The detection of endogenous steroid abuse in cattle: results from population studies in the UK James Philip Scarth, Adam Clarke, Phil Teale, Aileen Mill, Roy Macarthur, Jack Kay
To cite this version:
James Philip Scarth, Adam Clarke, Phil Teale, Aileen Mill, Roy Macarthur, et al.. The detection of endogenous steroid abuse in cattle: results from population studies in the UK. Food Additives and Contaminants, 2011, 28 (01), pp.44-61. 10.1080/19440049.2010.539628. hal-00660034
HAL Id: hal-00660034 https://hal.archives-ouvertes.fr/hal-00660034 Submitted on 15 Jan 2012
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For Peer Review Only
The detection of endogenous steroid abuse in cattle: results from population studies in the UK
Journal: Food Additives and Contaminants
Manuscript ID: TFAC-2010-283.R1
Manuscript Type: Original Research Paper
Date Submitted by the 25-Oct-2010 Author:
Complete List of Authors: Scarth, James; HFL, Drug Surveillance Clarke, Adam; HFL, Drug Surveillance Teale, Phil; HFL, Drug Surveillance Mill, Aileen; Fera MacArthur, Roy; Fera Kay, Jack; DEFRA, VMD
Chromatography - GC, Chromatography - GC/MS, Chromatography Methods/Techniques: - LC/MS, Chromatography - HPLC
Additives/Contaminants: Veterinary drug residues - anabolic steroids
Food Types: Animal
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1 2 3 4 The detection of endogenous steroid abuse in cattle: results 5 6 from population studies in the UK 7 8 9 James P. Scarth a,d , Adam Clarke a, Philip Teale a , Aileen Mill b, Roy Macarthur b 10 11 and Jack Kay c 12 13 14 a HFL Sport Science (A Quotient Bioresearch Ltd. Company), Newmarket 15 16 Road, Fordham,For Cambridgeshire, Peer Review CB7 5WW, UK. Only 17 b 18 The Food and Environment Research Agency, Sand Hutton, York, UK. 19 c 20 Veterinary Medicines Directorate, Woodham Lane, New Haw, Addlestone, 21 22 Surrey, KT15 3LS. UK. 23 d Corresponding author: [email protected] 24 25 26 27 28 29 Abstract 30 The use of steroids as growth-promoting agents in food production is banned 31 32 under European Union legislation. Detecting the abuse of testosterone, 33 34 nandrolone, boldenone, oestradiol and progesterone is complicated by the 35 36 fact these steroids are known to be endogenous in certain situations. In this 37 study, the concentrations of characteristic metabolites of each of these 38 39 steroids have been quantified in populations of untreated steers and heifers. 40 41 Steroid concentration population data were then used by a statistical model 42 43 (the Chebyshev inequality) to produce threshold concentrations for screening 44 45 and confirming the abuse of these steroids in steer and non-pregnant heifer 46 urine. In addition to thresholds based on testing one animal (a ‘1 out of 1’ 47 48 approach), new methods based on testing multiple animals from a herd (an ‘y 49 50 out of n’ approach) allowed threshold concentrations to be significantly 51 52 reduced and hence false compliances to be minimised. In the majority of 53 cases, the suggested thresholds were found to be capable of confirming the 54 55 abuse of endogenous steroids in steers and heifers. In the case of oestradiol 56 57 abuse in the female, however, confirmation based on a threshold is not 58 59 possible and alternative methods such as gas-combustion-isotope ratio mass 60 spectrometry are required. In addition to the steer and heifer populations, a
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1 2 3 small number of pregnant animals were also tested, yielding insights into the 4 5 biosynthetic pathways of some of the steroids. 6 7 8 9 Keywords:- Natural, endogenous, steroids, cattle, bovine, testosterone, 10 11 nandrolone, boldenone, progesterone, oestradiol, threshold 12 13 14 15 16 IntroductionFor Peer Review Only 17 18 19 The use of steroids and other growth promoting agents is prohibited in food 20 21 production according to European Union legislation (EU Council Directive 22 23 96/22/EC). The abuse of synthetic steroids can be detected by qualitative 24 25 assays, but the detection of endogenous steroid use is more difficult because 26 27 a simple qualitative determination of their presence is insufficient for 28 demonstrating abuse (Scarth et al. 2009a). The direct detection of steroid 29 30 esters in hair or plasma (Boyer et al. 2007, Gray et al. 2010) and the use of 31 32 combustion isotope ratio mass spectrometry have been developed for the 33 34 confirming the abuse of certain endogenous steroids (Prevost et al. 2004), but 35 these methods are not always suitable as screening approaches. Therefore, 36 37 the majority of methods that have been developed in related fields such as 38 39 human and animals sports rely on steroids exceeding concentration 40 41 thresholds as a means of detecting their abuse (Scarth et al. 2009a). 42 43 44 We previously published a validated method for quantifying target analytes 45 46 indicative of the abuse of testosterone, nandrolone, boldenone, progesterone 47 48 and oestradiol in the bovine using a combination of GC- and LC-MS/MS 49 50 (Scarth et al. 2009b). In the current study, we have applied this method to 51 populations of untreated bovine animals in the UK in order to establish urinary 52 53 screening and confirmatory thresholds able to detect the abuse of these 54 55 steroids. 56 57 58 5β-androstane-3α,17 β-diol (BAB-androstanediol - Figure 1) was chosen as 59 60 the testosterone target testosterone metabolite as this analyte has been
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1 2 3 shown to be increased in the urine of testosterone treated animals (Biddle et 4 5 al. 2003). 6 7 8 9 Since it is not known which metabolite will be most useful for detection 10 11 nandrolone abuse, epinandrolone, 19-noretiocholanolone and 5 α-estrane- 12 3β,17 α-diol (ABA-estranediol) were all quantified for threshold purposes 13 14 (Figure 1). 15 16 For Peer Review Only 17 18 In order to detect boldenone abuse, the boldenone metabolite 17 β-hydroxy- 19 5β-androst-1-ene-3-one was targeted (Figure 1) since it has been shown to 20 21 be present in the urine of boldenone treated animals, but absent in untreated 22 23 animals (Biddle et al. 2005). However, in order to assess whether any 24 25 absence of detectable 17 β-hydroxy-5β-androst-1-ene-3-one in bovine urine 26 27 was due to it’s absence as an endogenous compound (in all situations) or 28 instead only absent because there was no endogenous epiboldenone that 29 30 could be metabolized through to 17 β-hydroxy-5β-androst-1-ene-3-one in the 31 32 particular animal populations studied, epiboldenone was also quantified. 33 34 35 Published data regarding progesterone metabolism in the bovine is lacking. 36 37 Therefore, 5 α-pregnane-3β,20 α-diol (ABA-pregnanediol – Figure 1), was 38 39 chosen as the target analyte as preliminary analyses showed it to be detected 40 41 in the urine of bovine steers at concentrations that fell within the calibration 42 range that was used for the other steroids. In order to detect the abuse of 43 44 oestradiol, its major metabolite epioestradiol was chosen as the target analyte 45 46 (Figure 1). The concentrations of epioestradiol in urine are much higher than 47 48 oestradiol (Biddle et al. 2007), meaning that they more readily quantified and 49 50 are also more suitable for confirmation using an auxiliary techniques such as 51 gas-combustion-isotope-ratio-mass-spectrometry (GC-C-IRMS). The 52 53 significance of epioestradiol’s suitability for GC-C-IRMS lies in the prediction 54 55 that the observed endogenous urinary concentrations would overlap with 56 57 those resulting from oestradiol administration, therefore making any threshold 58 only suitable to an initial screening approach. 59 60
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1 2 3 Materials and methods 4 5 6 7 Chemicals and reagents 8 9 The chemicals and reagents used were as described in Scarth et al. 2009b, 10 11 with the exception of the origin of some of the urine samples, for which details 12 are given below: 13 14 15 16 Urine samplesFor were Peer obtained inReview the UK from both Only live animals (various 17 18 university and research institute owned herds) and from animals going for 19 slaughter (organically farmed animals slaughtered for food production). Whilst 20 21 it is theoretically possible that some of the organically farmed animals may 22 23 have been illicitly treated with steroids, this is considered unlikely because 24 25 consent was voluntarily given from each individual producer for analysis of 26 27 each animal’s urine. Also, the steroid free provenance of all samples obtained 28 from university or research institute owned herds was guaranteed because 29 30 the animals were controlled under home office license regulations. For 31 32 samples where ‘unusually’ high steroid concentrations were observed, 33 34 attempts were made to trace the history of the animal in order to ascertain 35 whether any causal explanations could be obtained. An alternative would 36 37 have been to analyse the samples using gas chromatography-carbon-isotope 38 39 ratio mass spectrometry (GC-C-IRMS). However, existing methods were not 40 41 available for the analysis of the majority of the steroids under study. 42 43 44 A total of 190 heifer urine samples (requested to be from non-pregnant 45 46 animals) were obtained from animals ranging from 12 to 151 months of age 47 48 and a total of 220 steer urine samples were obtained from animals ranging 49 50 from 7 to 30 months of age. Exact information regarding the breed of every 51 animals was not available, but for steers the samples obtained at slaughter 52 53 were from a mix of different ‘beef’ animals intended for meat consumption, 54 55 whereas samples from live animals were from a mix of ‘bbx’ and ‘dairy cross’ 56 57 animals. For heifers, the samples obtained at slaughter were from a mix of 58 different ‘beef’ animals intended for meat consumption, whereas samples 59 60 from line animals were from a mix of ‘bbx’, ‘simx’, ‘Holstein-Friesian’ and ‘Welsh Black animals. A selection of urine samples from pregnant dairy
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1 2 3 animals were also obtained, but the results of these will be discussed 4 5 separately from the other population data as animals are known to produce 6 7 high concentrations of steroid during gestation. No urine samples from intact 8 9 males were available. 10 11 12 Samples were collected and stored chilled on ice within 1-hour. Samples on 13 14 ice were then delivered by courier within 48-hours before being frozen at - 15 o 16 80 C until Forthe time ofPeer analysis. Review Only 17 18 19 Preparation of calibration lines and QCs 20 21 Calibration lines and QCs were constructed for each batch using pooled urine 22 23 from castrated male bovine animals as a surrogate matrix (as described in 24 25 Scarth et al. 2009b). QC concentrations for androgens/progestagens were; 26 27 the endogenous augmented with 100 pg/mL (E+100), the endogenous 28 augmented with 1000 pg/mL (E+1000) and the endogenous augmented with 29 30 2000 pg/mL (E+2000). QC concentrations for oestrogens were; the 31 32 endogenous augmented with 80 pg/mL (E+80), the endogenous augmented 33 34 with 800 pg/mL (E+800) and the endogenous augmented with 1600 pg/mL 35 (E+1600). Each analytical batch contained two QCs at each of the three 36 37 aforementioned levels (total n=6) and for a batch to pass QC criteria, at least 38 39 four of the six QCs needed to be within +/- 20% of the nominally spiked 40 41 concentration, with no more than one QC failing at any one concentration. 42 43 44 Extraction and analysis of samples for steroid concentration 45 46 Samples were extracted by a combination of enzyme hydrolysis, liquid-liquid 47 48 extraction and solid-phase extraction and were then analysed by GC-MS/MS 49 50 as their enol-TBDMS derivatives on a Varian 320-MS system (as described 51 and validated by Scarth et al. 2009b). The limits of detection and 52 53 quantification of the assay are summarised in Table 1. 54 55 56 57 Previous studies have shown that the majority of endogenous steroids in the 58 bovine are excreted in urine as glucuronide conjugates (Scarth et al. 2009). 59 60 Therefore, the current method used glucuronide conjugate hydrolysis using recombinantly produced β-glucuronidase enzyme to measure the combined
