Identification and confirmation of the presence of some steroid-like growth promoters in the urine of cattle and swine

Abstract

English: Anabolic steroids and/or growth promoters are used to improve growth rate and feed conversion efficiency of livestock. The residues of these anabolics, which are present in the meat, may have a pharmacological activity due to oral bioavailability, and pose a risk to the consumer. Certain anabolics can be given legally to farm animals in some countries, but are banned in most others because of their proved or alleged toxic and/or carcinogenic properties. The use of these substances is completely forbidden within the European Community (EC). Before meat products can be exported to any member state of the EC, it is compulsory for the exporting country to have a monitoring programme to test for illegal use of these anabolic substances. The necessity to test for illegal use or to determine residue levels after legal use, has led to a strong interest in developing analytical methods for the identification and confirmation of anabolic agents in biological samples. The objectives of this study were to develop suitable analytical methods with a view to identify residues of some growth-promoting veterinary drugs in the urine of cattle and/or swine, to confirm the presence of these veterinary drugs unequivocally in the urine and to examine the stability of these drugs in urine under different environmental storing conditions. The excretion of clenbuterol, diethylstilbestrol, nandrolone, trenbolone and zeranol from cattle and/or swine were studied. A thorough literature study was done on the published analytical methods as well as the metabolism and pharmacokinetics of these drugs in cattle and/or swine. Reference standards were used to develop a GC-MS screening method for the identification of these drugs and/or their metabolites in the urine of cattle and/or swine. Recoveries of 61-99% and detection limits of 0.9-2.1 ng/ml were obtained for the different analytes with the developed analytical method. Trials were conducted in which these drugs were administered to cattle and/or swine. Urine samples were collected at regular time intervals and stored immediately at -20°C until time of analysis. Reference standards were also used for the development of GC-MS-MS analytical methods to confirm the presence of these drugs and/or their metabolites in the urine of cattle and/or swine. Confirmation of the substances in urine was done by obtaining a MS-MS spectrum of the extract and comparing this with the MS-MS spectrum of a reference standard. The MS-MS spectra was obtained by using the ion-trap technique. Urine samples are often collected at different farms and transported to the laboratory for residue analysis. Although it is standard procedure to freeze samples immediately after collection, it is not always possible. The results of this study show that the analytes contained in urine samples that were stored frozen remained stable for at least 10 days. If urine samples are stored at ambient temperature, concentrations of the analytes can decrease with as much as 30% after 10 days. Epi-nandrolone (metabolite ofnandrolone in cattle) could however not be detected in the urine samples after 4 days. If urine samples are stored in direct sunlight, concentrations of the analytes can decrease with as much as 90% after 10 days. It can be concluded that a method was developed to identify residues of some growthpromoting veterinary drugs and/or their metabolites in the urine of cattle and/or swine, and to confirm the presence of these drugs unequivocally in the urine. It can further be concluded that urine samples should be frozen as soon as possible after collection to prevent false negative results.

Afrikaans: Anaboliese steroïede en/of groei promoters word gebruik om groeitempo en voeromsettingsdoeltreffendheid van lewende hawe te verbeter. Die residue van hierdie middels, wat in die vleis voorkom, mag 'n farmakologiese effek hê as gevolg van orale biobeskikbaarheid, en 'n risiko vir die verbruiker inhou. Sekere anaboliese middels kan tans wettig aan diere toegedien word in sommige lande, maar in die meeste ander lande word dit verbied as gevolg van die middels se bewese of beweerde toksiese en/of karsinogeniese eienskappe. Die gebruik van hierdie middels is totaal verbode binne die Europese Gemeenskap (EG). Voordat vleisprodukte na eruge lidland van die EG uitgevoer kan word, moet daardie uitvoerland 'n moniteringsprogram in plek hê om te toets vir die onwettige gebruik van hierdie anaboliese middels. Die noodsaaklikheid om te toets vir die onwettige gebruik van hierdie middels, of om residu vlakke te bepaal na wettige gebruik, het die belangstelling geprikkelom \./ analitiese metodes te ontwikkel vir die identifisering en bevestiging van anaboliese middels in biologiese monsters. Die doelstellings van hierdie studie was om geskikte analitiese metodes te ontwikkel waarmee residue van sekere anaboliese middels in die urien van beeste en/of varke aangetoon kan word, om die teenwoordigheid van hierdie middels onteenseglik in die urien te bewys en om die stabiliteit van hierdie middels te ondersoek onder verskillende omgewings bewaartoestande. Die uitskeiding van clenbuterol, diëtielstilbestrol, nandroloon, trenboloon en zeranol is in beeste en/of varke bestudeer. 'n Deeglike literatuurstudie is gedoen oor gepubliseerde analitiese metodes asook die metabolisme en farmakokinetika van hierdie middels in beeste en/of varke. Verwysingsstandaarde is gebruik om 'n GC-MS siftingsmetode te ontwikkel vir die identifisering van hierdie middels en/of hulle metaboliete in die urien van beeste en/of varke. Herwinnings van 61-99%, en deteksielimiete van 0.9-2.1 ng/ml is verkry vir die verskillende analiete met die analitiese metode wat ontwikkel is. Proewe is uitgevoer waarin hierdie middels aan beeste en/ofvarke toegdien is. Urienmonsters is gereeld versamel en dadelik gestoor by -20°C totdat dit geanaliseer is. Verwysingstandaarde is ook gebruik om GC-MS-MS analitiese metodes te ontwikkel vir die onteenseglike bewys van die teenwoordigheid van hierdie middels en/of hulle metaboliete in die urien van beeste en/of varke. Bevestiging van die middels in urien is gedoen deur' n MSMS spektrum van die ekstrak te verkry en dit te vergelyk met die MS-MS spektrum van 'n verwysingstandaard. Die MS-MS spektra is verkry deur die ioon-vangs tegniek te gebruik. Urienmonsters word gewoonlik by verskillende plase versamel en daarna versend na die laboratorium vir residu analise. Alhoewel dit standaard praktyk is om urienmonsters dadelik te vries nadat dit versamel is, is dit nie altyd prakties moontlik nie. Die resultate van hierdie studie toon dat analiete in urienmonsters wat dadelik gevries word, stabiel bly vir minstens 10 dae. Indien urienmonsters by kamertemperatuur gestoor word, kan die konsentrasies van die analiete met soveel as 30% afneem na 10 dae. Epi-nandroloon (metaboliet van nandroloon in beeste) kon egter na 4 dae nie meer in die urienmonsters aangetoon word nie. Indien die urienmonsters in direkte sonlig gestoor word, kan die konsentrasies van die analiete met soveel as 90% afneem na 10 dae. Die gevolgtrekking kan gemaak word dat 'n metode ontwikkel is om residue van sekere groei promoters en/of hulle metaboliete in die urien van beeste en/of varke aan te toon, en om die teenwoordigheid van hierdie middels onteenseglik in die urien te bewys. 'n Verdere gevolgtrekking kan gemaak word dat urienmonsters so gou as moontlik gevries moet word nadat dit versamel is, om vals negatiewe resultate te voorkom.