Fast analysis of 19 anabolic steroids in bovine tissues by high performance liquid chromatography with tandem mass spectrometry.

For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid-phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 µg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10-0.48 and 0.17-0.95 µg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients   > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.

Analysis of thyreostats in bovine feces using ultra high performance liquid chromatography with tandem mass spectrometry.

A method based on ultra-high performance liquid chromatography was developed and validated to detect six thyreostatic compounds: tapazole, thiouracil, methylthiouracil, dimethylthiouracil, propylthiouracil, and phenylthiouracil in faeces of bovine. Thyreostats were extracted from the matrix with a mixture of methanol and buffer (pH = 8). Next step was derivatization of analytes with 3-iodobenzylbromide. The liquid chromatographic separation of derivatives was obtained on a SB-C18 column (50 × 2.1 mm; 1.8 µm, Agilent) with gradient elution using a mobile phase consisting of acetonitrile/0.1% acetic acid within 7.5 min. The analysis was performed on a Shimadzu NEXERA X2 ultra-high performance liquid chromatograph with triple quadrupole MS 8050 instrument operating in positive electrospray ionization mode. Depending on the target compound, two or three diagnostic signals (selected reaction monitoring transitions) were monitored. The procedure was validated according to the Commission Decision 2002/657/EC. Recovery and repeatability met the performance criteria specified by this document for banned compounds. The recovery ranged from 97.5 to 110.5%, and repeatability did not exceed 14.1%. Decision limits and detection capabilities were below 10 µg/kg. The highest decision limits and detection capabilities concentrations were observed for phenylthiouracil of 3.48 and 6.96 µg/kg, respectively.

Co-ordinated Changes in the Accumulation of Metal Ions in Maize (Zea mays ssp. mays L.) in Response to Inoculation with the Arbuscular Mycorrhizal Fungus Funneliformis mosseae.

Arbuscular mycorrhizal symbiosis is an ancient interaction between plants and fungi of the phylum Glomeromycota. In exchange for photosynthetically fixed carbon, the fungus provides the plant host with greater access to soil nutrients via an extensive network of root-external hyphae. Here, to determine the impact of the symbiosis on the host ionome, the concentration of 19 elements was determined in the roots and leaves of a panel of 30 maize varieties, grown under phosphorus-limiting conditions, with or without inoculation with the fungus Funneliformis mosseae. Although the most recognized benefit of the symbiosis to the host plant is greater access to soil phosphorus, the concentration of a number of other elements responded significantly to inoculation across the panel as a whole. In addition, variety-specific effects indicated the importance of plant genotype to the response. Clusters of elements were identified that varied in a co-ordinated manner across genotypes, and that were maintained between non-inoculated and inoculated plants.

Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters.

Plant interactions with arbuscular mycorrhizal fungi have long attracted interest for their potential to promote more efficient use of mineral resources in agriculture. Their use, however, remains limited by a lack of understanding of the processes that determine the outcome of the symbiosis. In this study, the impact of host genotype on growth response to mycorrhizal inoculation was investigated in a panel of diverse maize lines. A panel of 30 maize lines was evaluated with and without inoculation with arbuscular mycorrhizal fungi. The line Oh43 was identified to show superior response and, along with five other reference lines, was characterized in greater detail in a split-compartment system, using 33 P to quantify mycorrhizal phosphorus uptake. Changes in relative growth indicated variation in host capacity to profit from the symbiosis. Shoot phosphate content, abundance of root-internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high phosphorus uptake by mycorrhizal plants. The data indicate that host genetic factors influence fungal growth strategy with an impact on plant performance.

Comparison of the Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes for Confirmation of Stilbenes in Bovine Urine Samples Using LC-MS/MS QTRAP® System.