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1 2 3 concentrations of free and glucuronide steroid. This is an important distinction, 4 5 since the use of alternative methods that hydrolyse all steroid conjugates 6 7 (including sulphates) could lead to higher calculated concentrations. If 8 9 applying the thresholds suggested in the current report, it will be necessary for 10 11 laboratories to measure ‘free and glucuronide’ concentrations in order to 12 ensure that their results are applicable. 13 14 15 16 ExtractionFor and analysis Peer of samples Review for creatinine Only concentration 17 18 In addition to quantifying the concentrations of steroid present, samples were 19 also analysed for creatinine content. Urinary creatinine was considered 20 21 potentially useful since it is a measure of the hydration status of the animal. It 22 23 was hypothesized that correction of urinary steroid concentration data for 24 25 creatinine content might lead to a reduction in the relative standard deviation 26 27 of the steroid population data and hence a reduction in the calculated 28 thresholds. An alternative to correcting steroid concentrations with creatinine 29 30 is to use the specific gravity (SG) of the sample instead; an approach already 31 32 applied in the human drug testing field (Mareck et al. 2007) and which is 33 34 currently being investigated for use in bovine drug residue surveillance 35 programmes in Australia (Wolfgang Korth, personal communication). 36 37 However, the creatinine approach was chosen for the current study as 38 39 preliminary experiments comparing the correlation of SG and creatinine with 40 41 the different steroid concentrations in a small sub-set of the samples 42 suggested that creatinine would be more suitable (data not shown). 43 44 45 46 Creatinine concentrations were quantified in each sample using a 47 48 manufacturer supplied assay kit on an Olympus AU640 automated clinical 49 50 analyser. The assay involves monitoring the absorbance at 520/800 nm 51 following the formation of a creatinine picrate complex after the addition of 52 53 picric acid (2.9 mmol/L) to samples in an alkaline medium (NaOH at 120 54 55 mmol/L. The rate of change of absorbance at this wavelength is proportional 56 57 to the creatinine concentration in the sample. 58 59 60
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1 2 3 Statistical analysis of the untreated animal population data . 4 5 Threshold calculations for a single animal within a herd 6 7 In order to control the abuse of each steroid, the statistical analysis set out to 8 9 suggest thresholds with various probabilities (1 in 20, 1 in 100, 1 in 1’000 and 10 11 1 in 10’000) of finding a larger value by chance in a natural population when 12 looking at the results from a single animal from a herd. Separate statistical 13 14 methods will be described in a later section that deals with thresholds based 15 16 on resultsFor of more thanPeer one animal Review within a herd. Fo Onlyr both types of threshold 17 18 calculation however, versions of the one-tailed version of the Chebyshev 19 inequality were used. This method is based on probability theory, makes 20 21 minimal assumptions about the distribution of the data and is described by 22 23 Estler (1997). Chebyshev confidence intervals are as far as possible from a 24 25 mean for a given standard deviation; they are distribution-independent 26 27 confidence intervals and are ideally suited to dealing with non-normally 28 distributed data such as those resulting from the current study. A one-tailed 29 30 Chebyshev confidence interval is given by: 31 32 33 1 34 p ≤ 35 1+ t 2 σ 2 36 37 38 Hence 39 40 2 σ 2 41 t ≤ −σ 42 p 43 44 Where , 45 46 47 t is the difference between the mean and concentration at the upper 48 confidence interval, 49 50 51 µ is the mean concentration, 52 2 53 σ is the variance (square of the standard deviation), 54 55 p is the probability that difference between the mean concentration and the 56 57 concentration of a sample taken at random is greater than t. 58 59 60
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1 2 3 To assess the extent to which concentration differed between steers and 4 5 heifers, a Monte Carlo permutation test was implemented (Manly, 2007). 6 7 Here, gender was permuted amongst individuals and the mean difference in 8 9 concentration of each steroid metabolite between steers and heifers was 10 11 calculated for each permutation. The observed difference was then compared 12 to the distribution of mean differences for the 1000 permutations and 13 14 significance assessed on the basis of the position of the observed mean in the 15 16 distribution.For Peer Review Only 17 18 19 20 Thresholds for results from more than one animal sampled from a herd 21 22 In addition to thresholds based on steroid concentration results from a single 23 24 animal, it is also possible to produce thresholds based on more than one 25 26 animal within a herd having a result above a certain steroid concentration i.e. 27 from ‘2 out of 2’ or ‘3 out of 3’ animals tested (‘y out of n’ animals). The 28 29 rationale for this approach is that if a steroid is being abused by a farmer, it is 30 31 likely to be the majority of the herd that are given the drug, not just a single 32 33 animal. Since threshold concentrations based on a ‘y out of n’ approach are 34 significantly lower than thresholds based on a single animal, the ‘y out of n’ 35 36 approach could lead to a lower false compliance rate for detecting steroid 37 38 abuse. Whilst this approach may be sufficiently robust for a screening 39 40 approach in all cases, using it for confirming steroid abuse requires that 41 steroid concentrations are not subject to ‘clustering’ within herds. Whilst the 42 43 authors are not aware of any evidence of steroid clustering within steer 44 45 populations, a biological phenomenon known as the ‘Whitten effect’ (Aron et 46 47 al. 1979) has been reported in female mammals. This phenomenon leads to 48 49 the synchronisation of oestrous in female animals and if it is accepted that 50 some of the high steroid concentration results within a population are caused 51 52 by oestrous, then this would class as clustering and could lead to false non- 53 54 compliance results using the ‘y out of n’ approach. Therefore, while ‘y out of n’ 55 56 calculations were performed for both steers and heifers for application as a 57 screening approach it is suggested that the resulting thresholds only be used 58 59 for steers where confirmation is concerned. 60
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1 2 3 In terms of the statistical methods, it is possible to derive thresholds which 4 5 apply to ’y out of n’ animals tested using a modification of the Chebyshev 6 7 method. For example, given a population containing analyte with mean µ and 8 9 standard deviation σ, the relation between the probability p1,1 of testing a 10 11 single animal and observing a result above x 1,1 12 13 14 2 15 σ 2 16 For Peerx 1,1 ≤Review + − σ Only 17 p 1,1 18 19 20 21 The upper limit to probability of observing at least y samples above x 1,1 out of 22 23 n tested is given by: 24 25 26 i=n n 27 i n−i p y,n = p 1,1 ()1− p 1,1 28 ∑ i= y i 29 30 with the particular case 31 32 33 n 1 n 34 p n,n = p 1,1 , p 1,1 = p n,n 35 36 37 38 Hence, if n animals out of n tested contain a concentration of analyte above 39 40 xn,n then this is evidence that the distribution is not consistent (with false non- 41 compliance rate p) with the population ( ,σ) where, 42 43 σ 2 44 x ≤ + −σ 2 45 n,n /1 n p 1,1 46 47 48 49 At this state, it is important to clarify that a positive ‘y out of n’ result such as 50 51 finding ‘2 out of 2’ samples above a threshold is different from finding 2 results 52 53 from a larger sampled population i.e. ‘2 out of 10’ above a threshold. If the 54 latter approach were used, then different statistical equations would be 55 56 required as this would change the probabilities. 57 58 59 60 Uncertainty of measurement at threshold concentrations
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1 2 3 For thresholds based on the single animal or the ‘y out of n’ approach, 4 5 uncertainty of measurement was calculated using a Monte Carlo sample 6 7 (Manly, 2007) from the distribution consistent with the observed result along 8 9 with the analytical performance observed at validation (Scarth et al. 2009b). 10 11 1000 samples were taken from these distributions and 1000 Chebyshev 12 threshold estimates were generated. The different probabilities of finding a 13 14 value by chance in a ‘natural’ population can then be reported at the upper 15 16 95% quantileFor results Peer after factoring Review in the determined Only method uncertainty. 17 18 Values for CC α and CC β on these probabilities (Commission Decision 19 2002/657/EC), again at the 95% confidence interval, were also calculated for 20 21 the analytical method used. 22 23 24 25 26 27 Results and discussion 28 29 30 31 All raw data and calculated threshold concentrations in the following section 32 are uncensored and are given to one decimal place. All other summary 33 34 statistics are presented to three significant figures. 35 36 37 38 Correlation of creatinine concentration with steroid concentrations 39 To assess whether creatinine would be a suitable indicator of hydration, the 40 41 concentration of each steroid was divided by the concentration of creatinine 42 43 measured. Figure 2 plots urinary steroid concentrations against creatinine 44 45 concentration while Table 2 displays the relative standard deviation of the 46 47 population data for each steroid with and without correction for creatinine 48 concentration. Since no reduction in relative standard deviation was observed 49 50 on correction of data for creatinine concentration, creatinine was not used in 51 52 any of the further statistical analyses. It is possible that creatinine is not a 53 54 good marker of hydration status or that variable hydration is less of a factor in 55 regions with a moderate climate, such as the UK, as compared to warmer 56 57 countries like Australia. It is also possible that the steroid concentrations 58 59 themselves are less influenced by hydration status as compared to other 60 determinants such as stress or oestrous.
