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  • Author: Ewelina Patyra x
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Ewelina Patyra and Krzysztof Kwiatek

Abstract

Introduction: The article presents a rapid and simple analytical procedure for determination of four tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) in animal medicated feedingstuffs. Material and Methods: Two-gramme samples were extracted by a Na2EDTA-McIlvaine buffer (pH 4)/methanol mixtures (40/60, v/v). The determination was achieved by liquid chromatography using a Zorbax Eclipse XDB C18 analytical column with mass spectrometer detection (LC-MS). Results: Recoveries of the antibiotics from spiked feed samples ranged from 78.2% to 113.5%. The LOD and LOQ for tetracyclines in feeds ranged from 2.8 to 4.2 and from 4.3 to 5.7 mg/kg, respectively. Conclusion: The method was successfully validated and proved to be efficient, precise, and useful for quantification of tetracyclines in medicated feedingstuffs.

Open access

Ewelina Patyra, Ewelina Kowalczyk and Krzysztof Kwiatek

Abstract

A chromatographic procedure for determination of oxytetracycline (OXT), tetracycline (TC), chlorotetracycline (CTC), and doxycycline (DC) in water samples was developed and was applied for the analysis of water samples collected from poultry and pig farms and environmental water samples. Samples were acidified with trifluoroacetetic acid to pH 3 and further purified by solid phase extraction using Oasis HLB cartridges. The samples were dried up and redissolved in the mixture of oxalic acid and methanol. Separation was performed on reserved phase column (Phenomenex column C18 , 250 mm × 4.6 mm, 5 μm) by multistep gradient elution, and detection was carried out at 360 nm for OTC and TC, 370 nm for CTC, and 350 nm for DC. The tetracyclines were eluted with the mobile phase of 0.05 M oxalic acid (pH 2.5), acetonitrile, and methanol. This method provided average recoveries of 83.53% to 108.59%, with coefficient of variations (CVs) of 2.41% to 8.64% in the range of 10 to 1000 μg/L OTC, TC, CTC, and DC in water. The linearity for the tetracyclines was determined by HPLC-DAD in the range 10 to 1000 μg/L, with the correlation coefficient (R) > 0.99. The LOD and LOQ for the tetracyclines in water samples ranged from 1.51 to 4.00 and 2.51 to 5.93 μg/L, respectively.

Open access

Ewelina Patyra, Ewelina Kowalczyk and Krzysztof Kwiatek

Abstract

High performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and optimised for the determination of tetracyclines (TCs) in medicated feedingstuffs. The extraction of the analyte from feedingstuffs was performed with Na2EDTA-McIlvaine buffer (pH 2.5 and pH 4). The extracts were cleaned up by solid phase extraction using octadecyl cartridges (C18). The samples were dried up and redissolved in the mixture of oxalic acid and methanol. Separation was performed on reserved phase column (Phenomenex C18, 250 x 4.6 mm, 5 μm) by multistep gradient elution, which provided better chromatographic separation. The analysis was performed at a wavelength of 390 nm. Validation study of the method revealed that all obtained calibration curves showed good linearity R= 0.9985 for doxycycline (DC) and R= 0.9981 for chlorotetracycline (CTC) over the range of 25-2,000 mg/kg. The analytical procedure was successfully adapted for quantitative determination of DC and CTC in medicated feedingstuff samples. Validation included determination of specificity, linearity, and repeatability. Mean recovery for spiked samples was 93.1% for CTC and 93.2% for DC. The results of validation of the analytical procedure proved that presented method is efficient, precise and useful for quantification of DC and CTC in medicated feedingstuffs.

Open access

E. Patyra, E. Kowalczyk, A. Grelik, M. Przeniosło-Siwczyńska and K. Kwiatek

Abstract

A liquid chromatography – diode array detector (HPLC-DAD) procedure has been developed for the determination of oxytetracycline (OTC), tetracycline (TC), chlorotetracycline (CTC), doxycycline (DC), enrofloxacin (ENR), ciprofloxacin (CIP), sarafloxacin (SAR) and flumequine (FLU) residues in animal drinking water. This method was applied to animal drinking water. Solid-phase extraction (SPE) clean-up on an Oasis HLB cartridge allowed an extract suitable for liquid chromatographic analysis to be obtained. Chromatographic separation was carried out on a C18 analytical column, using gradient elution with 0.1% trifluoroacetic acid – acetonitrile – methanol at 30°C. The flow-rate was 0.7 mL/min and the eluate was analysed at 330 nm. The whole procedure was evaluated according to the requirements of the Commission Decision 2002/657/EC, determining specificity, decision limit (CCα), detection capacity (CCβ), limit of detection (LOD), limit of quantification (LOQ), precision and accuracy during validation of the method. The recoveries of TCs and FQs from spiked samples at the levels of 10, 100 and 1000 μg/L were higher than 82%. The developed method based on HPLC-DAD has been applied for the determination of four tetracyclines and four fluoroquinolones in animal drinking water samples.

Open access

E. Kowalczyk, E. Patyra, A. Grelik and K. Kwiatek

Abstract

A high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) method was developed for determination of five ergot alkaloids (EA): ergometrine, ergotamine, ergocornine, ergocrypine and ergocristine in animal feedingstuffs. The method was based on the application of QuEChERS salts for extraction and modified QuEChERS dispersive SPE for the cleanup step. Alkaloids separation was performed on a C18, 250 mm x 4.6 mm, 5 μm column with the mobile phase containing ammonium carbonate and acetonitrile. The excitation and emission wavelengths were 330 and 420 nm respectively. The method was validated according to the Commission Decision 2002/657/EC and all parameters are in agreement with the requirements of the Decision. Linearity was determined for the concentration range of 25-400 μg/kg. The coefficient of determination (R2) for all curves was from 0.985 to 0.996. The limit of detection (LOD) was in the range 3.23 to 6.53 μg/kg and the limit of quantification (LOQ) from 11.78 to 13.06 μg/kg. The decision limit (CCα) ranged from 29.56 to 43.08 μg/kg and detection capability (CCβ) from 40.65 to 51.01 μg/kg. The highest coefficient of variation (CV) for repeatability was 14.3% and for reproducibility 15.4%.

Open access

Monika Przeniosło-Siwczyńska, Ewelina Patyra, Maja Chyłek-Purchała, Beata Kozak and Krzysztof Kwiatek

Abstract

The paper describes a microbiological method for the detection of antibacterial substances in feedingstuffs. The method allowed detection of the main antibiotic groups, including tetracyclines. In 2013-2014, a total of 171 feed samples were analysed to determine antibacterial substances. Among the analysed samples 84 (49.1%) were suspected to contain tetracyclines. Out of the 84 feeds analysed using chromatography, 28 (33.3%) contained undeclared tetracyclines, which were identified at concentrations ranging from 0.32 mg kg-1 to 48.98 mg kg-1.