In accordance with Commission Decision 2002/657/EC, confirmatory methods for the detection of prohibited substances should comply with specific requirements, including the criteria for confirmation. Two strategies: multiple reaction monitoring (MRM) and enhanced product ion (EPI) scanning functions were compared for confirming the anabolic compounds from synthetic stilbenes group in bovine urine samples. In the research, twenty samples fortified at the Recommended Concentration (RC) of 1 µg L-1 with diethylstilbestrol, dienestrol and hexestrol were analyzed by liquid chromatography-tandem mass spectrometry on a QTRAP 5500 instrument. The analytical procedure, validated in accordance with the Commission Decision 2002/657/EC, used in the official control of hormones in Poland was applied. The validation parameters were in agreement with 2002/657/EC performance criteria. The effectiveness of MRM and EPI scanning modes for confirmation purposes was evaluated based on the percentage of the results confirmed. In all urine samples recorded in the MRM mode, the confirmation criteria (retention time, relative intensities between transitions) have been fulfilled. The presence of stilbenes in all urine samples using EPI scan mode was confirmed too as evidenced by a good matching of stilbenes spectra in the samples to the reference spectra with critical match factor above 0.7. The results of the research show that EPI scanning function provides the same effectiveness for confirmation of banned compounds as the mostly used MRM scan mode and can be an additional tool to confirm the doubtful case results in the analysis of hormones residues, even at such low concentration levels.

LC-MS/MS fast analysis of androgenic steroids in urine.

The liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated to detect androgenic steroids: trenbolone, nortestosterone, boldenone, methylboldenone, testosterone, methyltestosterone, 17ß-1-testosterone and their metabolites in bovine urine. Sample preparation before LC-MS/MS analysis involved an enzymatic hydrolysis with glucuronidase AS-HP, isolation of free hormones from urine on C(18) SPE column and purification of the extract using liquid-liquid extraction with n-pentane and SPE NH(2) column. For the chromatographic separation of steroids, the Poroshell 120-EC C18 column (150 × 2.1 mm, 2.7 µm) was used. Mass spectrometric measurement was achieved using the API 4000 triple quadrupole (QqQ) instrument with a TurboIon-Spray source operating in positive electrospray ionization mode. The procedure was validated according to the Decision 2002/657/EC. Recovery ranged from 76.5% to 118.9% for all examined compounds. The repeatability was below 20% and reproducibility did not exceed the 25%. The linearity was good for all analytes in the whole range of tested concentrations, as proved by the correlation coefficients greater than 0.99. The decision limit (CCα) ranged from 0.10 to 0.17 µg L(-1) for all analytes, whereas the detection capability (CCß) ranged from 0.17 to 0.29 µg L(-1). The application of an innovative Poroshell column allowed for very good chromatographic separation of steroids with a much shorter time of analysis.

Factors affecting work ability and influencing early retirement decisions of older employees: an attempt to integrate the existing approaches.

The article has several objectives. First, it aims to discuss, based on the international literature analysis, theoretical models and factors influencing early retirement decisions, and limiting work ability and employability of older people. Second, the concept of (and the factors related to) work ability as well as the role of age management in improving employability and sustaining work activity of older adults are analyzed against the backdrop of the Polish context. Cultural (ageism) and organizational (e.g., a lack of appropriate age management in companies) factors influencing work activity in the oldest age groups are taken into account. Third, the authors present some solutions and recommendations coming from the international Participation To Healthy Workplaces And inclusive Strategies in the Work Sector (PATHWAYS) project. Finally, theoretical models, concepts and solutions are integrated into a model elucidating the conditions (cultural, social, organizational, individual) influencing work ability of older adults. The model aims to take into account theories and factors identified in the literature review and grouped into several categories. The analysis highlights the role of appropriate age management in companies in the process of sustaining work ability of older employees. Int J Occup Med Environ Health. 2022;35(5):509-26.

Polybrominated diphenyl ethers (PBDEs) in raw milk from different animal species and in infant formula. Occurrence and risk assessment.

Polybrominated diphenyl ethers (PBDEs) are widespread, persistent in the environment, and classified as global pollutants. Their presence has been confirmed in various types of food which adversely affect human health when consumed in sufficient amounts. Although milk has advantageous nutritional qualities and there are health benefits associated with its consumption, it could also contain toxic PBDEs. The aim of the study was the determination of the concentrations of ten congeners (BDE -28, -47, -49, -99, -100, -138, -153, -154, -183, and 209) in cow's, sheep's, and goat's milk obtained from Polish farms and their determination in infant formula. A total of 103 samples of raw milk and infant formula were tested using an accredited high-resolution gas chromatography-high-resolution mass spectrometry method. PBDEs were detected in all analyzed samples, the highest concentration being found in sheep's milk (11.9 ng g-1 fat), and cow's milk containing the least contamination. BDE-209 makes the predominant contribution to the sum of the ten congeners, constituting at least 38%. The profiles of PBDEs were dependent on the milk type and the differences between its varieties are discussed. The highest median concentration of the sum of ten PBDEs (0.473 ng g-1 fat) was determined in infant formula, which was identified as an important source of infants' exposure (5.48 ng kg-1 b.w. day-1 calculated based on P95 concentration). Milk is a source of PBDE in the diet; however, considered in isolation its consumption does not pose a risk to either adults' or children's health.