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1 2 3 4 5 6 7 8 9 Population steroid data and determined thresholds 10 11 12 Population results 13 14 Summary statistics for the determined concentrations of each steroid in steer 15 16 and heiferFor populations Peer are given Review in Table 3, while histogramsOnly of the data are 17 18 shown in Figures 3 to 10. 19 20 21 For all steroids other than 17 β-hydroxy-5β-androst-1-ene-3-one, mean and 22 23 maximum steroid concentrations in both steers and heifer were above the 24 25 limits of detection and quantification and hence it was possible to produce well 26 27 characterised population histograms. The mean concentrations of 28 epioestradiol, ABA-estranediol, BAB-androstanediol, epiboldenone and ABA- 29 30 pregnanediol were determined to be statistically different between steer and 31 32 heifer populations (Monte carlo permutation test P<0.05, 1000 permutations). 33 34 19-noretiocholanolone and epinandrolone concentrations, however, were 35 determined not to be statistically significantly different between steers and 36 37 heifers (Monte carlo permutation test P<0.05, 1000 permutations). Although 38 39 the mean concentrations of these two steroids were deemed not to be 40 41 statistically significantly different, there may still have been differences in the 42 relative standard deviations for the sexes. However, this situation is difficult to 43 44 test when dealing with non-parametric data such as these. This is especially 45 46 important considering that the Chebyshev method used to calculate the 47 48 thresholds is heavily influenced by the variance within the data. 49 50 51 The most obvious differences in steroid concentrations between the sexes 52 53 were for BAB-androstanediol, epioestradiol and ABA-pregnanediol. BAB- 54 55 androstanediol concentrations were much higher and more variable in steers, 56 57 while epioestradiol and ABA-pregnanediol concentrations were much higher 58 and more variable in heifers (Table 3). For epioestradiol and ABA- 59 60 pregnanediol, this was an expected outcome since females produce large quantities of oestrogens and progestagens at certain stages of oestrous
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1 2 3 (Hadley and Levine, 2006). For BAB-androstanediol in the steers however, 4 5 the higher concentrations were unexpected, since these males were reported 6 7 to be castrated and hence androgen output would theoretically have been 8 9 restricted to adrenal output. It is possible, however, that some of the ‘steers’ 10 11 may have retained residual gonadal tissue, such as an undescended testes, 12 and therefore occasionally registered a high BAB-androstanediol 13 14 concentration. 15 16 For Peer Review Only 17 18 For 17 β-hydroxy-5β-androst-1-ene-3-one, all but one determined 19 concentration were below the limit of detection and all sample concentrations 20 21 were well below the lower limit of quantification (LLOQ). In the case of the one 22 23 sample that was above the limit of detection of 160.7 pg/mL, its concentration 24 25 was determined at only 161.4 pg/mL. Because this determined concentration 26 27 is below the LLOQ and is so close to the LOD, it is possible that the result is a 28 false non-compliance caused by analytical background noise. The later 29 30 section on screening and threshold approaches will discuss the implication of 31 32 this finding in more detail. 33 34 35 Other than in pregnant animals, the highest concentrations of several of the 36 37 steroids were observed in one of the non-pregnant heifers and one of the 38 39 steers from a research owned herd (therefore guaranteeing the untreated 40 41 status of the animals). The owners of the herd were contacted in order to 42 clarify that the information provided about the animal was correct. It was 43 44 confirmed that the sex and other information regarding these animals was 45 46 correct and that the heifer was not pregnant at the time of sampling. However, 47 48 while the heifer was not considered unfit for consumption at the time of 49 50 sampling, it was found to suffering from an unknown illness several months 51 later. Since it would have been treated the same as any other animal as far as 52 53 routine urine sampling was concerned, the results from this animals were 54 55 included within the population dataset for threshold determination purposes. 56 57 58 In pregnant animals, the concentrations of all steroids other than 17 β-hydroxy- 59 60 5β-androst-1-ene-3-one were considerably higher than the non-pregnant animals (not determined statistically – Table 3). This was an expected
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1 2 3 outcome, since steroid output during pregnancy is known to be increased (De 4 5 Brabander et al. 1994). An interesting observation was that epiboldenone 6 7 concentrations in the pregnant animals were far in excess of those in the non- 8 9 pregnant animals. This may reflect an increased endogenous production of 1- 10 11 dehydro-steroids during pregnancy, possibly related to the process of 12 aromatization. Alternatively, it may reflect an increased conversion in the gut 13 14 due to an increased availability of precursor steroids for bacteria to act upon. 15 16 Even thoughFor the concentrationsPeer Review of epiboldenone wereOnly high in the pregnant 17 18 animals (reaching 3’725.0 pg/mL), there were no results above the limit of 19 detection for 17 β-hydroxy-5β-androst-1-ene-3-one, thus supporting the 20 21 suitability of this analyte as an indicator of boldenone administration only. 22 23 24 25 Screening and confirmatory threshold approaches 26 27 The general principle of adopting a threshold concentration to confirm the 28 abuse of an endogenous compound is an accepted method in both food 29 30 production (Heitzman, 1994) and animals sports (Houghton and Crone 2000). 31 32 If used formally to confirm the abuse of a proven endogenous steroid, the 33 34 standard approach is to set the threshold at a statistical probability of finding a 35 false non-compliance at a rate of 1 in 10’000 in a natural population 36 37 (Houghton and Crone 2000), since this is considered to offer a sufficiently 38 39 large safety margin to prevent the occurrence of false non-compliances. 40 41 42 Thresholds for screening may be set at a lower probability, but there then 43 44 needs to be a secondary mechanism for confirmation if the confirmatory 45 46 threshold is not also breeched. Typically, this may include follow-up analyses 47 48 using gas chromatography carbon isotope mass spectrometry (GC-C-IRMS) 49 50 (Prévost et al. 2004), detection of an intact steroid ester in hair or plasma 51 (Boyer et al. 2007, Gray et al. 2010) or an on-farm inspection (Jack Kay, 52 53 personal observation). In an ideal world, a confirmatory threshold would also 54 55 be suitable as a screening threshold, but this requires that the threshold is 56 57 able to produce both low rates of false compliance and non-compliance; an 58 ideal that is seldom achieved. 59 60
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1 2 3 If using the ‘2 out of 2’ instead of the traditional ‘1 out of 1’ (single animal) 4 5 approach for screening, it is suggested that a 1 in 10’000 probability be used 6 7 since the use of a lower probability such as 1 in 1’000 might lead to Minimum 8 9 Residue Performance Limits (MRPLs) that are below many laboratory’s 10 11 capabilities or concentrations that cannot be followed by GC-C-IRMS. 12 13 14 For all steroids where endogenous concentrations were quantified above the 15 16 LLOQ, thereforeFor excluding Peer 17 β -hydroxy-5Reviewβ-androst-1-ene-3-one, Only a threshold 17 18 for confirmation at a false probability of 1 in 10’000 is suggested as this is in 19 line with the approach in other industries as described above. For all steroids 20 21 other than 17 β-hydroxy-5β-androst-1-ene-3-one, steer and heifer thresholds 22 23 were calculated separately, regardless of whether there were any statistically 24 25 significant differences between the sexes. The reason for this approach is 26 27 because there are known physiological variations between males and females 28 (Hadley and Levine, 2006) that could theoretically lead to different steroid 29 30 concentrations and a simple comparison of the mean endogenous 31 32 concentrations of the sexes is not sufficient alone to justify grouping them 33 34 together. 35 36 37 The fact that 17 β-hydroxy-5β-androst-1-ene-3-one concentrations in all 38 39 samples were below the LLOQ was a desirable outcome since it was 40 41 hypothesized that this analyte would be seen following the administration of 42 boldenone but not in untreated populations. A simple approach to 43 44 confirmation of abuse would then be to say that because no concentrations 45 46 were detected above the LLOQ, then the presence of any concentration of 47 48 this analyte above the LLOQ in real samples could be used as demonstration 49 50 of boldenone abuse. However, a safer approach for a potentially endogenous 51 analyte such as 17 β-hydroxy-5β-androst-1-ene-3-one would be to treat the 52 53 determined concentrations (which may effectively be thought of as 54 55 instrumental noise since the majority are below the LOD and all are below the 56 57 LLOQ) according to the Chebyshev threshold approach. Applying this ‘ultra- 58 safe’ Chebyshev threshold approach to the analytical noise, a probability of 1 59 60 in 1’000, rather than the 1 in 10’000 used for the other analyte where endogenous concentrations are proven to exist above the LLOQ, is
