956 respectively. These methods are tested by analyzing synthetic mixtures of the two drugs and they are applied to their pharmaceutical veterinary preparation. Methods are validated according to the ICH guidelines and accuracy, precision and repeatability are found to be within the acceptable limit.

Spectrofluorimetric analysis of ethopabate in veterinary formulations with application to residue determination in chicken muscles and liver.

Ethopabate is a veterinary drug used in the prophylaxis and treatment of coccidiosis in chickens. The presence of drug residues in edible tissues can be dangerous to human consumers. It may cause direct toxic effects, allergic reactions and increased bacterial resistance. A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of ethopabate in its veterinary formulations. The proposed method is based on measuring the native fluorescence of ethopabate in water at 364 nm after excitation at 270 nm. The fluorescence-concentration plot was rectilinear over the range of 2-100 ng/mL, with a limit of detection of 2.9 ng/g and a limit of quantification of 9.8 ng/g for ethopabate. The method was successfully applied to the analysis of ethopabate in its commercial veterinary formulations and the results were in good agreement with those obtained with the reference method. The method was extended to the determination of ethopabate residues in chicken muscles and liver, and the results were satisfactory. The recoveries obtained were in the 108.36-113.42% range. No organic solvents are used in the procedure, so it can be considered a type of 'green' chemistry.

Validation of a high-performance liquid chromatographic method with UV detection for the determination of ethopabate residues in poultry liver.

Ethopabate is frequently used in the prophylaxis and treatment of coccidiosis in poultry. Residues of this drug in food present a potential risk to consumers. A simple, rapid, and sensitive column high-performance liquid chromatographic (HPLC) method with UV detection for determination of ethopabate in poultry liver is presented. The drug is extracted with acetonitrile. After evaporation, the residue is dissolved with an acetone-hexane mixture and cleaned up by solid-phase extraction using Florisil columns. The analyte is then eluted with methanol. LC analysis is carried out on a C18 5 microm Gemini column, 15 cm x 4.6 mm. Ethopabate is quantified by means of UV detection at 270 nm. Parameters such as decision limit, detection capability, precision, recovery, ruggedness, and measurement uncertainty were calculated according to method validation guidelines provided in 2002/657/EC and ISO/IEC 17025:2005. Decision limit and detection capability were determined to be 2 and 3 microg/kg, respectively. Average recoveries from poultry samples fortified with 10, 15, and 20 microg/kg levels of ethopabate were 100-105%. A complete statistical analysis was performed on the results obtained, including an estimation of the method uncertainty. The method is to be implemented into Brazil's residue monitoring and control program for ethopabate.

High performance liquid chromatographic assay of amprolium and ethopabate in chicken feed using solid-phase extraction.

A method for the assay of mixtures of amprolium and ethopabate in chicken feed was developed utilizing reversed-phase high-performance liquid chromatography (HPLC) after sample clean-up of a methanolic extract by solid-phase extraction using CN cartridges. HPLC was done with benzocaine as internal standard on a C-8 column with methanol-water 40:60, containing octanesulfonic acid, triethylamine and acetic acid, as mobile phase. Eluate was monitored at 274 nm. Baseline separation was achieved with retention times of approximately 7.5, 9.4, and 10.4 min, for amprolium, benzocaine, and ethopabate respectively. Feed constituents did not give peaks after 6.5 min. Peak area ratios were linear over 10-180 ng of amprolium, and 2-18 ng of ethopabate injected. Limits of quantitation at AUFS 0.05 were 0.5 and 0.3 ng respectively. Recovery studies from spiked feed (n = 9), covering +/- 30% of usual doses in feed, gave percent recoveries (+/- SD) of 99.4 +/- 1.4% for amprolium and 100.5 +/- 2.6% for ethopabate. Applying the method to two different batches of commercial feed gave results which were comparable to those obtained by the AOAC spectrofluorometric methods.

Sensitive determination of ethopabate residues in chicken tissues by liquid chromatography with fluorometric detection.

A liquid chromatographic (LC) method is described for determination of ethopabate residues in chicken tissues. The drug is extracted from tissues with acetonitrile, and the extract is concentrated to 2-3 mL. This aqueous solution is rinsed with ethyl acetate and cleaned up by Florisil column chromatography. LC analysis is carried out on a Zorbax ODS column, and ethopabate is quantitated by using a fluorometric detector set at 306 nm (excitation) and 350 nm (emission). Recoveries of ethopabate added to chicken tissues at levels of 0.01 and 0.05 ppm were 87.8 and 92.7%, respectively. The detection limit was 100 pg for ethopabate standard, and 0.5 ppb in chicken tissues.

Sample preparation of carbadox, furazolidone, nitrofurazone, and ethopabate in medicated feeds for high pressure liquid chromatography.

Medicated feeds (pelleted or mash) containing guarantees of carbadox, furazolidone, nitrofurazone, and ethopabate are pretreated with water, extracted with 95% dimethylformamide overnight at room temperature, cleaned up on a column of alumina, and injected into a high pressure liquid chromatograph for quantitative measurement. Carbadox, nitrofurazone, and furazolidone can be separated; chromatograms show excellent baseline resolution, and results are in good agreement with colorimetric methods. The same extraction and cleanup can be used to improve colorimetric methods for furazolidone and nitrofurazone.

Gas-liquid chromatographic determination of ethopabate in feed premixes.

A gas-liquid chromatographic method is presented for determining ethopabate in 0.8 and 8.0% premixes. A sample is extracted with tetrahydrofuran containing an internal standard, by sonication or overnight soaking. The extract is clarified by centrifugation, diluted if necessary, and injected into a gas chromatograph equipped with a flame ionization detector. Average per cent recoveries for spiked blank samples were 100.6 at the 0.8% level and 100.4 at the 8.0% level. Precision, as indicated by replicate analyses of several premixes, ranged from 0.5 to 1.7% relative standard deviation.

High pressure liquid chromatographic determination of ethopabate in feeds.

A high pressure liquid chromatographic (HPLC) method is described for determining ethopabate in poultry feeds. Feed samples containing ethopabate are finely ground, extracted 30 min by sonification with methanol-water (80 + 20), and filtered. The extract is cleaned up on an alumina column, and the first 4 ml eluate collected is analyzed. The drug is eluted through a muBondapak C18 column with acetonitrile-water (30 + 70) at a flow rate of 1.4 ml/min and detected by ultraviolet absorption at 280 nm. Chromatography is complete in 7 min, and detector response is linear. The drug is quantitated by peak height ratios. The procedure described is reproducible and shows good agreement with the official AOAC colorimetric method. The lower limit of detection is 2 ng by HPLC, making the method applicable to residue analyses.