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.
Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.
Material and Methods
PAs were extracted with sulphuric acid and purified with cation exchange cartridges. A newly developed solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis.
The developed method was validated according to SANTE/11945/2015 guidelines. The recovery was from 84.1% to 112.9%, the repeatability ranged from 3.0% to 13.6%, and the reproducibility was from 4.8% to 18.9%.
A sensitive and selective method for determination of PAs in feed materials has been developed and validated. All evaluated validation parameters were in accordance with EU Reference Laboratories document no. SANTE/11945/2015. Almost 41% of the analysed feed samples were positive for the presence of at least one PA.
Pyrrolizidine alkaloids (PAs) are probably the most widespread toxins of natural origin. More than 6,000 plant species produce these toxic compounds. Bees can forage on flowers of plants producing PAs, which leads to contamination of honey with the toxic compounds. To determine the contamination of honey with PAs, a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.
Material and Methods
PAs were extracted with 0.05 M sulphuric acid and purified with MCX cartridges. A solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine (8:1:1:0.1:0.1, v/v) was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC–MS analysis.
The developed method was validated according to SANTE/11945/2015 requirements. The recovery was from 80.6% to 114.5%. The repeatability ranged from 2.3% to 14.6%, and the reproducibility was from 4.9% to 17.7%.
A new method for the determination of PAs in honey has been developed and validated. All evaluated parameters were in accordance with the SANTE/11945/2015 guidance document. Out of 50 analysed honey samples, 16 (32%) were positive for the content of at least one PA.
Effect of Genetically Modified Feeds on Physico-Chemical Properties of Pork
The objective of this study was to evaluate the effects of genetically modified (GM), insect-resistant Bt maize (MON810) and the meal made of glyphosate-tolerant soybean (Roundup Ready MON40-3-2) used as the dietary components for pigs on the physico-chemical properties of meat. Forty-eight fatteners derived from Polish Landrace x Polish Large White sows mated to a Duroc x Pietrain boar were used. All animals received isonitrogenous and isoenergetic diets containing or not containing the genetically modified components. The design of the experiment was as follows: group I (control) - non-modified soybean meal and maize; group II - GM soybean meal and non-modified maize; group III - non-modified soybean meal and GM maize; group IV - GM soybean meal and GM maize. The examination of the pH values of loin and neck muscles indicated no statistically significant differences between pigs fed diets containing non-transgenic or transgenic feeds. No statistical differences were observed for water holding capacity (WHC) within dietary treatments. The introduction of transgenic maize and soybean meal into pig diets did not significantly affect the a* colour parameter of loin as well as neck muscles. The use of transgenic maize or soybean meal did not cause significant changes in the L* colour value of loin. Results obtained for neck muscles were more differentiated, possibly due to the natural heterogeneity of this primal cut. Pigs which had consumed the transgenic diet exhibited slightly decreased lipid stability of loin, as indicated by thiobarbituric acid reactive substances (TBARS). The decrease was statistically significant only in the case of muscles from group II. The addition of feeds derived from genetically modified crops into pig diets did not significantly affect the stability of neck muscle lipids; however, TBARS values of these muscles were twice those of loin muscles.
The paper presents the first results of a study on the contamination of honey produced in the Republic of Kazakhstan with C. botulinum spores known to pose a potential infection threat to infants. During microbiological analysis, culturing methods with TPGY, Willis-Hobbs agar, FAA agar connected with PCR, sequencing, and a mouse bioassay were used. The C. botulinum contamination rate of honey was relatively low as determined, at 0.91%. Nonetheless, the potential danger of the bacteria to childrens’ health should not be neglected
Introduction: The aim of this study was to evaluate the prevalence of Clostridium botulinum and Clostridium perfringens in food samples purchased from Polish producers. Material and Methods: The analyses were performed on 260 food samples collected in Lublin and Subcarpathian regions: 56 of smoked meat, 21 of pork meat, 20 of dairy products, 26 of vegetable and fruit preserves, 40 of ready-to-eat meals, 27 of fish preserves, and 70 of honey collected directly from apiaries. Results: C. botulinum strains were isolated from 2.3% (6/260) of samples and the isolates were classified as toxin types A (4/260) and B (2/260). C. perfringens strains were isolated from 14% (37/260) of samples. All the isolates were classified as toxin type A, 28 of them were able also to produce α toxin and 9 - β2 toxin. Conclusion: On the basis of the obtained results it could be suggested that risk assessment, especially regarding the entire honey harvesting process, should be provided in order to ensure the microbiological safety of the products to be consumed by infants and people with a weakened immune system.
The authorisation of tylosin as feed additive was withdrawn for reasons of human health concerning resistance of pathogenic bacteria. An analytical method for the identification and quantification of tylosin in animal feed was developed and validated.
Material and Methods
The samples were extracted using an acidified methanol:water mixture and solid-phase extraction was employed for the isolation of the antibiotic from diluted feed samples. Tylosin was analysed by liquid chromatography with electrospray ionisation mass spectrometric detection. The method’s performance was evaluated in adherence to the Commission Decision 2002/657/EC.
The recovery of the analyte from spiked samples was determined to be in the range from 78.9% to 108.3% depending on tylosin concentrations. The CCα and CCβ values for tylosin in feeds were determined at 0.085 mg kg-1 and 0.091 mg kg-1, respectively. The method detection limit was found to be 0.035 mg kg-1 and the quantification limit 0.05 mg kg-1. The applicability of the developed method was tested by analysing real feed samples.
A reliable LC-MS method was developed to identify and quantify tylosin in animal feed with a good repeatability and a high specificity and sensitivity. Because of these characteristics, the proposed method is applicable and could be deemed necessary within the field of feed control and safety.
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.
The aim of this paper was to present the results of comparative evaluation of the usefulness of PCR method for the detection and identification of bovine DNA in feeds. In the validation study, the limit of detection for PCR was determined as 0.05% for bovine meat and bone meal (MBM). Among 132 feed samples, bovine DNA was detected in eight (6.06%) samples. In the next stage of the study, sediment and flotate from the investigated samples were examined with PCR. Out of 132 sediment and flotate samples, bovine DNA was detected in eight (6.06%) and nine (6.82%) samples, respectively. On the basis of the results obtained with the use of the PCR, it is possible to state that the molecular biology methods can, at present, be used as supplementary tools for detection and identification of bovine MBM.
The aim of this study was to perform an in-house validation of multiplex PCR method for C. botulinum detection in food and feed samples. The study was carried out on food and feed matrixes artificially contaminated by spores of C. botulinum reference strains. The following characteristic parameters for qualitative detection were estimated: limit of detection expressed as LOD50 according to the Spearman-Kärber formula, specificity, sensitivity, and accuracy according to the PN-EN ISO 16140:2004. The validated method showed high specificity. Specific PCR products were revealed only for DNA obtained from samples contaminated with C. botulinum spores. PCR inhibition was observed, especially during examination of contaminated feed. The calculated LOD50 for feed was nearly 10 times higher than for food. The implemented method enables to obtain test results during 3 d without time- consuming process of isolation and proving the ability of strains to produce botulinum toxins.