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1 2 3 considered more than adequate. Because no concentrations of this analyte 4 5 were above the LLOQ of the assay, the male and female data (uncensored for 6 7 concentrations below the LOD and LLOQ) were treated as one population for 8 9 the purposes of the threshold calculations. 10 11 12 The estimated Chebyshev thresholds of finding a value by chance in a 13 14 ‘natural’ population at different probabilities are given in Tables 4, 5 and 6 and 15 16 are reportedFor as the Peer ‘95% confidence Review interval,’ sign Onlyifying that they are the 17 18 upper estimates for the thresholds after factoring in the method’s uncertainty 19 of measurement as described in the methods section. Table 5 also lists the 20 21 CC α and CC β values (decision and detection limits respectively) relating to 22 23 these thresholds when using the analytical method presented here. 24 25 26 27 For all the analytes, the use of the ‘2 out of 2’ and ‘3 out of 3’ animal 28 approaches lead to concentration thresholds approximately 10-and 20-fold 29 30 lower respectively compared to the traditional ‘1 out of 1’ (single animal) 31 32 approach. However, although the larger ‘y out of n’ approach allows for a 33 34 reduction in the concentration threshold, the risk of false compliances 35 increases with n due to potential inter-individual variation in excretion profiles. 36 37 Therefore, it is suggested that only the ‘2 out of 2 ’approach be used to 38 39 supplement the traditional‘1 out of 1’ methods as this is predicted to offer the 40 41 best balance of a low enough concentration threshold without risking false 42 compliances due to inter-individual variation. 43 44 45 46 In order to use these threshold data to design pragmatic screening and 47 48 confirmatory approaches, it is necessary to relate the threshold 49 50 concentrations to those obtained following the administration of different 51 steroids in order to ascertain whether a sufficiently low rate of false 52 53 compliances can be achieved. Steroid ‘post-administration’ data for the 54 55 different metabolites were available for boldenone, nandrolone, oestradiol and 56 57 testosterone, but not for progesterone. 58 59 60
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1 2 3 Threshold approaches for regulating boldenone abuse 4 5 Biddle et al. reported a quantitative boldenone metabolism study in the bovine 6 7 in 2005. Two steers were treated with a 700 mg boldenone undecylenate 8 9 intra-muscular (IM) injection and urine collected for 56 days. The 10 11 concentrations of epiboldenone were quantified throughout the excretion 12 study. Since the concentrations of 17 β-hydroxy-5β-androst-1-ene-3-one could 13 14 not be quantified in this previous study due to a lack of availability of reference 15 16 standards,For three of thePeer post administration Review urine samples, Only those collected pre- 17 18 dose and then at 2 and 12 days post-dose from both animals, were re- 19 analysed in the current study in order to quantify both 17 β-hydroxy-5β- 20 21 androst-1-ene-3-one and epiboldenone. 22 23 24 25 The pre-dose concentrations of epiboldenone as determined by Biddle et al. 26 27 in 2005 were http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 17 of 46 Food Additives and Contaminants 1 2 3 hydroxy-5β-androst-1-ene-3-one respectively. The fact that the majority of 4 5 these metabolite concentrations were conjugated is significant since previous 6 7 studies (European Commission, 2003) have suggested that boldenone 8 9 metabolites arising from faecal contamination are uncojugated. 10 11 12 The suggested screening and confirmatory Chebyshev thresholds for 13 14 regulating boldenone abuse are given in Tables 7 and 8 respectively. All 15 16 these 17 βFor-hydroxy-5 Peerβ-androst-1-ene-3-one Review and epiboldenone Only thresholds are 17 18 significantly lower than those observed post boldenone-administration, 19 meaning a low false compliance rate in a real screening environment is likely. 20 21 22 23 For confirmation of boldenone abuse using the threshold approach, it is 24 25 suggested that the 1 in 1’000 threshold for ‘1 out of 1’ animals with 17 β- 26 27 hydroxy-5β-androst-1-ene-3-one as the target analyte be employed and that 28 the concentration be determined as the amount of conjugated steroid in order 29 30 to prevent any false non-compliances caused by the potential presence of 31 32 free analyte through faecal contamination. The use of the ‘2 out of 2’ 33 34 approach for confirming boldenone abuse is not recommended as clustering 35 of findings of metabolites related to boldenone has been reported in the past 36 37 (De Brabander et al. 2004). The use of epiboldenone as a confirmatory 38 39 analyte is not recommended as the endogenous nature of this analyte is still 40 41 subject to debate (European Commission, 2003). If the concentration 42 thresholds are adopted as screening thresholds only, and if confirmation is 43 44 based on GC-C-IRMS, then the use of epiboldenone as the target analyte for 45 46 the GC-C-IRMS analysis is suggested since it is the metabolite of the highest 47 48 abundance. 49 50 51 Threshold approaches for regulating nandrolone abuse 52 53 Biddle et al. reported a quantitative nandrolone metabolism study in the 54 55 bovine in 2003. Three steers and three heifers were treated with a 200 mg 56 57 nandrolone laurate intra-muscular (IM) injection and urine collected for 24 58 days. The concentrations of epinandrolone, ABA-estranediol and 19- 59 60 noretiocholanolone were quantified in heifers and epinandrolone and ABA- estranediol in steers throughout the excretion study. In both heifers and http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 18 of 46 1 2 3 steers, the relative concentrations of the different analytes varied between 4 5 individuals such that epinandrolone was the major metabolite in some animals 6 7 and ABA-estranediol or 19-noretiocholanolone the major metabolite in others. 8 9 However, within the excretion profile of each individual animal, the relative 10 11 proportions of the different metabolites stayed relatively constant as the 12 concentrations dropped off over the urine collection period. Peak 13 14 concentrations of each analyte were generally achieved after around 3 days 15 16 and then Forstayed level Peer or only tailed Review off very slowly forOnly the following 2-weeks, 17 18 before dropping off at a faster rate between weeks 2 and 3. Up to 2-weeks 19 post-dose, concentrations of epinandrolone in heifers varied from around 20 21 15’000 to 50,000 pg/mL, while in males varied from around 6’000 to 23’000 22 23 pg/mL. Concentrations of ABA-estranediol in heifers varied from around 24 25 11’000 to 25’000 pg/mL, while in males varied from around 6’000 to 13’000 26 27 pg/mL. Concentrations of 19-noretiocholanolone in heifers varied from around 28 5’000 to 19’000 pg/mL, while in males the concentrations were not quantified. 29 30 31 32 The suggested screening and confirmatory Chebyshev thresholds for 33 34 regulating nandrolone abuse are given in Tables 7 and 8 respectively. By 35 monitoring all three nandrolone metabolites, whose relative abundance varies 36 37 between different individuals, these screening and confirmatory approaches 38 39 were able to detect nandrolone abuse in each of the animals reported by the 40 41 Biddle et al. 2003 study, meaning a low false compliance rate in a real 42 screening environment is likely. 