The use of growth promoters in animal husbandry to increase weight gain and efficiency of feed conversion into muscle has been banned in the European Union since 1988, and under Directive 96/23/EC, surveillance for anabolic steroid hormones is obligatory. The hormones present in animal tissues may be of endogenous origin or may result from illegal administration. Steps have been taken to determine selected steroids in the form of esters in the alternative matrix of animal hair. Their detection in biological material is direct proof of the illegal use of anabolics.
Material and Methods
The procedure for the determination of steroid esters in animal hair, based on digestion, extraction, purification, and liquid chromatography with tandem mass spectrometry was validated under the current regulations. In total, 348 samples of animal hair were examined using this method.
Good recoveries and precision values (RSD) were obtained during validation. Decision limits (CCα) and detection capabilities (CCβ) were in the ranges of 2.57–4.18 μg kg−1 and 4.38–7.12 μg kg−1, respectively. The method met the criteria for confirmation techniques with respect to Commission Decision 2002/657/EC.
Testing for steroid esters in animal hair was introduced into the National Residue Control Programme in 2017. Steroid esters were not found in any hair samples above the CCα, which indicates that illegal use of anabolics was not confirmed.
This paper describes the quantitative method of determination of chosen substances from resorcylic acid lactones group: zeranol, taleranol, α-zearalenol, β-zearalenol, and zearalanone in bovine muscle tissue. The presented method is based on double diethyl ether liquid-liquid extraction (LLE), solid phase extraction (SPE) clean up, and gas chromatography mass spectrometry (GC-MS) analysis. The residues were derivatised with a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide, ammonium iodide, and DL-dithiothreitol (1,000:2:5, v/w/w). The GC-MS apparatus was operated in positive electron ionisation mode. The method was validated according to the European Union performance criteria pointed in Decision Commission 2002/657/EC. The average recoveries of all analytes at 1 μg kg-1 level were located between 83.7% and 94.5% values with the coefficients of variation values <25%. The decision limits (CCα) and detection capabilities (CCβ) for all analytes ranged from 0.58 to 0.82 μg kg-1 and from 0.64 to 0.94 μg kg-1, respectively. The procedure has been accredited and is used as a screening and confirmatory method in control of hormone residues in animal tissues.
Introduction: Studies of anabolic hormone residues in the tissues of slaughter animals have been carried out in Poland for more than 25 years. During the period of 2011 to 2015, a total of 35 387 samples from different animal species were tested in the National Residue Control Programme for the presence of residues of compounds that cause hormonal effects, as listed in Annex 1 of Directive 96/23/EC.
Material and Methods: The research was conducted in the National Reference Laboratory and eight regional laboratories in departments of veterinary hygiene located throughout the country. Urine, muscle tissue, serum, kidney fat, and drinking water were the targeted matrices. Test methods based on instrumental techniques such as gas and liquid chromatography coupled with mass spectrometry were applied, as well as enzyme-linked immunosorbent assays (ELISA).
Results: The concentration of detected hormones exceeded the decision limits in 30 samples, the consequence of which was 41 non-compliances with current applicable criteria. The hormones found present pseudo-endogenous (nortestosterone and boldenone) only, while synthetic hormones were not identified.
Conclusion: The non-compliant findings constitute a small percentage (0.085%) of the five-year analysis compilation. On this basis the related food produced in Poland can be accepted as safe for human consumption with regard to the hormone residues tested.
Introduction: The present study is a comprehensive overview of the natural occurrence of 17β-oestradiol and testosterone in serum of cattle in Poland. Material and Methods: The serum samples (n = 826) were collected from cattle within five years. The samples were examined for the presence of oestradiol and testosterone using ELISA or gas chromatography with mass spectrometry. Results: In 98 samples (24%) 17β-oestradiol was detected above decision limits of applied methods, including five samples over the recommended concentration of 0.1 μg L-1. Of the serum samples taken from cows (≤18 months of age), 95 and 99 percentiles of the animals had 17β-oestradiol concentration below 0.027 and 0.086 μg L-1 and of samples from cows over 18 months of age - below 0.059 and 0.125 μg L-1 respectively. Calculated values for bulls (≤18 months of age) were 0.025 and 0.034 μg L-1 and for the animals older than 18 months of age - 0.035 and 0.041 μg L-1. The natural presence of testosterone was detected in 201 serum samples (48.7%). According to the obtained data, 95% and 99% of cows (≤18 months of age) serum samples had testosterone concentration below 0.05 and 0.23 μg L-1 and the animals over 18 months of age - 0.30 and 0.49 μg L-1, respectively. For bulls these values did not depend on the age of the animals and were in the ranges of 5 - 6.3 μg L-1 (95%) and 11.4 - 12.1 μg L-1 (99%). Conclusion: Our study showed that the threshold values for these hormones in plasma of cattle designated years ago are correct, but they need to be supplemented for animals older than 18 months.
Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level.
Material and Methods: 17β-testosterone and internal standards of 17β-testosterone-d2 were extracted from serum samples with a mixture of tert-butyl methyl ether/petroleum ether and were directly analysed by an LC/MS/MS on QTRAP 5500 instrument with a TurboIon-Spray source operating in a positive ionisation mode. Chromatographic separation was achieved on the analytical column Inertsil® ODS-3 with an isocratic elution using mobile phase consisting of acetonitrile, methanol, and water. Method validation has been carried out in accordance with the Commission Decision 2002/657/EC.