Risk factors for infectious complications after retrograde intrarenal surgery - a systematic review and narrative synthesis.

INTRODUCTION: Infectious complications are among the most frequent and significant complications in retrograde intrarenal lithotripsy. To date, review articles have covered complications after a ureteroscopy, but not after retrograde intrarenal surgery (RIRS), specifically. Because the complications and risk factors are different for a ureteroscopy and RIRS, we aimed to identify variables related to the occurrence of infectious complications post-RIRS. MATERIAL AND METHODS: This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement. We included original studies that described 100 or more procedures published in 2014-2021. We extracted data and performed a narrative synthesis to explore and interpret differences between the studies. RESULTS: We selected 17 studies for analysis, including 10 from 2019-2021. Infectious complications after RIRS were observed in 2.8-7.5% of patients (mean 7.1%). We found seven independent risk factors associated with infectious complications after RIRS: long operative time, recent history of positive urine culture or urinary tract infection or antibiotic use, pyuria/nitrites, small caliber of ureteral access sheath, struvite stone, high irrigation rate, and comorbidities. CONCLUSIONS: If an increased rate of infectious complications is found at a RIRS center, countermeasures should include restrictions on operative time and irrigation rate, and consideration of larger access sheaths, especially for patients with abnormal urine results or with struvite stones or with a history of urinary tract infection or co-morbidities.

Determination of selected testosterone esters in blood serum of slaughter animals by liquid chromatography with tandem mass spectrometry.

Anabolic hormones, which cause muscle growth, have been banned for anabolic purposes in animal husbandry in Europe since the 1980s. Control of hormones from the list of Annex I to Directive 96/23/EC is mandatory in the European Union. The presence of hormones in samples of animal origin may be due to their endogeneous nature or illegal use. One way to distinguish their origin is to study hormones, particularly steroids in the form of ester derivatives. In the body synthetic hormone esters could be only exogenous therefore their detection in animal tissues is the undisputed evidence of illegal administration. The analytical procedure involves the extraction of esters from serum with organic solvents, derivatisation with methoxyamine and detection by liquid chromatography tandem mass spectrometry. The method was approved in accordance with the applicable legislative criteria and its effectiveness was verified in the proficiency test. The research material consisted of bovine serum samples officially taken. During the validation good apparent recovery, precision, decision limits and detection capabilities in the range 0.006-0.012 µg L-1 and 0.010-0.020 µg L-1 respectively were obtained. The developed method met the criteria for confirmation set out in Commission Decision 2002/657/EC. Since the inclusion of serum in 2018 for testing for testosterone esters in the National Residue Control Program, 130 samples have been examined. In none of the serum samples, esters above the decision limits were found. The control of animals and food of animal origin for hormone esters will be continued to ensure the health and safety of consumers.

Evaluation of the Effect of Surgical and Immunological Castration of Male Pigs on Boar Taint Compounds in Oral Fluid and Fat Tissue by LC-MS/MS Method.

INTRODUCTION: An effective way of preventing undesirable boar taint in pork meat caused by the presence of androstenone, skatole and indole is surgical castration of piglets. This, however, arouses growing social opposition. An alternative method of inhibiting the development of unpleasant odour is immune castration. The aim of the study was to compare the effectiveness of both methods of castration for the elimination of the compounds responsible and to assess the suitability of oral fluid for pre-slaughter predictive testing for boar taint. MATERIAL AND METHODS: The research material was pooled oral fluid and fat samples taken from gilts and surgically and immunologically castrated piglets. The samples were tested with a liquid chromatography- tandem mass spectrometry method developed in this research. RESULTS: The compounds giving rise to boar taint were found only sporadically above the accepted limits; only one sample of oral fluid contained skatole at a concentration above 200 µg L-1 and one contained indole more concentrated than 100 µg L-1. Indole above the limit value was also detected in one fat sample. In none of the tested samples was androstenone found. CONCLUSION: The results indicate the similar effectiveness of both methods of piglet castration on the reduction of compounds generating boar taint. The usefulness of testing oral fluid for the ante-mortem prediction of boar taint has not been fully confirmed and further investigation is needed.