43 44 45 46 For confirmation of nandrolone abuse in steers using the threshold approach, 47 48 it is suggested that the 1 in 10’000 thresholds for both the ‘1 out of 1’ and ‘2 49 50 out of 2’ approach for each of epinandrolone, ABA-estranediol or 19- 51 noretiocholanolone be adopted. It would only be necessary for a sample or 52 53 pair of samples to register a concentration above any one of these thresholds 54 55 in order to trigger a positive confirmatory, thus allowing for any variation in 56 57 metabolite profiles between individuals. However, it is now know that bovine 58 animals may excrete increased concentrations of nandrolone and 59 60 epinandrolone following casualty and during pregnancy (Kennedy et al. 2009). It is therefore recommended that such animals not be subjected to the http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 19 of 46 Food Additives and Contaminants 1 2 3 threshold approaches for nandrolone unless additional marker metabolites 4 5 which are specific for nandrolone administration also be detected. In this 6 7 respect, Pinel et al. (2010) have recently discovered that 5 α-estrane-3β,17 β- 8 9 diol (ABB-estranediol) and 5 β-estrane-3α,17 β-diol (BAB-estranediol) are 10 11 detected following administration of nandrolone, but not during pregnancy or 12 following injury (Gaud Pinel, personal communication). It is therefore 13 14 recommended that these additional ‘marker’ metabolites should also be 15 16 detected qualitativelyFor Peer in pregnant Review or injured anima lsOnly whose samples exceed 17 18 one of the specified nandrolone thresholds before the result is considered 19 non-compliant. 20 21 22 23 If the concentration thresholds are adopted as screening thresholds only, and 24 25 if confirmation is based on GC-C-IRMS instead, then the use of epinandrolone 26 27 as the target analyte for the GC-C-IRMS analysis is suggested since it is the 28 metabolite of the highest abundance. 29 30 31 32 Threshold approaches for regulating testosterone abuse 33 34 iddle et al. reported a quantitative testosterone metabolism study in the bovine 35 in 2003. Three steers and three heifers were treated with a 200 mg 36 37 testosterone propionate sub-cutaneous ear implant and urine collected for 24 38 39 days. The concentrations of several testosterone metabolites, including BAB- 40 41 androstanediol in male samples only, were quantified throughout the excretion 42 study. In steers, the concentrations of BAB-androstanediol, BBA- 43 44 androstanediol and epietiocholanolone appeared to increase slightly following 45 46 testosterone administration, reaching maximum concentrations of 5’720, 47 48 5’110 and 3’520 pg/mL respectively. Following this previous study, BAB- 49 50 androstanediol was chosen as a target analyte for the current study as a 51 deuterated internal standard analogue was available. In females, the 52 53 concentrations of the different metabolites did not increase significantly 54 55 relative to pre-dose concentrations, but BAB-androstanediol was not 56 57 quantified, so no post-administration data is available for this analyte. The 58 lack a significant increase in testosterone metabolite concentrations in the 59 60 previous study was unexpected, since the dose of testosterone given was relatively large (200 mg). Although these results may reflect a ‘typical’ post- http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 20 of 46 1 2 3 testosterone administration, it is also possible that the particular steroid 4 5 treatment did not work correctly on this occasion and therefore lead to atypical 6 7 results. Further testosterone administration studies would be required to 8 9 clarify the situation. 10 11 12 The suggested screening and confirmatory Chebyshev thresholds for 13 14 regulating testosterone abuse are given in Tables 7 and 8 respectively. Until 15 16 further testosteroneFor Peer studies are Review carried out and th eOnly resulting urine samples 17 18 analysed for BAB-androstanediol concentrations, it is not possible to give a 19 definitive answer as to the percentage false compliance rate of using BAB- 20 21 androstanediol as a marker analyte. 22 23 24 25 Threshold approaches for regulating progesterone abuse 26 27 The suggested screening and confirmatory Chebyshev thresholds for 28 regulating testosterone abuse are given in Tables 7 and 8 respectively. 29 30 Published data regarding progesterone metabolism in the bovine is lacking. 31 32 Therefore, 5 α-pregnane-3β,20 α-diol was chosen as the target analyte as 33 34 preliminary analyses showed it to be detected in the urine of bovine steers at 35 concentrations that fell within the calibration range that was used for the other 36 37 steroids. However, until progesterone studies are carried out and the resulting 38 39 urine samples analysed for ABA-pregnanediol concentrations, it is not 40 41 possible to give a definitive answer as to the percentage false compliance rate 42 of using ABA-pregnanediol as a marker analyte. 43 44 45 46 Threshold approaches for regulating oestradiol abuse 47 48 Biddle et al. reported a quantitative oestradiol metabolism study in the bovine 49 50 in 2003. Three steers and three heifers were treated with a 43.9 mg oestradiol 51 sub-cutaneous ear implant and urine collected for 24 days. The 52 53 concentrations of epioestradiol were quantified throughout the excretion 54 55 study. In steers, time of peak epioestradiol concentration varied between day 56 57 0 and day 3 post-dose. Two of the three animals produced peak 58 concentrations of 14’452.0 and 22’450.8 pg/mL, but in the third animal 59 60 reached only 4’668.8 pg/mL. In the two animals with the highest peak epioestradiol concentrations, levels remained above 10’000 pg/mL for two http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 21 of 46 Food Additives and Contaminants 1 2 3 days, before then dropping down to around 6’000 pg/mL by day 23. However, 4 5 for the third animal, epioestradiol concentrations dropped off rapidly such that 6 7 by day 15 they were only 103.5 pg/mL. As with the testosterone implant data 8 9 described earlier, it is possible that in this third animal the implant was less 10 11 successfully inserted, thus leading to a different profile compared to the other 12 two animals. In the female animals, time of peak epioestradiol concentration 13 14 varied between day 2 and day 8 post-dose, with concentrations of 21’713.0, 15 16 29’962.1 For and 39’743.1 Peer pg/mL Review in the three different Only animals respectively. 17 18 Concentrations then dropped off relatively slowly, such that by day 24, 19 concentrations in the three animals were 12’201.6, 2’650.8 and 21’848.8 20 21 pg/mL respectively. 22 23 24 25 The suggested screening and confirmatory Chebyshev thresholds for 26 27 regulating oestradiol abuse are given in Tables 7 and 8 respectively. Unlike 28 the previous analytes, the ‘two of two’ screening thresholds for epioestradiol 29 30 are not derived from 1 in 10’000 probabilities since these may lead to an 31 32 unacceptably high level of false compliances. A pragmatic approach based on 33 34 setting the threshold at the highest level that is predicted to be capable of 35 detecting abuse was therefore adopted based on knowledge of epioestradiol 36 37 post-administration concentrations. In this respect, the screening thresholds 38 39 given in Table 7 would be able to detect the abuse of epioestradiol in the 40 41 majority of the post-administration samples for several days post-dose and 42 would still be predicted to lead to a relatively low false non-compliance follow- 43 44 up rate. 45 46 47 48 In terms of confirming oestradiol abuse, the approaches given in table 8 for 49 50 steers are likely to detect a proportion of abuse cases for several days post- 51 dose, but the threshold concentrations in females are unlikely to ever be 52 53 reached following oestradiol administration. This is due to the extremely high 54 55 concentrations of epioestradiol found naturally in some animals and is most 56 57 likely due to the increase in females during the oestrous cycle. Therefore, in 58 the opinion of the authors, the most pragmatic approach for females would be 59 60 to screen according to the thresholds given in Table 7 and then confirm using GC-C-IRMS. As with all the other analytes, the chances of detecting http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 22 of 46 1 2 3 oestradiol abuse in ‘real’ populations would be greatly enhanced by 4 5 conducting ‘on-farm’ sampling rather than ‘at slaughter’ testing. That way, the 6 7 chances of detecting the abuse of a steroid closest in time to the point of 8 9 administration of the drug, where urinary concentrations would be highest, 10 11 would be maximized. 12 13 14 Factors for consideration when applying suggested threshold concentrations 15 16 Pregnant Foranimals and Peer intact male Review samples were not Onlyincluded in the population 17 18 statistics, so the approaches suggested in this report should only be applied 19 to steers and heifers. The ages of the animals used varied from 7 to 30 20 21 months for steers and from 12 to 151 months for heifers, so these are the age 22 23 ranges for which the determined threshold concentrations are applicable. 