Results: The method was characterised by good recovery (82%) and precision (R.S.D 17 %). Decision limit (CCα) and detection capability (CCβ) was 0.05 μg L−1 and 0.09 μg L−1 respectively. The method met the criteria set out in Commission Decision 2002/657/EC for the purpose of confirmation in terms of retention time and ion ratio in the whole range of its application.
Conclusions: The developed method is specific and sensitive, suitable for measuring the natural level of testosterone in blood of cattle and for use in routine control programme for the detection of this hormone in bovine serum.
Sensitive and selective methods for the screening (GC-MS) and confirmatory analysis (GC-MS/MS) of 17α- and 17β- trenbolone in bovine urine were developed. In the first stage of the analysis, the enzymatic hydrolysis of trenbolone metabolites with glucuronidase AS-HP in acetate buffer (pH 5.2) solution was carried out. Free compounds were extracted from urine with diethyl ether. For the purification of the extract solid phase, extraction with C18 and NH2 columns was applied. The evaporated extract was subjected to two derivatisation steps; the first with MSTFA/I2 solution and second with MSTFA. The separation of the analytes on HP-5 ms capillary column was conducted. The methods were validated according to the Commission Decision 2002/657/EC. For GC-MS method, CCα and CCβ were 0.21-0.36 μg L−1 for 17α- trenbolone and 0.20-0.34 μg L−1 for 17β- trenbolone, while for GC-MS/MS method the values were lower and amounted to 0.15-0.25 μg L−1 for 17α- trenbolone and 0.20-0.34 μg L−1 for 17β-trenbolone. Method recoveries in spiked samples ranged from 86%-111% with standard deviation lower than 25% for both detection techniques.
A simple and sensitive gas chromatography method was developed to determine a group of oestrogens in surface water. In the first stage of analysis, enzymatic hydrolysis of oestrogen metabolites with glucuronidase AS-HP was performed. Free compounds were extracted from 200 mL of water sample on C18 SPE column (6 mL, 1000 mg). The evaporated extract was subjected to derivatisation with a mixture of MSTFA/NH4I/DTT (1000:2:5, v/w/w). The separation of the analytes on HP-5ms capillary column was conducted. The method was validated according to the Commission Decision 2002/657/EC. Recovery in spiked samples ranged from 90% to 120 % with standard deviation lower than 30% for all examined compounds. The decision limit and detection capability of five oestrogens were in the range of 0.3-0.6 ng L-1 and 0.5-0.9 ng L-1, respectively. Nineteen water samples collected from different sites of several Polish rivers and lakes were tested for the presence of oestrogens. Some target compounds such as 17α-oestradiol, 17β-oestradiol, oestrone, oestriol, and 17α-ethynyloestradiol were found in trace amounts in the analysed samples. The highest concentration observed for oestradiol reached 23 ng L-1.
A rapid liquid chromatography tandem mass spectrometry method was developed and validated to detect and confirm five thyreostatic drugs: tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil in animal feeding stuff samples. Thyreostats were extracted from feed with methanol, and then degreasing of the extract with petroleum ether was performed, followed by the derivatisation of the compounds with 3-iodobenzylbromide in basic medium (pH 8.0). The derivatives were extracted with diethyl ether and analysed by gradient elution on a Poroshell 120-EC C18 column with triple quadrupole MS detection with turbo spray source in positive ionisation mode. The method was validated in accordance with the Commission Decision 2002/657/EC. For validation level of 10 ļig kg-1, the recovery ranged from 82% to 97.5% for all examined compounds. The repeatability and reproducibility did not exceed the limit of 20% for all analytes. The linearity was good for all thyreostats in the whole range of tested concentrations, as proved by the correlation coefficients greater than 0.99. The decision limits (CCa) ranged from 1.63 ļig kg-1 to 3.95 ļig kg-1, whereas the detection capabilities (CCß) ranged from 2.74 ļig kg-1 to 6.73 ļig kg-1. The developed analysis is sensitive and robust, and therefore useful for quantification and confirmation of thyreostats in residue control programme.
Introduction: In the European Union, the use of thyreostatic drugs for fattening slaughter animals has been banned since 1981 under Council Directive 81/602/EEC. For protection of consumer health against unwanted residues and in compliance with Directive 96/23, each EU country must monitor thyreostats in samples of animal origin. This paper presents the results of research on thyreostatic residues carried out in Poland in 2011–2017.
Material and Methods: The material for testing was urine (n = 3,491), drinking water (n = 127), and muscle samples (n = 349) officially collected by Veterinary Sanitary Inspectors in slaughterhouses and farms throughout the country in accordance with the national residue control plan. The samples were examined for the presence of tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil using liquid chromatography tandem mass spectrometry through an accredited method.
Results: In four bovine and three porcine urine samples, the permissible thiouracil concentration was exceeded. In one sample of porcine urine, methyl- and propylthiouracil were found. The presence of thiouracil and its derivatives in urine samples is most likely due to feeding animals diet containing cruciferous plants.
Conclusions: The results of research indicate that thyreostats are not used for anabolic purposes in slaughter animals in Poland.