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.