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

J Vet Res 63, 561-572, 2019 DOI:10.2478/jvetres-2019-0061 Determination of steroid esters in hair of slaughter animals by liquid chromatography with tandem mass spectrometry Iwona Matraszek-Żuchowska, Barbara Woźniak, Katarzyna Sielska, Andrzej Posyniak Department of Pharmacology and Toxicology, National Veterinary Research Institute, 24-100 Puławy, Poland [email protected] Received: February 8, 2019 Accepted: September 16, 2019 Abstract 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 (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. 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. Keywords: bovines, swine, hair, steroid esters, liquid chromatography with tandem mass spectrometry. Introduction having anabolic effect included in Annex 1 of Council Directive 96/23/EC. In line with the recommendations Consumer safety and health should be a priority of the designated European Union Reference for services dealing with the examination of animals Laboratories (EURLs) which oversee and are and food of animal origin, and there is an obligation responsible for the testing of prohibited substances in upon those services to detect residues of hazardous the EU, their residues in biological material of animal substances that can be administered to animals for origin should be monitored (9). The EURLs in various reasons and could potentially pose a threat. Germany, the Netherlands, and France responsible for Undoubtedly, one of the sources of consumer exposure specific groups of veterinary medicinal products have to veterinary medicinal products is carelessness at the proposed recommended concentrations (RC) for stage of food production and non-compliance with the individual hormones in biological matrices for the prescribed safety measures by the farmers. purpose of residue monitoring in a joint document The use of hormonal substances for fattening referring to analytical techniques used in National purposes in livestock has been banned in the European Residue Control Programmes (NRCPs) (10). The Union (EU) since 1988, according to Council EURLs in consort, as the institution supervising the Directives 96/22/EC and 2003/74/EC (7, 8). The range of hormone tests in EU Member States, steroid esters that belong to the A3 group of banned recommended adopting an RC value of 10 µg kg−1 for substances should not be present in biological matrices steroid esters, although the cited legal document does of animal origin, just as other unauthorised substances not include these compounds in the hair matrix. © 2019 I. Matraszek-Żuchowska et al. This is an open access article distributed under the Creative Commons Attribution- NonCommercial-NoDerivs license (http://creativecommons.org/licenses/by-nc-nd/3.0/) 562 I. Matraszek-Żuchowska et al./J Vet Res/63 (2019) 561-572 Synthetic steroid hormones are usually research on threshold values for specific biomarkers administered to animals in the form of ester derivatives, (species and sex dependent) and collecting further data which thanks to the ester chains more easily reach their from NRCPs (27). target sites in the animal’s body. This form of Although steroid hormones are found in different administration also allows their easier absorption and matrices, of which the most common is urine, it is transport to the target organs. Due to their chemical difficult to distinguish their origin and to prove abuse. structures, steroid esters differ in their properties, e.g. Therefore, one alternative way to confirm illegal use is volatility. The esters can be administered to animals in the study and detection of these synthetic steroid the form of pour-on hormone cocktails in places from hormones in the form in which they are directly the neck to the tail and in the form of intramuscular administered both to animals and humans, i.e. as injections. After administration (especially by steroid esters (28). Steroid hormones do not naturally injection) they are hydrolysed or epimerised and occur in the form of esters; therefore the detection of transported with body fluids to peripheral blood. The steroid esters gives incontrovertible proof of the illegal length of the carbon chain in the steroid ester molecule use of anabolics. However, the detection of abuse of determines its solubility in water; compounds with synthetic steroid esters in routine urine testing of short chains are quickly transported from the injection animals or athletes is practically impossible, because site in the muscles to the blood, resulting in higher within a few hours of administration hormone esters are concentrations; esters with long side chains, in contrast, quickly hydrolysed to parental synthetic steroid are transported slowly, are present in lower hormones. These are mainly excreted in urine and concentrations, but last for a long time. Unlike animals, have a structure analogous to pseudoendogeneous in humans oral administration predominates by compounds also present in the urine, and the synthetic a wide margin (28). As compared to administration by and pseudoendogeneous are difficult to distinguish other means, e.g. orally in non-esterified form, from each other by origin. For this reason, steroid administration of steroid hormones intramuscularly in esters became attractive to athletes or cyclists as early the form of esters is likely to prolong the effect of their as the 1980s when it was noticed that they are action and improve the bioavailability in the body (16). undetectable in urine (15). There are many well-known medicinal preparations The detection and confirmation of steroid esters in available on the market, the main components of which biological samples of animal origin is one of the oldest are steroid esters, e.g. Omnadren, Sustanon, Testoviron analytical approaches for proving abuse of natural Depot (all with testosterone propionate), Deca- hormones. However, for many years its applicability Durabolin (with nandrolone decanoate), Durabolin was limited to the analyses of alleged injection sites; (with nandrolone phenylpropionate), Boldane, the tissues for testing were taken from the places where Equipoise, Parenabol, and Vebonol (all with boldenone the injection was most likely given while the analyses undecylenate), as well as a wide range of other were carried out using chromatographic techniques less products. sensitive in relation to more advanced current methods, The hormones present in animal tissues may be of for example using ultraviolet/diode array detection endogeneous origin or may have been illegally (UV/DAD) (6, 21, 29, 30, 31). Based on new know- administered. The concentration of hormones occurring how for the incorporation of steroid ester tests on naturally in living organisms differs from animal to alternative biological matrices of animal origin and animal depending on the species, sex, age, normality of new technical detection capabilities, in recent years physiological and individual development, and the state steroid esters have become more popular again in the of health and acquired injuries (16). The natural analysis of residues of anabolic hormones. Currently, hormones are present in almost all matrices. Some determining whether the hormone present in the urine naturally occurring steroid hormones like boldenone, is endogeneous or whether it is a result of nortestosterone (nandrolone), and testosterone as well administration can be carried out through studies of as their metabolites may have the same chemical synthetic esters in hair of animal origin. Steroid esters structure as their synthetic analogues (11, 17, 27). A are not metabolised in hair. The residues of different large number of studies in scientific centres were compounds including steroid esters in the hair of carried out on populations of treated and untreated animals persist even up to several months after animals of different species to learn the steroid profile administration, therefore the presence of steroid esters of hormones. Acknowledging the positive results for in the hair of animals indicates without ambiguity the steroid hormones in EU countries over the years, the illegal administration of anabolics. reasons for the pseudoendogeneous hormone Drugs can penetrate into the hair of animals from occurrences were

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