Resorcylic acid lactones in urine samples of slaughtered animals resulting from potential feed contamination with zearalenone.

Resorcylic Acid Lactones, including zeranol, anabolics listed in the group A4 of Directive 96/23/EC, are banned in Europe for use in animals since 1985. Zeranol, after administration to animals, is metabolized to taleranol and zearalanone. It can also naturally occur in the urine due to conversion of zearalenone that may be present in animal feed. In 2010-2017, in Poland, 7746 animal samples were tested for zeranol residues within the official monitoring program. In 13, zeranol was detected after screening. Re-examinations confirmed resorcylic acid lactones in six samples. The recommendations state that only the presence of zeranol and/or taleranol gives the basis for non-compliance. Confirmation should cover the entire profile of six resorcylic acid lactones. In case of detection, the relationship ratio should be verified. Following the proposed criteria, it could be concluded that zeranol detected in urine samples in Poland originated from contamination of feed with mycotoxin, not from illegal use.

Determination of Steroid Esters in Hair of Slaughter Animals by Liquid Chromatography with Tandem Mass Spectrometry.

INTRODUCTION: 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. RESULTS: Good recoveries and precision values (RSD) were obtained during validation. Decision limits (CCα) and detection capabilities (CCß) 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. CONCLUSION: 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.

Difficult entry and return to the labor market: on the professional (re)integration of people with disabilities and chronic health conditions in Poland (The PATHWAYS research project)

Objectives: The objective of this study is to present the barriers existing in Poland to professional integration and reintegration of people with chronic diseases, including mental health conditions, and discuss them on the basis of expert opinions and in comparison with the situation in other European countries. Material and Methods: The research methodology was based on a mixed-methods approach, including literature review, in-depth expert interviews, and a survey (based on a structured questionnaire) performed with experts. Results: The mapping of policies, systems and services facilitating the integration and reintegration of people with chronic diseases in Poland, as well as expert opinions, made it possible to identify many barriers regarding access to support, such as an obligation to have a certificate of disability, and a system of certification by 2 institutions. There are also barriers regarding the support itself, including problems with subsidies and with professional (re)integration services. Conclusions: Poland has a multi-level and multi-sector system of public institutions, which should provide support for people with disabilities and chronic diseases. Nevertheless, the research conducted in Poland indicates an insufficient implementation of the existing solutions. Int J Occup Med Environ Health. 2019;32(4):475­88

The effect of diet enriched with rapeseed meal on endogenous thiouracil contents in urine and milk of cattle.

Thyreostatic compounds, such as thiouracil, are orally active drugs that can be used to increase the weight of cattle before slaughter. Due to potentially teratogenic and carcinogenic effects of their residues on public health, the use of thyreostats in animal production has been banned in the European Union since 1981. Systematic detection of low concentrations of thiouracil in the urine of livestock in many countries is believed to be of endogenous origin due to the use of Brassicaceae plants in the animal diet. Therefore, the purpose of the study was to determine the effects of diets rich in rapeseed meal on formation of thiouracil in urine and milk of dairy cows. For two weeks three cows were subjected to a diet supplemented with rapeseed at 30%, compared to the control cattle diet which contained up to 11% rapeseed. During the experiment, samples of urine and milk were collected and analysed by LC-MS/MS. The increase and decrease of thiouracil concentration in urine samples in different animals was individual and cyclic. The highest concentration of natural thiouracil determined in urine was 3.61 µg l-1. It has been found that endogenous thiouracil exists in two tautomeric forms. A few days of storage of frozen urine samples affected the stability of natural thiouracil, whereas an acidic medium improved the stability of the compound and its isomer, which remained stable even after two months of storage at temperatures below -18°C. Due to the instability of thiouracil, urine samples upon sampling should be delivered to the laboratory as soon as possible or properly preserved. In milk samples, thiouracil was not found above the decision limit of the applied method of 0.63 µg l-1. Preliminary studies have shown that faecal examination for banned thiouracil can be a complementary test for urine samples, and may be helpful in determining the origin of the compound present in urine.