24 25 26 27 Animals from a wide range of breeds from within the UK were analysed in the 28 current study. Considering the large degree of separation between the highest 29 30 concentrations observed in these animals and the resulting Chebyshev 31 32 threshold concentrations, it would seem reasonable to apply the thresholds to 33 34 animals of all breeds within the UK. However, one previous study has shown 35 a marginal, but statistically significant, difference in testosterone 36 37 concentrations for one breed. Plusquellec et al. (2001) showed that lactating 38 39 animals of the Herens breed, which were artificially selected for fighting ability, 40 41 had higher median plasma testosterone concentrations than animals of the 42 Brune des Alpes breed, with concentrations of 0.21 and 0.11 ng/mL 43 44 respectively. It would therefore seem sensible to exclude animals of the 45 46 Herens breed, since there is a scientific rationale for why these may have 47 48 increased androgen concentrations. 49 50 51 The current method used glucuronide conjugate hydrolysis using 52 53 recombinantly produced β-glucuronidase enzyme to measure the combined 54 55 concentrations of free and glucuronide steroid. Therefore, when applying the 56 57 thresholds suggested in the current report, it will be necessary for laboratories 58 to measure ‘free and glucuronide’ concentrations in order to ensure that their 59 60 results are applicable. Laboratories will also need to calculate their own CC α and CC β values before applying the methods. http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 23 of 46 Food Additives and Contaminants 1 2 3 4 5 6 7 Conclusion 8 9 In this study, an extensive population study of natural steroid concentrations 10 11 in bovine urine samples obtained in the UK has been conducted and 12 strategies for screening and confirming the abuse of boldenone, nandrolone, 13 14 testosterone, progesterone and oestradiol in the bovine have been suggested 15 16 (Tables 7For and 8). Peer The threshold Review approach is predict Onlyed to be useful for 17 18 confirming the abuse of nandrolone and boldenone in steers and heifers and 19 oestradiol in steers, but can only be used as a screening indication in the case 20 21 of oestradiol in heifers. More data regarding the post-administration 22 23 concentrations of testosterone and progesterone metabolites are required 24 25 before the usefulness of the proposed thresholds for these steroids can be 26 27 fully tested. 28 29 30 When using the data presented, two broad choices are available. If a 31 32 regulatory body chooses to use the thresholds as a screen only, then the 33 34 threshold concentrations suggested in Table 7 could be followed up by 35 confirmation with GC-C-IRMS (Prevost et al. 2004), an on-farm inspection 36 37 and/or another non-threshold approach such as the detection of intact steroid 38 39 esters in hair or plasma (Boyer et al. 2007, Gray et al. 2010). If, however, a 40 41 regulatory body chooses to base both screening and confirmation on the 42 concentration threshold approach, then the threshold concentrations 43 44 suggested in Table 8 could be applied. It would, of course, be possible for 45 46 regulatory bodies to use screening thresholds lower than those given in Table 47 48 7 in order to further minimize the number of false compliances (using the data 49 50 in Tables 4 to 6 as a guide), but this would be likely to lead to an increased 51 number of false non-compliances that could not be confirmed using GC-C- 52 53 IRMS or other techniques. 54 55 56 57 In line with approaches used in other industries (Houghton and Crone 2000), 58 it is suggested that the 1 in 10’000 false non-compliance probability approach 59 60 for ‘1 out of 1’ and ‘2 out of 2’ animal approaches be used for confirmation according to the thresholds given in Table 8. The only exception to the 1 in http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 24 of 46 1 2 3 10’000 probability would be for 17 β-hydroxy-5β-androst-1-ene-3-one, where it 4 5 is suggested that only a 1 in 1’000 probability is required due to the already 6 7 ‘safe’ nature of applying the Chebyshev threshold approach to a population 8 9 where all sample concentrations were below the LLOQ. 10 11 12 In addition to comparing the concentration of each individual steroid against 13 14 the thresholds, it is suggested that the overall steroid profile of the animal 15 16 should alsoFor be inspected Peer as an Reviewadditional safety ch eckOnly to rule out false non- 17 18 compliances due to unusual medical conditions etc. In this context, an 19 unusual finding would be one where a large number of steroids are high in 20 21 concentration; a situation that would not be expected following the 22 23 administration of one or two different steroids. Specifically relating to 24 25 nandrolone, either 5 α-estrane-3β,17 β-diol (ABB-estranediol) or 5 β-estrane- 26 27 3α,17 β-diol (BAB-estranediol) should also be detected qualitatively in 28 pregnant or injured animals whose samples exceed one of the specified 29 30 nandrolone thresholds before the result is considered non-compliant. 31 32 33 34 Acknowledgements 35 This study was funded by the Department for Environment, Food and Rural 36 37 Affairs. 38 39 40 41 References 42 43 44 Aron, C. (1979). Mechanisms of control of the reproductive function by 45 46 olfactory stimuli in female mammals. Physiological Reviews, 59 (2), 229-284. 47 48 49 Biddle, S., Teale, P., Robinson, A., Bowman, J., & Houghton, E. (2007). Gas 50 51 chromatography-mass spectrometry/mass spectrometry analysis to determine 52 53 natural and post-administration levels of oestrogens in bovine serum and 54 55 urine. Analytica Chimica Acta, 586 (1-2 SPEC. ISS.), 115-121. 56 57 58 Biddle, S. et al. (2003). Unpublished studies on the natural occurrence of 59 60 androgens and estrogens in bovine plasma, urine and bile and the effect of http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 25 of 46 Food Additives and Contaminants 1 2 3 exogenous steroid administration on these profiles (HFL study HFL086). Work 4 5 carried out at HFL ltd. UK. 6 7 8 9 Biddle, S. et al. (2005). Unpublished studies on the natural occurrence of 10 11 boldenone in bovine urine and the metabolism of boldenone after 12 administration (HFL study HFL1382). Work carried out at HFL ltd. UK. 13 14 15 16 Boyer, S.,For Garcia, Peer P., Popot, M.Review A., Steiner, V. Only and Lesieur, M. (2007). 17 18 Detection of testosterone propionate administration in horse hair samples. 19 Journal of Chromatography B. 852, 684-688. 20 21 22 23 De Brabander, H. F., van Hende, J., Batjoens, P., Hendriks, L., Raus, J., & 24 25 Smets, F. et al. (1994). Endogenic nortestosterone in cattle? Analyst, 119 (12), 26 27 2581-2585. 28 29 30 De Brabander, H. F., Poelmans, S., Schilt, R., Stephany, R. W., Le Bizec, B., 31 32 & Draisci, R. et al. (2004). Presence and metabolism of the anabolic steroid 33 34 boldenone in various animal species: A review. Food Additives and 35 Contaminants, 21 (6), 515-525. 36 37 38 39 Estler, W. T. (1997). A distribution-independent bound on the level of 40 41 confidence in the result of a measurement. Journal of Research of the 42 National Institute of Standards and Technology, 102 (5), 587-588. 43 44 45 46 European Commission, Health and Consumer Protection Directorate-General, 47 48 Outcome of the experts meeting on the control of Boldenone in veal calves, 49 th 50 Brussels, 30 September 2003, pp. 1-3. 51 52 53 European Union. 1996a. Council Directive 96/22/EC.Off J Eur Union L125 23 54 55 May 1996. Council Directive96/22/EC of 29 April 1996 concerning the 56 57 prohibition on the use in stock farming of certain substances having a 58 hormonal or thyrostatic action and of beta-agonists, and replacing Directives 59 60 81/602/EEC, 88/146/EEC and88/299/EEC. p. 3–9. http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 26 of 46 1 2 3 European Union. 2002. Council Decision 2002/57/EC. Off. J Eur Comm L221. 4 5 Commission Decision (2002/57/EC) of 12 August 2002. p. 8–36. 6 7 8 9 Gray, B., Pearce, C. and Teale, P. (2010). The development of a screening 10 th 11 method for anabolic steroid esters in plasma. In: Proceedings of the 18 12 International Conference of Racing Analysts and Veterinarians (in press). 13 14 15 th 16 Hadley, M.For E. and Levine,Peer J. (2006). Review Endocrinology. Only6 edition. Prentice Hall. 17 18 ISBN: 0131876066. 19 20 21 Heitzman, R. J. (1994). In Veterinary Drug Residues: residues in food 22 23 producing animals and their products: reference materials and methods. 24 25 Blackwell Scientific Publications. ISBN: 0-632-03786-5. 26 27 28 Houghton, E. and Crone, D. L. (2000). The approaches adopted by the racing 29 30 industry to address endogenous substances and substances of dietary origin. 31 th 32 Proceedings of the 13 International Conference of Racing Analysts and 33 34 Veterinarians, Cambridge, UK. Pg. 23-26. 35 36 37 Kennedy, G. D., Shortt, H. D., Crooks, S. R. H., Young, P. B., Price, H. J., 38 39 Smyth, W. G., et al. (2009). Occurrence of α- and β-nortestosterone residues 40 41 in the urine of injured male cattle. Food Additives and Contaminants - Part A 42 Chemistry, Analysis, Control, Exposure and Risk Assessment, 26 (5), 683- 43 44 691. 45 46 47 48 Manly, BJF. (2007) Randomization, bootstrap and Monte Carlo methods in 49 50 Biology. Chapman and Hall. Third Edition. 