Control of Residues of Thyreostats in Slaughter Animals in Poland in 2011-2017.

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.

Development of LC-MS/MS Confirmatory Method for the Determination of Testosterone in Bovine Serum.

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: 17ß-testosterone and internal standards of 17%-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 (CCß) 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.

Toward a new European threshold to discriminate illegally administered from naturally occurring thiouracil in livestock.

Thiouracil is a thyrostat inhibiting the thyroid function, resulting in fraudulent weight gain if applied in the fattening of livestock. The latter abuse is strictly forbidden and monitored in the European Union. Recently, endogenous sources of thiouracil were identified after frequently monitoring low-level thiouracil positive urine samples and a "recommend concentration" (RC) of 10 µg/L was suggested by the EURL to facilitate decision-making. However, the systematic occurrence of urine samples exceeding the RC led to demands for international surveys defining an epidemiologic threshold. Therefore, six European member states (France, Poland, The Netherlands, United Kingdom, Norway, and Belgium) have shared their official thiouracil data (2010-2012) collected from bovines, porcines, and small livestock with 95 and 99% percentiles of 8.1 and 18.2 µg/L for bovines (n = 3894); 7.4 and 13.5 µg/L for porcines (n = 654); and 7.4 µg/L (95% only) for small livestock (n = 85), respectively. Bovine percentiles decreased with the animal age (nonadults had significantly higher levels for bovines), and higher levels were observed in male bovines compared to female bovines.

Evaluation of estrogenic activity in animal diets using in vitro assay.

A yeast estrogen bioassay (RIKILT REA) was in-house validated for feed on the 5µg 17ß-estradiol-equivalents per kg level according to EC Decision 2002/657/EC. All the performance characteristics met the criteria as defined in the Decision and the REA is able to detect 17ß-estradiol in animal feed at a low level of 1.15-2µgkg(-1). Subsequently, the developed and validated procedure was applied to determine the estrogenic activity in 24 feed samples intended for food producing animals, pets and laboratory animals. Two batches of rodent diet Murigran and one dog feed have been presented as a suspect, i.e. gave responses above the determined decision limit (CCα) and detection capability (CCß). In assessing the performance of the estrogenic activity in these diets evaluated by comparison with the 17ß-estradiol calibration curve, 17ß-estradiol-equivalence levels of 7.07µg EEQkg(-1) and 9.54µg EEQkg(-1) in two batches of rodent diet and 5.3µg EEQkg(-1) in dog feed have been established. The activities observed in the rodent feed could be explained by chemical analysis, revealing high amounts of genistein, daidzein and trace amounts of zearalenone. In addition, the estrogenic activity in one of rodent feed was above the established CCα, but below the CCß values established and all other samples showed no estrogenic activity with responses below the CCα value, which corresponds to levels below 2µg EEQkg(-1).

Determination of stilbenes and resorcylic acid lactones in bovine, porcine and poultry muscle tissue by liquid chromatography-negative ion electrospray mass spectrometry and QuEChERS for sample preparation.

Liquid chromatography tandem mass spectrometry method was developed and validated to confirm of resorcylic acid lactones: zeranol, taleranol, zearalanone, zearalenone, α and ß-zearalenol and stilbenes in muscle tissue. The compounds were analyzed by LC-MS/MS QTrap 5500 apparatus in negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 (150mm×2.1mm, 2.7µm) column was achieved at 45°C using isocratic elution of mobile phase - methanol/water (65:35, v/v). For the treatment of tissue samples prior to analysis, QuEChERS method was applied based on the extraction of analytes from muscle samples with ethyl acetate, separation of the aqueous and organic phases with application of magnesium sulphate and sodium acetate, the purification of the extract obtained by dispersive SPE with the use of sorbent C18, PSA and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. Good recoveries were obtained (from 83% to 115%) as well as acceptable within-lab reproducibility (<22%). The values of the decision limit CCα and the detection capability CCß for individual compounds are found to be below the recommended concentration set at 1µgkg(-1) and not exceed 0.23µgkg(-1) and 0.39µgkg(-1), respectively. The elaborated method meets the criteria for confirmatory methods and is used in the official control of hormones.