51 52 53 Mareck, U., Geyer, H., Flenker, U., Piper, T., Thevis, M., & Schänzer, W. 54 55 (2007). Detection of dehydroepiandrosterone misuse by means of gas 56 57 chromatography-combustion-isotope ratio mass spectrometry. European 58 Journal of Mass Spectrometry, 13 (6), 419-426. 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 27 of 46 Food Additives and Contaminants 1 2 3 Pinel, G., Rambaud, L., Monteau, F., Elliot, C., & Le Bizec, B. Estranediols 4 5 profiling in calves' urine after 17 β-nandrolone laureate ester administration. 6 7 (2010). Journal of Steroid Biochemistry and Molecular Biology (in press). 8 9 10 11 Plusquellec, P., & Bouissou, M. -. (2001). Behavioural characteristics of two 12 dairy breeds of cows selected (Hé rens) or not (brune des alpes) for fighting 13 14 and dominance ability. Applied Animal Behaviour Science, 72 (1), 1-21. 15 16 For Peer Review Only 17 18 Pré vost, S., Buisson, C., Monteau, F., Andre, F. and Le Bizec, B. (2004). Is 19 GC-C-IRMS a possible analytical approach to clear up misuse situations for 20 21 forbidden natural substances in edible tissues? Euroresidue 5 . 777-782. 22 23 24 25 Scarth, J., Akre, C., Van Ginkel, L., Le Bizec, B., De Brabander, H., Korth, W., 26 27 Points, J., Teale, P. and Kay, J. (2009a). The presence and metabolism of 28 endogenous androgenic-anabolic steroid hormones in meat producing 29 30 animals. A review. Food Additives and Contaminants: part A. Vol. 26(5), 640- 31 32 671. 33 34 35 Scarth, J., Clarke, A., Hands, J., Teale, P. and Kay, J. (2009b). Validation of a 36 37 quantitative multi-residue urinary assay for the detection of androgen, 38 39 estrogen and progestagen abuse in the bovine. Submitted to 40 41 Chromatographia in September 2009. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 28 of 46 1 2 3 4 5 6 Figure legends 7 8 Figure 1 – structures of the metabolites studied (name in brackets indicates 9 the parent steroid). 10 11 12 Figure 2 – plots of creatinine ( mol/L) versus steroid concentration (pg/mL) for 13 the different analytes quantified. 14 15 Figure 3 – histogram of ABA-pregnanediol concentrations (pg/mL) in A) heifer 16 and B) steerFor urine. Peer Review Only 17 18 19 Figure 4 – histogram of BAB-androstanediol concentrations (pg/mL) in A) 20 heifer and B) steer urine. 21 22 Figure 5 – histogram of ABA-estranediol concentrations (pg/mL) in A) heifer 23 24 and B) steer urine. 25 26 Figure 6 – histogram of 19-noretiocholanolone concentrations (pg/mL) in A) 27 heifer and B) steer urine. 28 29 Figure 7 – histogram of epinandrolone concentrations (pg/mL) in A) heifer and 30 31 B) steer urine. 32 33 Figure 8 – histogram of epiboldenone concentrations (pg/mL) in A) heifer and 34 B) steer urine. 35 36 37 Figure 9 – histogram of 17 β-hydroxy-5β-androst-1-ene-3-one concentrations 38 (pg/mL) in heifer and steer urine (combined population). 39 40 Figure 10 – histogram of epioestradiol concentrations (pg/mL) in A) heifer and 41 B) steer urine. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 29 of 46 Food Additives and Contaminants 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Fig 1 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 30 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 Fig 2 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 31 of 46 Food Additives and Contaminants 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig 3 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 32 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig 4 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 33 of 46 Food Additives and Contaminants 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig 5 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 34 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig 6 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 35 of 46 Food Additives and Contaminants 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig 7 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 36 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig 8 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 37 of 46 Food Additives and Contaminants 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Fig 9 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 38 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Fig 10 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 39 of 46 Food Additives and Contaminants 1 2 3 4 5 Steroid Limit of Detection Limit of Quantification 6 (pg/mL)* (pg/mL)** 7 8 ABA-pregnanediol 12.2 1’168.5 9 BAB-androstanediol 94.1 449.3 10 ABA-estranediol 10.9 178.7 11 Epinandrolone 19.8 81.7 12 19-noretiocholanolone 32.4 82.7 13 Epiboldenone 10.5 55.9 14 17 β-hydroxy-5Forβ-androst-1-ene-3-one Peer Review 160.7 Only 250 15 Epioestradiol 14.8 475.1 16 17 Table 1 – limits of detection and quantification for the steroid metabolites. 18 19 * The limits of detection were calculated during the validation (Scarth et al. 2009b). 20 21 ** The limits of quantification were calculated during the validation (Scarth et al. 2009b). These 22 equated to the endogenous concentrations from a castrated male bovine urine pool that was 23 used to prepare the calibration line and QCs. In the case of 17 β-hydroxy-5β-androst-1-ene-3-one 24 (not endogenous at the limits of detection applied), the limit of quantification was the lowest 25 calibrant that could be quantified with a relative error of <25%. Although these values are the 26 formal limits of quantification, because this method uses a standard addition approach, the true 27 limits of quantification may in some cases be much lower than those given above. 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 40 of 46 1 2 3 4 5 6 7 % RSD of steroid only % RSD of steroid adjusted for creatinine concentration 8 9 ABA-pregnanediol 108 165 10 BAB-androstanediol 444 590 11 ABA-estranediol 126 160 12 Epinandrolone 160 243 13 19-noretiocholanolone 125 178 14 EpiboldenoneFor Peer Review 153 Only 270 15 17B-OH-androst-1-en-3-one 239 382 16 Epioestradiol 736 792 17 18 19 Table 2 – relative standard deviations (RSDs) of steroid concentrations with and 20 without adjustment for creatinine concentration. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 41 of 46 Food Additives and Contaminants 1 2 3 4 5 Steroid Mean SD RSD Min Max 6 (pg/mL) (pg/mL) (%) (pg/mL) (pg/mL) 7 8 9 Steer (n=220) 10 ABA-pregnanediol 1’039.4 1’180 114 40 41 * Indicates that the concentration of epioestradiol in two of the six pregnant animal urines were determined to be above 42 the validated calibration range and are therefore omitted from the results in the above table. It was not possible to re- 43 analyse these samples with dilution because the remaining volume of urine was required for other assays. The true mean 44 concentration for this steroid may therefore be significantly higher than that shown. 45 46 47 Table 3 – summary statistics for determined steroid concentrations in steer and 48 heifer urine. 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 42 of 46 1 2 3 4 5 6 No. of samples 1 2 of 2 3 of 3 7 8 False non-compliance 1 in 1 in 1 in 9 1 in 20 1 in 100 1 in 1’000 10’000 10’000 10’000 probability 10 11 12 ABA-pregnanediol 95% quantile 6’531.0 13’557.7 40’778.9 126’767.2 13’599.6 6’777.6 13 (pg/mL) CC α 7’575.7 15’461.3 46’009.9 142’508.6 15’508.4 7’852.4 14 For CCPeerβ 9’018.3 Review 17’986.8 52’780.2 Only 162’713.7 18’040.4 9’332.5 15 16 BAB-androstanediol 95% quantile 12’039.6 26’732.2 83’643.4 263’355.9 26’861.1 12’502.6 17 (pg/mL) CC α 13’757.7 30’246.1 94’113.7 295’792.8 30’390.8 14’277.3 18 CC β 16’047.7 34’823.7 107’580.4 337’342.4 34’988.5 16’639.1 19 20 ABA-estranediol 95% quantile 1’286.1 2’711.6 8’261.0 25’777.5 2’714.2 1’329.6 21 (pg/mL) CC α 1’689.8 3’289.4 9’517.2 29’174.8 3’292.4 1’738.5 22 CC β 2’462.6 4’180.3 11’224.0 33’603.4 4’183.7 2’510.8 23 24 Epinandrolone 95% quantile 891.2 1’913.4 5’870.1 18’364.7 1’906.5 921.3 25 (pg/mL) CC α 1’246.6 2’393.7 6’834.0 20’855.9 2’386.0 1’280.3 26 CC β 2’073.3 3’195.9 8’176.8 24’129.0 3’187.7 2’097.9 27 28 19-noretiocholanolone 95% quantile 799.0 1’732.1 5’339.0 16’731.1 1’745.1 832.2 29 (pg/mL) CC α 1’143.1 2’190.3 6’238.1 19’022.6 2’204.9 1’180.4 30 CC β 2’007.8 2’977.9 7’501.2 22’041.4 2’993.5 2’029.3 31 32 Epiboldenone 95% quantile 873.4 1’893.9 5’848.9 18’337.3 1’900.5 906.4 33 (pg/mL) CC α 1’226.5 2’371.9 6’810.2 20’825.1 2’379.3 1’263.7 34 CC β 2’059.3 3’172.4 8’149.8 24’093.9 3’180.4 2’085.6 35 36 Epioestradiol 95% quantile 4’734.1 10’210.1 31’416.8 98’374.2 10’248.2 4’894.4 37 (pg/mL) CC α 5’559.1 11’704.6 35’503.3 110’645.1 11’747.3 5’739.1 38 CC β 6’732.5 13’711.5 40’812.0 126’413.5 13’760.1 6’936.2 39 40 41 42 Table 4 – the 95% quantile thresholds (the upper threshold estimate after 43 factoring in uncertainty of measurement) and associated CC α/β concentrations 44 (P=0.05) for target analytes in steer urine at different false non-compliance 45 46 probability rates. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 43 of 46 Food Additives and Contaminants 1 2 3 4 5 6 No. of samples 1 2 of 2 3 of 3 7 8 False non- 1 in 1 in 9 1 in 20 1 in 100 1 in 1’000 1 in 10’000 10’000 10’000 compliance 10 probability 11 12 13 ABA-pregnanediol 95% 14 Forquantile Peer 11’769.9 Review 23’873.1 70’744.1 Only 218’744.1 23’823.2 12’143.7 15 (pg/mL) CC α 13’455.0 27’037.6 79’637.7 245’728.0 26’981.6 13’874.4 16 CC β 15’703.2 31’169.2 91’088.9 280’306.0 31’105.4 16’180.6 17 18 BAB- 95% 19 quantile 20 androstanediol 3’009.2 6’539.6 20’218.2 63’451.3 6’549.3 3’134.0 21 (pg/mL) CC α 3’623.4 7’585.3 22’935.9 71’453.5 7’596.3 3’763.5 β 22 CC 4’552.8 9’029.2 26’497.8 81’765.4 9’041.6 4’709.4 23 95% 24 ABA-estranediol quantile 1’311.0 2’821.2 8’665.0 27’124.6 2’839.0 1’356.9 25 (pg/mL) CC α 1’717.7 3’412.5 9’970.6 30’686.5 3’432.4 1’769.2 26 CC β 2’490.1 4’317.3 11’739.4 35’325.3 4’339.5 2’541.4 27 28 95% 29 Epinandrolone quantile 1’231.9 2’707.9 8’430.2 26’467.7 2’713.7 1’284.9 30 (pg/mL) CC α 1’628.9 3’285.3 9’707.0 29’949.3 3’291.8 1’688.3 31 CC β 2’403.5 4’175.7 11’439.7 34’485.7 4’182.9 2’461.2 32 33 19- 95% 34 noretiocholanolone quantile 942.6 2’034.0 6’281.6 19’694.0 2’026.8 973.1 35 (pg/mL) CC α 1’304.2 2’529.0 7’295.9 22’347.7 2’521.0 1’338.4 36 CC β 2’116.1 3’342.6 8’700.7 25’827.9 3’333.8 2’143.2 37 38 Epiboldenone 95% 39 quantile 1’159.3 2’502.4 7’714.4 24’178.7 2’479.7 1’196.9 40 (pg/mL) CC α 1’547.5 3’054.7 8’903.7 27’380.5 3’029.2 1’589.6 41 CC β 2’326.3 3’919.8 10’526.7 31’559.8 3’891.6 2’365.9 42 43 Epioestradiol 95% 44 quantile 159’826.5 356’583.3 1’118’757.3 3’525’690.1 356’134.4 166’179.6 45 (pg/mL) CC α 179’608.8 400’415.5 1’255’751.0 3’956’888.0 399’911.7 186’738.5 46 CC β 204’979.9 456’533.9 1’430’977.0 4’508’254.0 455’960.0 213’102.4 47 48 49 50 Table 5 – the 95% quantile thresholds (the upper threshold estimate after 51 factoring in uncertainty of measurement) and associated CC α/β concentrations 52 53 (P=0.05) for target analytes in heifer urine at different false non-compliance 54 probability rates. 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 44 of 46 1 2 3 4 5 No. of samples 1 2 of 2 3 of 3 6 7 False non- 1 in 1 in 8 1 in 20 1 in 100 1 in 1’000 1 in 10’000 10’000 10’000 9 compliance 10 probability 11 12 13 17 β-OH-androst-1- 95% 14 en-3-one Forquantile Peer 414.1 Review 904.7 2’810.2 Only 8’822.9 905.2 425.5 15 (pg/mL) CC α 711.1 1’261.7 3’400.1 10’147.8 1’262.3 723.9 16 CC β 2’516.2 2’084.2 4’303.5 11’940.8 2’084.6 2’424.5 17 18 19 20 21 Table 6 – the 95% quantile thresholds (the upper threshold estimate after 22 factoring in uncertainty of measurement) and associated CC α/β concentrations 23 (P=0.05) for target analytes in combined steer and heifer urine for 17 β-OH- 24 androst-1-en-3-one at different false non-compliance probability rates. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 45 of 46 Food Additives and Contaminants 1 2 Steroid Steers* Heifers* 3 4 1) ABA-pregnanediol >13’599.6 pg/mL in two of 1) ABA-pregnanediol >23’823.2 pg/mL in two of 5 two samples or two samples or 6 Progesterone 2) >40’778.9 pg/mL in one sample. 2) >70’744.1 pg/mL in one sample 7 8 Suggested GC-C-IRMS confirmatory analyte: Suggested GC-C-IRMS confirmatory analyte: 9 ABA-pregnanediol ABA-pregnanediol 10 11 1) BAB-androstanediol >26’861.1 pg/mL in two 1) BAB-androstanediol >6’549.3 pg/mL in two of 12 of two samples or two samples or 13 Testosterone 2) BAB-androstanediol >83’643.4 pg/mL in one 2) BAB-androstanediol >20’218.2 pg/mL in one 14 sampleFor Peer Reviewsample Only 15 16 Suggested GC-C-IRMS confirmatory analyte: Suggested GC-C-IRMS confirmatory analyte: 17 Epitestosterone Epitestosterone 18 19 1) Epinandrolone, ABA-estranediol or 1) Epinandrolone, ABA-estranediol or 20 19-noretiocholanolone >1’906.5, >2’714.2 or 19-noretiocholanolone >2’713.7, >2’839.0 or 21 >1’745.1 pg/mL respectively in two of two >2’026.8 pg/mL respectively in two of two 22 Nandrolone samples samples or 23 2) >5’870.1, >8’261.0 or >5’339.0 pg/mL 2) >8’430.2, >8’665.0 or >6’281.6 pg/mL 24 respectively in one sample respectively in one sample 25 26 Suggested GC-C-IRMS confirmatory analyte: Suggested GC-C-IRMS confirmatory analyte: 27 Epinandrolone Epinandrolone 28 29 30 1) Conjugated epiboldenone or 17B-OH- 1) Conjugated epiboldenone or 17B-OH- 31 androst-1-en-3-one >1’900.5 or 905.2 pg/mL androst-1-en-3-one >2’479.7 or 905.2 pg/mL respectively in two of two samples or respectively in two of two samples or 32 Boldenone 2) >5’848.9 or 2’810.2 pg/mL respectively in 2) >7’714.4 or 2’810.2 pg/mL respectively in 33 one sample one sample 34 35 Suggested GC-C-IRMS confirmatory analyte: Suggested GC-C-IRMS confirmatory analyte: 36 Epiboldenone Epiboldenone 37 38 39 1) Epioestradiol >5’000 pg/mL** in two of two 1) Epioestradiol >10’000 pg/mL** in two of two 40 samples or samples or 41 Estradiol 2) >31’416.8 pg/mL in one sample 2) >1’118’757.3 pg/mL in one sample 42 43 Suggested GC-C-IRMS confirmatory analyte: Suggested GC-C-IRMS confirmatory analyte: 44 Epioestradiol Epioestradiol 45 46 Table 7 – suggested strategy for screening for the abuse of natural steroids in 47 the urine of non-pregnant animals (not including bulls) where confirmation is 48 49 based on GC-C-IRMS, an on-farm visit and/or another non-threshold method. 50 Unless stated otherwise, ‘one sample’ thresholds are derived at 1 in 1’000 51 probabilities and ‘two of two’ thresholds at 1 in 10’000 probabilities. 52 * In addition to the concentration of an individual steroid, the overall steroid profile of the animal should be inspected as an 53 additional safety check to rule out false non-compliances due to unusual medical conditions etc. In this context, an 54 unusual finding would be one where a large number of steroids are high in concentration; a situation that would not be expected following the administration of one or two different steroids. 55 56 ** The ‘two of two’ thresholds for epioestradiol are not derived from 1 in 10’000 probabilities since these may lead to an 57 unacceptably high level of false compliances. A pragmatic approach based on setting the threshold at the highest level 58 that is predicted to detect abuse based on knowledge of epioestradiol post-administration concentrations was therefore adopted (see more details in the text). 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 46 of 46 1 2 3 Steroid Steers* Heifers * 4 5 6 1) ABA-pregnanediol >13’599.6 pg/mL in two of 7 1) ABA-pregnanediol concentration Progesterone two samples or 8 >218’744.1 pg/mL in one sample 2) >126’767.2 pg/mL in one sample 9 10 11 1) BAB-androstanediol >26’861.1 pg/mL in two 12 1) BAB-androstanediol concentration Testosterone of two samples or 13 >63’451.3 pg/mL in one sample 2) >263’355.9 pg/mL in one sample 14 For Peer Review Only 15 16 1) Epinandrolone, ABA-estranediol or 17 19-noretiocholanolone >1’906.5, >2’714.2 or 18 1) Epinandrolone, ABA-estranediol or Nandrolone*** >1’745.1 pg/mL respectively in two of two 19 19-noretiocholanolone >26’467.7, >27’124.6 or samples or 20 >19’694.0 pg/mL respe ctively in one sample 2) Epinandrolone, ABA-estranediol or 21 19-noretiocholanolone >18’364.7, >25’777.5 or 22 >16’731.1 pg/mL respectively in one sample 23 24 25 26 Boldenone 1) 17B-OH-androst-1-en-3-one conjugates 1) 17B-OH-androst-1-en-3-one conjugates 27 >2’810.2** pg/mL in one sample > 2’810.2 ** pg/mL in one sample 28 29 30 1) epioestradiol >10’248.2 pg/mL in two of two 1) epioestradiol concentration 31 Estradiol samples or >3’525’6 90.1 pg/mL in one sample. 32 2) >98’374.2 pg/mL in one sample 33 34 35 36 37 Table 8 – suggested strategy for confirming the abuse of natural steroids in the 38 urine of non-pregnant animals (not including bulls) where confirmation is based 39 on urinary concentration thresholds. Unless stated otherwise, all thresholds are 40 derived at 1 in 10’000 probabilities. 41 42 * In addition to confirming the concentration of an individual steroid, the overall steroid profile of the animal should be 43 inspected as an additional safety check to rule out false non-compliances due to unusual medical conditions etc. In this context, an unusual finding would be one where a large number of steroids are high in concentration; a situation that 44 would not be expected following the administration of one or two different steroids. 45 46 ** Thresholds for 17B-OH-androst-1-en-3-one are derived at a 1 in 1’000 probability. See text for a full explanation. 47 *** Either 5α-estrane-3β,17 β-diol (ABB-estranediol) or 5 β-estrane-3α,17 β-diol (BAB-estranediol) should also be detected 48 qualitatively in pregnant or injured animals whose samples exceed one of the specified nandrolone thresholds before the 49 result is considered non-compliant (see text for full explanation). 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected]