James P Scarth
email: jscarth@hfl.co.uk

Adam Clarke

Philip Teale

Aileen Mill

Roy Macarthur

Jack Kay

The detection of endogenous steroid abuse in cattle: results from population studies in the UK

Keywords: Chromatography -GC, Chromatography - Chromatography -HPLC Additives/Contaminants: Veterinary drug residues -anabolic steroids Natural, endogenous, steroids, cattle, bovine, testosterone, nandrolone, boldenone, progesterone, oestradiol, threshold

The use of steroids as growth-promoting agents in food production is banned under European Union legislation. Detecting the abuse of testosterone, nandrolone, boldenone, oestradiol and progesterone is complicated by the fact these steroids are known to be endogenous in certain situations. In this study, the concentrations of characteristic metabolites of each of these steroids have been quantified in populations of untreated steers and heifers.

Steroid concentration population data were then used by a statistical model (the Chebyshev inequality) to produce threshold concentrations for screening and confirming the abuse of these steroids in steer and non-pregnant heifer urine. In addition to thresholds based on testing one animal (a '1 out of 1' approach), new methods based on testing multiple animals from a herd (an 'y out of n' approach) allowed threshold concentrations to be significantly reduced and hence false compliances to be minimised. In the majority of cases, the suggested thresholds were found to be capable of confirming the abuse of endogenous steroids in steers and heifers. In the case of oestradiol abuse in the female, however, confirmation based on a threshold is not possible and alternative methods such as gas-combustion-isotope ratio mass spectrometry are required. In addition to the steer and heifer populations, a

Introduction

The use of steroids and other growth promoting agents is prohibited in food production according to European Union legislation (EU Council Directive 96/22/EC). The abuse of synthetic steroids can be detected by qualitative assays, but the detection of endogenous steroid use is more difficult because a simple qualitative determination of their presence is insufficient for demonstrating abuse (Scarth et al. 2009a). The direct detection of steroid esters in hair or plasma [START_REF] Boyer | Detection of testosterone propionate administration in horse hair samples[END_REF][START_REF] Gray | The development of a screening method for anabolic steroid esters in plasma[END_REF]) and the use of combustion isotope ratio mass spectrometry have been developed for the confirming the abuse of certain endogenous steroids (Prevost et al. 2004), but these methods are not always suitable as screening approaches. Therefore, the majority of methods that have been developed in related fields such as human and animals sports rely on steroids exceeding concentration thresholds as a means of detecting their abuse (Scarth et al. 2009a).

We previously published a validated method for quantifying target analytes indicative of the abuse of testosterone, nandrolone, boldenone, progesterone and oestradiol in the bovine using a combination of GC-and LC-MS/MS (Scarth et al. 2009b). In the current study, we have applied this method to populations of untreated bovine animals in the UK in order to establish urinary screening and confirmatory thresholds able to detect the abuse of these steroids.

5β-androstane-3α,17β-diol (BAB-androstanediol -Figure 1) was chosen as the testosterone target testosterone metabolite as this analyte has been shown to be increased in the urine of testosterone treated animals [START_REF] Biddle | Unpublished studies on the natural occurrence of androgens and estrogens in bovine plasma, urine and bile and the effect of exogenous steroid administration on these profiles (HFL study HFL086[END_REF]).

Since it is not known which metabolite will be most useful for detection nandrolone abuse, epinandrolone, 19-noretiocholanolone and 5α-estrane-3β,17α-diol (ABA-estranediol) were all quantified for threshold purposes (Figure 1).

In order to detect boldenone abuse, the boldenone metabolite 17β-hydroxy-5β-androst-1-ene-3-one was targeted (Figure 1) since it has been shown to be present in the urine of boldenone treated animals, but absent in untreated animals [START_REF] Biddle | Unpublished studies on the natural occurrence of boldenone in bovine urine and the metabolism of boldenone after administration (HFL study HFL1382[END_REF]. However, in order to assess whether any absence of detectable 17β-hydroxy-5β-androst-1-ene-3-one in bovine urine was due to it's absence as an endogenous compound (in all situations) or instead only absent because there was no endogenous epiboldenone that could be metabolized through to 17β-hydroxy-5β-androst-1-ene-3-one in the particular animal populations studied, epiboldenone was also quantified.

Published data regarding progesterone metabolism in the bovine is lacking.

Therefore, 5α-pregnane-3β,20α-diol (ABA-pregnanediol -Figure 1), was chosen as the target analyte as preliminary analyses showed it to be detected in the urine of bovine steers at concentrations that fell within the calibration range that was used for the other steroids. In order to detect the abuse of oestradiol, its major metabolite epioestradiol was chosen as the target analyte (Figure 1). The concentrations of epioestradiol in urine are much higher than oestradiol [START_REF] Biddle | Gas chromatography-mass spectrometry/mass spectrometry analysis to determine natural and post-administration levels of oestrogens in bovine serum and urine[END_REF], meaning that they more readily quantified and are also more suitable for confirmation using an auxiliary techniques such as gas-combustion-isotope-ratio-mass-spectrometry (GC-C-IRMS). The significance of epioestradiol's suitability for GC-C-IRMS lies in the prediction that the observed endogenous urinary concentrations would overlap with those resulting from oestradiol administration, therefore making any threshold only suitable to an initial screening approach.

Materials and methods

Chemicals and reagents

The chemicals and reagents used were as described in Scarth et al. 2009b, with the exception of the origin of some of the urine samples, for which details are given below:

Urine samples were obtained in the UK from both live animals (various university and research institute owned herds) and from animals going for slaughter (organically farmed animals slaughtered for food production). Whilst it is theoretically possible that some of the organically farmed animals may have been illicitly treated with steroids, this is considered unlikely because consent was voluntarily given from each individual producer for analysis of each animal's urine. Also, the steroid free provenance of all samples obtained from university or research institute owned herds was guaranteed because the animals were controlled under home office license regulations. For samples where 'unusually' high steroid concentrations were observed, attempts were made to trace the history of the animal in order to ascertain whether any causal explanations could be obtained. An alternative would have been to analyse the samples using gas chromatography-carbon-isotope ratio mass spectrometry (GC-C-IRMS). However, existing methods were not available for the analysis of the majority of the steroids under study.

A total of 190 heifer urine samples (requested to be from non-pregnant animals) were obtained from animals ranging from 12 to 151 months of age and a total of 220 steer urine samples were obtained from animals ranging from 7 to 30 months of age. Exact information regarding the breed of every animals was not available, but for steers the samples obtained at slaughter were from a mix of different 'beef' animals intended for meat consumption, whereas samples from live animals were from a mix of 'bbx' and 'dairy cross' animals. For heifers, the samples obtained at slaughter were from a mix of different 'beef' animals intended for meat consumption, whereas samples from line animals were from a mix of 'bbx', 'simx', 'Holstein-Friesian' and animals were also obtained, but the results of these will be discussed separately from the other population data as animals are known to produce high concentrations of steroid during gestation. No urine samples from intact males were available.

Samples were collected and stored chilled on ice within 1-hour. Samples on ice were then delivered by courier within 48-hours before being frozen at -80 o C until the time of analysis.

Preparation of calibration lines and QCs

Calibration lines and QCs were constructed for each batch using pooled urine from castrated male bovine animals as a surrogate matrix (as described in Scarth et al. 2009b). QC concentrations for androgens/progestagens were;

the endogenous augmented with 100 pg/mL (E+100), the endogenous augmented with 1000 pg/mL (E+1000) and the endogenous augmented with 2000 pg/mL (E+2000). QC concentrations for oestrogens were; the endogenous augmented with 80 pg/mL (E+80), the endogenous augmented with 800 pg/mL (E+800) and the endogenous augmented with 1600 pg/mL (E+1600). Each analytical batch contained two QCs at each of the three aforementioned levels (total n=6) and for a batch to pass QC criteria, at least four of the six QCs needed to be within +/-20% of the nominally spiked concentration, with no more than one QC failing at any one concentration.

Extraction and analysis of samples for steroid concentration

Samples were extracted by a combination of enzyme hydrolysis, liquid-liquid extraction and solid-phase extraction and were then analysed by GC-MS/MS as their enol-TBDMS derivatives on a Varian 320-MS system (as described and validated by Scarth et al. 2009b). The limits of detection and quantification of the assay are summarised in Table 1.

Previous studies have shown that the majority of endogenous steroids in the bovine are excreted in urine as glucuronide conjugates (Scarth et al. 2009).

Therefore, the current method used glucuronide conjugate hydrolysis using recombinantly produced β-glucuronidase enzyme to measure the combined concentrations of free and glucuronide steroid. This is an important distinction, since the use of alternative methods that hydrolyse all steroid conjugates (including sulphates) could lead to higher calculated concentrations. If applying the thresholds suggested in the current report, it will be necessary for laboratories to measure 'free and glucuronide' concentrations in order to ensure that their results are applicable.

Extraction and analysis of samples for creatinine concentration

In addition to quantifying the concentrations of steroid present, samples were also analysed for creatinine content. Urinary creatinine was considered potentially useful since it is a measure of the hydration status of the animal. It was hypothesized that correction of urinary steroid concentration data for creatinine content might lead to a reduction in the relative standard deviation of the steroid population data and hence a reduction in the calculated thresholds. An alternative to correcting steroid concentrations with creatinine is to use the specific gravity (SG) of the sample instead; an approach already applied in the human drug testing field [START_REF] Mareck | Detection of dehydroepiandrosterone misuse by means of gas chromatography-combustion-isotope ratio mass spectrometry[END_REF]) and which is currently being investigated for use in bovine drug residue surveillance programmes in Australia (Wolfgang Korth, personal communication).

However, the creatinine approach was chosen for the current study as preliminary experiments comparing the correlation of SG and creatinine with the different steroid concentrations in a small sub-set of the samples suggested that creatinine would be more suitable (data not shown).

Creatinine concentrations were quantified in each sample using a manufacturer supplied assay kit on an Olympus AU640 automated clinical analyser. The assay involves monitoring the absorbance at 520/800 nm following the formation of a creatinine picrate complex after the addition of picric acid (2.9 mmol/L) to samples in an alkaline medium (NaOH at 120 mmol/L. The rate of change of absorbance at this wavelength is proportional to the creatinine concentration in the sample.

Threshold calculations for a single animal within a herd

In order to control the abuse of each steroid, the statistical analysis set out to suggest thresholds with various probabilities (1 in 20, 1 in 100, 1 in 1'000 and 1 in 10'000) of finding a larger value by chance in a natural population when looking at the results from a single animal from a herd. Separate statistical methods will be described in a later section that deals with thresholds based on results of more than one animal within a herd. For both types of threshold calculation however, versions of the one-tailed version of the Chebyshev inequality were used. This method is based on probability theory, makes minimal assumptions about the distribution of the data and is described by [START_REF] Estler | A distribution-independent bound on the level of confidence in the result of a measurement[END_REF]. Chebyshev confidence intervals are as far as possible from a mean for a given standard deviation; they are distribution-independent confidence intervals and are ideally suited to dealing with non-normally distributed data such as those resulting from the current study. A one-tailed Chebyshev confidence interval is given by:

2 2 1 1 σ t p + ≤ Hence 2 2 σ σ - ≤ p t
Where , t is the difference between the mean and concentration at the upper confidence interval, µ is the mean concentration, σ 2 is the variance (square of the standard deviation), p is the probability that difference between the mean concentration and the concentration of a sample taken at random is greater than t. To assess the extent to which concentration differed between steers and heifers, a Monte Carlo permutation test was implemented [START_REF] Manly | Randomization, bootstrap and Monte Carlo methods in Biology[END_REF].

Here, gender was permuted amongst individuals and the mean difference in concentration of each steroid metabolite between steers and heifers was calculated for each permutation. The observed difference was then compared to the distribution of mean differences for the 1000 permutations and significance assessed on the basis of the position of the observed mean in the distribution.

Thresholds for results from more than one animal sampled from a herd

In addition to thresholds based on steroid concentration results from a single animal, it is also possible to produce thresholds based on more than one animal within a herd having a result above a certain steroid concentration i.e. from '2 out of 2' or '3 out of 3' animals tested ('y out of n' animals). The rationale for this approach is that if a steroid is being abused by a farmer, it is likely to be the majority of the herd that are given the drug, not just a single animal. Since threshold concentrations based on a 'y out of n' approach are significantly lower than thresholds based on a single animal, the 'y out of n' approach could lead to a lower false compliance rate for detecting steroid abuse. Whilst this approach may be sufficiently robust for a screening approach in all cases, using it for confirming steroid abuse requires that steroid concentrations are not subject to 'clustering' within herds. Whilst the authors are not aware of any evidence of steroid clustering within steer populations, a biological phenomenon known as the 'Whitten effect' [START_REF] Aron | Mechanisms of control of the reproductive function by olfactory stimuli in female mammals[END_REF]) has been reported in female mammals. This phenomenon leads to the synchronisation of oestrous in female animals and if it is accepted that some of the high steroid concentration results within a population are caused by oestrous, then this would class as clustering and could lead to false noncompliance results using the 'y out of n' approach. Therefore, while 'y out of n' calculations were performed for both steers and heifers for application as a screening approach it is suggested that the resulting thresholds only be used for steers where confirmation is concerned. In terms of the statistical methods, it is possible to derive thresholds which apply to 'y out of n' animals tested using a modification of the Chebyshev method. For example, given a population containing analyte with mean µ and standard deviation σ, the relation between the probability 1 , 1 p of testing a single animal and observing a result above x 1,1

2 1 , 1 2 1 , 1 σ σ µ - + ≤ p x
The upper limit to probability of observing at least y samples above x 1,1 out of n tested is given by:

( ) ∑ = = - -         = n i y i i n i n y p p i n p 1 , 1 1 , 1 , 1 with the particular case n 1 , 1 n , n p p = , n 1 n , n 1 , 1 p p =
Hence, if n animals out of n tested contain a concentration of analyte above

x n,n then this is evidence that the distribution is not consistent (with false noncompliance rate p) with the population ( )

σ , µ
where,

2 / 1 1 , 1 2 , σ σ µ - + ≤ n n n p x
At this state, it is important to clarify that a positive 'y out of n' result such as finding '2 out of 2' samples above a threshold is different from finding 2 results from a larger sampled population i.e. '2 out of 10' above a threshold. If the latter approach were used, then different statistical equations would be required as this would change the probabilities. For thresholds based on the single animal or the 'y out of n' approach, uncertainty of measurement was calculated using a Monte Carlo sample [START_REF] Manly | Randomization, bootstrap and Monte Carlo methods in Biology[END_REF] from the distribution consistent with the observed result along with the analytical performance observed at validation (Scarth et al. 2009b). 1000 samples were taken from these distributions and 1000 Chebyshev threshold estimates were generated. The different probabilities of finding a value by chance in a 'natural' population can then be reported at the upper 95% quantile results after factoring in the determined method uncertainty.

Uncertainty of measurement at threshold concentrations

Values for CCα and CCβ on these probabilities (Commission Decision 2002/657/EC), again at the 95% confidence interval, were also calculated for the analytical method used.

Results and discussion

All raw data and calculated threshold concentrations in the following section are uncensored and are given to one decimal place. All other summary statistics are presented to three significant figures.

Correlation of creatinine concentration with steroid concentrations

To assess whether creatinine would be a suitable indicator of hydration, the concentration of each steroid was divided by the concentration of creatinine measured. Figure 2 plots urinary steroid concentrations against creatinine concentration while Table 2 displays the relative standard deviation of the population data for each steroid with and without correction for creatinine concentration. Since no reduction in relative standard deviation was observed on correction of data for creatinine concentration, creatinine was not used in any of the further statistical analyses. It is possible that creatinine is not a good marker of hydration status or that variable hydration is less of a factor in regions with a moderate climate, such as the UK, as compared to warmer countries like Australia. It is also possible that the steroid concentrations themselves are less influenced by hydration status as compared to other determinants such as stress or oestrous.

Population steroid data and determined thresholds

Population results

Summary statistics for the determined concentrations of each steroid in steer and heifer populations are given in Table 3, while histograms of the data are shown in Figures 3 to 10.

For all steroids other than 17β-hydroxy-5β-androst-1-ene-3-one, mean and maximum steroid concentrations in both steers and heifer were above the limits of detection and quantification and hence it was possible to produce well characterised population histograms. The mean concentrations of epioestradiol, ABA-estranediol, BAB-androstanediol, epiboldenone and ABApregnanediol were determined to be statistically different between steer and heifer populations (Monte carlo permutation test P<0.05, 1000 permutations).

19-noretiocholanolone and epinandrolone concentrations, however, were determined not to be statistically significantly different between steers and heifers (Monte carlo permutation test P<0.05, 1000 permutations). Although the mean concentrations of these two steroids were deemed not to be statistically significantly different, there may still have been differences in the relative standard deviations for the sexes. However, this situation is difficult to test when dealing with non-parametric data such as these. This is especially important considering that the Chebyshev method used to calculate the thresholds is heavily influenced by the variance within the data.

The most obvious differences in steroid concentrations between the sexes were for BAB-androstanediol, epioestradiol and ABA-pregnanediol. BABandrostanediol concentrations were much higher and more variable in steers, while epioestradiol and ABA-pregnanediol concentrations were much higher and more variable in heifers (Table 3). For epioestradiol and ABApregnanediol, this was an expected outcome since females produce large quantities of oestrogens and progestagens at certain stages of oestrous [START_REF] Hadley | Endocrinology[END_REF]. For BAB-androstanediol in the steers however, the higher concentrations were unexpected, since these males were reported to be castrated and hence androgen output would theoretically have been restricted to adrenal output. It is possible, however, that some of the 'steers' may have retained residual gonadal tissue, such as an undescended testes, and therefore occasionally registered a high BAB-androstanediol concentration.

For 17β-hydroxy-5β-androst-1-ene-3-one, all but one determined concentration were below the limit of detection and all sample concentrations were well below the lower limit of quantification (LLOQ). In the case of the one sample that was above the limit of detection of 160.7 pg/mL, its concentration was determined at only 161.4 pg/mL. Because this determined concentration is below the LLOQ and is so close to the LOD, it is possible that the result is a false non-compliance caused by analytical background noise. The later section on screening and threshold approaches will discuss the implication of this finding in more detail.

Other than in pregnant animals, the highest concentrations of several of the steroids were observed in one of the non-pregnant heifers and one of the steers from a research owned herd (therefore guaranteeing the untreated status of the animals). The owners of the herd were contacted in order to clarify that the information provided about the animal was correct. It was confirmed that the sex and other information regarding these animals was correct and that the heifer was not pregnant at the time of sampling. However, while the heifer was not considered unfit for consumption at the time of sampling, it was found to suffering from an unknown illness several months later. Since it would have been treated the same as any other animal as far as routine urine sampling was concerned, the results from this animals were included within the population dataset for threshold determination purposes.

In pregnant animals, the concentrations of all steroids other than 17β-hydroxy-5β-androst-1-ene-3-one were considerably higher than the non-pregnant animals (not determined statistically -Table 3). This was an expected outcome, since steroid output during pregnancy is known to be increased [START_REF] De Brabander | Endogenic nortestosterone in cattle?[END_REF]). An interesting observation was that epiboldenone concentrations in the pregnant animals were far in excess of those in the nonpregnant animals. This may reflect an increased endogenous production of 1dehydro-steroids during pregnancy, possibly related to the process of aromatization. Alternatively, it may reflect an increased conversion in the gut due to an increased availability of precursor steroids for bacteria to act upon.

Even though the concentrations of epiboldenone were high in the pregnant animals (reaching 3'725.0 pg/mL), there were no results above the limit of detection for 17β-hydroxy-5β-androst-1-ene-3-one, thus supporting the suitability of this analyte as an indicator of boldenone administration only.

Screening and confirmatory threshold approaches

The general principle of adopting a threshold concentration to confirm the abuse of an endogenous compound is an accepted method in both food production [START_REF] Heitzman | In Veterinary Drug Residues: residues in food producing animals and their products: reference materials and methods[END_REF] and animals sports [START_REF] Houghton | The approaches adopted by the racing industry to address endogenous substances and substances of dietary origin[END_REF].

If used formally to confirm the abuse of a proven endogenous steroid, the standard approach is to set the threshold at a statistical probability of finding a false non-compliance at a rate of 1 in 10'000 in a natural population [START_REF] Houghton | The approaches adopted by the racing industry to address endogenous substances and substances of dietary origin[END_REF], since this is considered to offer a sufficiently large safety margin to prevent the occurrence of false non-compliances.

Thresholds for screening may be set at a lower probability, but there then needs to be a secondary mechanism for confirmation if the confirmatory threshold is not also breeched. Typically, this may include follow-up analyses using gas chromatography carbon isotope mass spectrometry (GC-C-IRMS) (Prévost et al. 2004), detection of an intact steroid ester in hair or plasma [START_REF] Boyer | Detection of testosterone propionate administration in horse hair samples[END_REF][START_REF] Gray | The development of a screening method for anabolic steroid esters in plasma[END_REF] or an on-farm inspection (Jack Kay, personal observation). In an ideal world, a confirmatory threshold would also be suitable as a screening threshold, but this requires that the threshold is able to produce both low rates of false compliance and non-compliance; an ideal that is seldom achieved. If using the '2 out of 2' instead of the traditional '1 out of 1' (single animal) approach for screening, it is suggested that a 1 in 10'000 probability be used since the use of a lower probability such as 1 in 1'000 might lead to Minimum Residue Performance Limits (MRPLs) that are below many laboratory's capabilities or concentrations that cannot be followed by GC-C-IRMS.

For all steroids where endogenous concentrations were quantified above the LLOQ, therefore excluding 17β-hydroxy-5β-androst-1-ene-3-one, a threshold for confirmation at a false probability of 1 in 10'000 is suggested as this is in line with the approach in other industries as described above. For all steroids other than 17β-hydroxy-5β-androst-1-ene-3-one, steer and heifer thresholds were calculated separately, regardless of whether there were any statistically significant differences between the sexes. The reason for this approach is because there are known physiological variations between males and females [START_REF] Hadley | Endocrinology[END_REF] that could theoretically lead to different steroid concentrations and a simple comparison of the mean endogenous concentrations of the sexes is not sufficient alone to justify grouping them together.

The fact that 17β-hydroxy-5β-androst-1-ene-3-one concentrations in all samples were below the LLOQ was a desirable outcome since it was hypothesized that this analyte would be seen following the administration of boldenone but not in untreated populations. A simple approach to confirmation of abuse would then be to say that because no concentrations were detected above the LLOQ, then the presence of any concentration of this analyte above the LLOQ in real samples could be used as demonstration of boldenone abuse. However, a safer approach for a potentially endogenous analyte such as 17β-hydroxy-5β-androst-1-ene-3-one would be to treat the determined concentrations (which may effectively be thought of as instrumental noise since the majority are below the LOD and all are below the LLOQ) according to the Chebyshev threshold approach. Applying this 'ultrasafe' Chebyshev threshold approach to the analytical noise, a probability of 1 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 The estimated Chebyshev thresholds of finding a value by chance in a 'natural' population at different probabilities are given in Tables 4, 5 and 6 and are reported as the '95% confidence interval,' signifying that they are the upper estimates for the thresholds after factoring in the method's uncertainty of measurement as described in the methods section. Table 5 also lists the CCα and CCβ values (decision and detection limits respectively) relating to these thresholds when using the analytical method presented here. For all the analytes, the use of the '2 out of 2' and '3 out of 3' animal approaches lead to concentration thresholds approximately 10-and 20-fold lower respectively compared to the traditional '1 out of 1' (single animal) approach. However, although the larger 'y out of n' approach allows for a reduction in the concentration threshold, the risk of false compliances increases with n due to potential inter-individual variation in excretion profiles. Therefore, it is suggested that only the '2 out of 2 'approach be used to supplement the traditional'1 out of 1' methods as this is predicted to offer the best balance of a low enough concentration threshold without risking false compliances due to inter-individual variation.

In order to use these threshold data to design pragmatic screening and confirmatory approaches, it is necessary to relate the threshold concentrations to those obtained following the administration of different steroids in order to ascertain whether a sufficiently low rate of false compliances can be achieved. Steroid 'post-administration' data for the different metabolites were available for boldenone, nandrolone, oestradiol and testosterone, but not for progesterone. The results from the current study show that pre-dose concentrations of 17βhydroxy-5β-androst-1-ene-3-one were <LOD in both animals 1 and 2, but rose to 9'316 pg/mL and 6'446 respectively at day 2 and were still at 7'660 and 5'164 pg/mL respectively at day 12. Since the concentrations of 17β-hydroxy-5β-androst-1-ene-3-one were not quantified in the samples during the original study by [START_REF] Biddle | Unpublished studies on the natural occurrence of boldenone in bovine urine and the metabolism of boldenone after administration (HFL study HFL1382[END_REF], the results from the current study cannot be compared to the original study to test the stability of this analyte.

The samples from day 2 and day 12 from animal 1 were also analysed without hydrolysis in order to ascertain the conjugation status of the two boldenone metabolites. Uncojugated epiboldenone concentrations were calculated to be only 3.2 and 0.6 % of the total excreted epiboldenone at days 2 and 12 respectively, while uncojugated 17β-hydroxy-5β-androst-1-ene-3-one concentrations were calculated to be only 13.9 and 0.0 % of the total 17β- hydroxy-5β-androst-1-ene-3-one respectively. The fact that the majority of these metabolite concentrations were conjugated is significant since previous studies (European Commission, 2003) have suggested that boldenone metabolites arising from faecal contamination are uncojugated.

The suggested screening and confirmatory Chebyshev thresholds for regulating boldenone abuse are given in Tables 7 and8 respectively. All these 17β-hydroxy-5β-androst-1-ene-3-one and epiboldenone thresholds are significantly lower than those observed post boldenone-administration, meaning a low false compliance rate in a real screening environment is likely.

For confirmation of boldenone abuse using the threshold approach, it is suggested that the 1 in 1'000 threshold for '1 out of 1' animals with 17βhydroxy-5β-androst-1-ene-3-one as the target analyte be employed and that the concentration be determined as the amount of conjugated steroid in order to prevent any false non-compliances caused by the potential presence of free analyte through faecal contamination. The use of the '2 out of 2' approach for confirming boldenone abuse is not recommended as clustering of findings of metabolites related to boldenone has been reported in the past [START_REF] De Brabander | Presence and metabolism of the anabolic steroid boldenone in various animal species: A review[END_REF]). The use of epiboldenone as a confirmatory analyte is not recommended as the endogenous nature of this analyte is still subject to debate (European Commission, 2003). If the concentration thresholds are adopted as screening thresholds only, and if confirmation is based on GC-C-IRMS, then the use of epiboldenone as the target analyte for the GC-C-IRMS analysis is suggested since it is the metabolite of the highest abundance. 15'000 to 50,000 pg/mL, while in males varied from around 6'000 to 23'000 pg/mL. Concentrations of ABA-estranediol in heifers varied from around 11'000 to 25'000 pg/mL, while in males varied from around 6'000 to 13'000 pg/mL. Concentrations of 19-noretiocholanolone in heifers varied from around 5'000 to 19'000 pg/mL, while in males the concentrations were not quantified.

Threshold approaches for regulating nandrolone abuse

The suggested screening and confirmatory Chebyshev thresholds for regulating nandrolone abuse are given in Tables 7 and8 respectively. By monitoring all three nandrolone metabolites, whose relative abundance varies between different individuals, these screening and confirmatory approaches were able to detect nandrolone abuse in each of the animals reported by the [START_REF] Biddle | Unpublished studies on the natural occurrence of androgens and estrogens in bovine plasma, urine and bile and the effect of exogenous steroid administration on these profiles (HFL study HFL086[END_REF] study, meaning a low false compliance rate in a real screening environment is likely.

For confirmation of nandrolone abuse in steers using the threshold approach, it is suggested that the 1 in 10'000 thresholds for both the '1 out of 1' and '2 out of 2' approach for each of epinandrolone, ABA-estranediol or 19noretiocholanolone be adopted. It would only be necessary for a sample or pair of samples to register a concentration above any one of these thresholds in order to trigger a positive confirmatory, thus allowing for any variation in metabolite profiles between individuals. However, it is now know that bovine animals may excrete increased concentrations of nandrolone and epinandrolone following casualty and during pregnancy [START_REF] Kennedy | Occurrence of α-and β-nortestosterone residues in the urine of injured male cattle[END_REF].

It is therefore recommended that such animals not be subjected to the days. The concentrations of several testosterone metabolites, including BABandrostanediol in male samples only, were quantified throughout the excretion study. In steers, the concentrations of BAB-androstanediol, BBAandrostanediol and epietiocholanolone appeared to increase slightly following testosterone administration, reaching maximum concentrations of 5'720, 5'110 and 3'520 pg/mL respectively. Following this previous study, BABandrostanediol was chosen as a target analyte for the current study as a deuterated internal standard analogue was available. In females, the concentrations of the different metabolites did not increase significantly relative to pre-dose concentrations, but BAB-androstanediol was not quantified, so no post-administration data is available for this analyte. The lack a significant increase in testosterone metabolite concentrations in the previous study was unexpected, since the dose of testosterone given was relatively large (200 mg). Although these results may reflect a 'typical' post- testosterone administration, it is also possible that the particular steroid treatment did not work correctly on this occasion and therefore lead to atypical results. Further testosterone administration studies would be required to clarify the situation.

The suggested screening and confirmatory Chebyshev thresholds for regulating testosterone abuse are given in Tables 7 and8 respectively. Until further testosterone studies are carried out and the resulting urine samples analysed for BAB-androstanediol concentrations, it is not possible to give a definitive answer as to the percentage false compliance rate of using BABandrostanediol as a marker analyte.

Threshold approaches for regulating progesterone abuse

The suggested screening and confirmatory Chebyshev thresholds for regulating testosterone abuse are given in Tables 7 and8 respectively.

Published data regarding progesterone metabolism in the bovine is lacking.

Therefore, 5α-pregnane-3β,20α-diol was chosen as the target analyte as preliminary analyses showed it to be detected in the urine of bovine steers at concentrations that fell within the calibration range that was used for the other steroids. However, until progesterone studies are carried out and the resulting urine samples analysed for ABA-pregnanediol concentrations, it is not possible to give a definitive answer as to the percentage false compliance rate of using ABA-pregnanediol as a marker analyte. days, before then dropping down to around 6'000 pg/mL by day 23. However, for the third animal, epioestradiol concentrations dropped off rapidly such that by day 15 they were only 103.5 pg/mL. As with the testosterone implant data described earlier, it is possible that in this third animal the implant was less successfully inserted, thus leading to a different profile compared to the other two animals. In the female animals, time of peak epioestradiol concentration varied between day 2 and day 8 post-dose, with concentrations of 21'713.0, 29'962.1 and 39'743.1 pg/mL in the three different animals respectively.

Threshold approaches for regulating oestradiol abuse

Concentrations then dropped off relatively slowly, such that by day 24, concentrations in the three animals were 12'201.6, 2'650.8 and 21'848.8 pg/mL respectively.

The suggested screening and confirmatory Chebyshev thresholds for regulating oestradiol abuse are given in Tables 7 and8 respectively. Unlike the previous analytes, the 'two of two' screening thresholds for epioestradiol are not derived from 1 in 10'000 probabilities since these may lead to an unacceptably high level of false compliances. A pragmatic approach based on setting the threshold at the highest level that is predicted to be capable of detecting abuse was therefore adopted based on knowledge of epioestradiol post-administration concentrations. In this respect, the screening thresholds given in Table 7 would be able to detect the abuse of epioestradiol in the majority of the post-administration samples for several days post-dose and would still be predicted to lead to a relatively low false non-compliance followup rate.

In terms of confirming oestradiol abuse, the approaches given in table 8 for steers are likely to detect a proportion of abuse cases for several days postdose, but the threshold concentrations in females are unlikely to ever be reached following oestradiol administration. This is due to the extremely high concentrations of epioestradiol found naturally in some animals and is most likely due to the increase in females during the oestrous cycle. Therefore, in the opinion of the authors, the most pragmatic approach for females would be 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 oestradiol abuse in 'real' populations would be greatly enhanced by conducting 'on-farm' sampling rather than 'at slaughter' testing. That way, the chances of detecting the abuse of a steroid closest in time to the point of administration of the drug, where urinary concentrations would be highest, would be maximized.

Factors for consideration when applying suggested threshold concentrations

Pregnant animals and intact male samples were not included in the population statistics, so the approaches suggested in this report should only be applied to steers and heifers. The ages of the animals used varied from 7 to 30 months for steers and from 12 to 151 months for heifers, so these are the age ranges for which the determined threshold concentrations are applicable.

Animals from a wide range of breeds from within the UK were analysed in the current study. Considering the large degree of separation between the highest concentrations observed in these animals and the resulting Chebyshev threshold concentrations, it would seem reasonable to apply the thresholds to animals of all breeds within the UK. However, one previous study has shown a marginal, but statistically significant, difference in testosterone concentrations for one breed. Plusquellec et al. (2001) showed that lactating animals of the Herens breed, which were artificially selected for fighting ability, had higher median plasma testosterone concentrations than animals of the Brune des Alpes breed, with concentrations of 0.21 and 0.11 ng/mL respectively. It would therefore seem sensible to exclude animals of the Herens breed, since there is a scientific rationale for why these may have increased androgen concentrations.

The current method used glucuronide conjugate hydrolysis using recombinantly produced β-glucuronidase enzyme to measure the combined concentrations of free and glucuronide steroid. Therefore, when applying the thresholds suggested in the current report, it will be necessary for laboratories to measure 'free and glucuronide' concentrations in order to ensure that their results are applicable. Laboratories will also need to calculate their own CCα and CCβ values before applying the methods. (Tables 7 and8). The threshold approach is predicted to be useful for confirming the abuse of nandrolone and boldenone in steers and heifers and oestradiol in steers, but can only be used as a screening indication in the case of oestradiol in heifers. More data regarding the post-administration concentrations of testosterone and progesterone metabolites are required before the usefulness of the proposed thresholds for these steroids can be fully tested.

When using the data presented, two broad choices are available. If a regulatory body chooses to use the thresholds as a screen only, then the threshold concentrations suggested in Table 7 could be followed up by confirmation with GC-C-IRMS (Prevost et al. 2004), an on-farm inspection and/or another non-threshold approach such as the detection of intact steroid esters in hair or plasma [START_REF] Boyer | Detection of testosterone propionate administration in horse hair samples[END_REF][START_REF] Gray | The development of a screening method for anabolic steroid esters in plasma[END_REF]). If, however, a regulatory body chooses to base both screening and confirmation on the concentration threshold approach, then the threshold concentrations suggested in Table 8 could be applied. It would, of course, be possible for regulatory bodies to use screening thresholds lower than those given in Table 7 in order to further minimize the number of false compliances (using the data in Tables 4 to 6 as a guide), but this would be likely to lead to an increased number of false non-compliances that could not be confirmed using GC-C-IRMS or other techniques.

In line with approaches used in other industries [START_REF] Houghton | The approaches adopted by the racing industry to address endogenous substances and substances of dietary origin[END_REF], it is suggested that the 1 in 10'000 false non-compliance probability approach for '1 out of 1' and '2 out of 2' animal approaches be used for confirmation according to the thresholds given in 10'000 probability would be for 17β-hydroxy-5β-androst-1-ene-3-one, where it is suggested that only a 1 in 1'000 probability is required due to the already 'safe' nature of applying the Chebyshev threshold approach to a population where all sample concentrations were below the LLOQ.

In addition to comparing the concentration of each individual steroid against the thresholds, it is suggested that the overall steroid profile of the animal should also be inspected as an additional safety check to rule out false noncompliances 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 would not be expected following the ** The limits of quantification were calculated during the validation (Scarth et al. 2009b). These equated to the endogenous concentrations from a castrated male bovine urine pool that was used to prepare the calibration line and QCs. In the case of 17β-hydroxy-5β-androst-1-ene-3-one (not endogenous at the limits of detection applied), the limit of quantification was the lowest calibrant that could be quantified with a relative error of <25%. Although these values are the formal limits of quantification, because this method uses a standard addition approach, the true limits of quantification may in some cases be much lower than those given above.

F

  adequate. Because no concentrations of this analyte were above the LLOQ of the assay, the male and female data (uncensored for concentrations below the LOD and LLOQ) were treated as one population for the purposes of the threshold calculations.

  Threshold approaches for regulating boldenone abuseBiddle et al. reported a quantitative boldenone metabolism study in the bovine in 2005. Two steers were treated with a 700 mg boldenone undecylenate intra-muscular (IM) injection and urine collected for 56 days. The concentrations of epiboldenone were quantified throughout the excretion study. Since the concentrations of 17β-hydroxy-5β-androst-1-ene-3-one could not be quantified in this previous study due to a lack of availability of reference standards, three of the post administration urine samples, those collected predose and then at 2 and 12 days post-dose from both animals, were reanalysed in the current study in order to quantify both 17β-hydroxy-5βandrost-1-ene-3-one and epiboldenone.The pre-dose concentrations of epiboldenone as determined by[START_REF] Biddle | Unpublished studies on the natural occurrence of boldenone in bovine urine and the metabolism of boldenone after administration (HFL study HFL1382[END_REF] were <LOD and 260 pg/mL in animals 1 and 2 respectively, but rose to 99'990 and 131'610 pg/mL respectively at day 2 and were 103'860 and 107'690 pg/mL respectively at day 12. The concentrations of epiboldenone determined in the samples re-analysed during the current study were within 12% of the concentrations obtained in the original study, thus demonstrating the stability of this analyte in urine when frozen at -80 o C.

  Biddle et al. reported a quantitative nandrolone metabolism study in the bovine in 2003. Three steers and three heifers were treated with a 200 mg nandrolone laurate intra-muscular (IM) injection and urine collected for 24 days. The concentrations of epinandrolone, ABA-estranediol and 19noretiocholanolone were quantified in heifers and epinandrolone and ABAestranediol in steers throughout the excretion study. In both heifers and relative concentrations of the different analytes varied between individuals such that epinandrolone was the major metabolite in some animals and ABA-estranediol or 19-noretiocholanolone the major metabolite in others. However, within the excretion profile of each individual animal, the relative proportions of the different metabolites stayed relatively constant as the concentrations dropped off over the urine collection period. Peak concentrations of each analyte were generally achieved after around 3 days and then stayed level or only tailed off very slowly for the following 2-weeks, before dropping off at a faster rate between weeks 2 and 3. Up to 2-weeks post-dose, concentrations of epinandrolone in heifers varied from around

  nandrolone unless additional marker metabolites which are specific for nandrolone administration also be detected. In this respect,Pinel et al. (2010) have recently discovered that 5α-estrane-3β,17βdiol (ABB-estranediol) and 5β-estrane-3α,17β-diol (BAB-estranediol) are detected following administration of nandrolone, but not during pregnancy or following injury (Gaud Pinel, personal communication). It is therefore recommended that these additional 'marker' metabolites should also be detected qualitatively in pregnant or injured animals whose samples exceed one of the specified nandrolone thresholds before the result is considered non-compliant.If the concentration thresholds are adopted as screening thresholds only, and if confirmation is based on GC-C-IRMS instead, then the use of epinandrolone as the target analyte for the GC-C-IRMS analysis is suggested since it is the metabolite of the highest abundance.Threshold approaches for regulating testosterone abuseiddle et al. reported a quantitative testosterone metabolism study in the bovine in 2003. Three steers and three heifers were treated with a 200 mg testosterone propionate sub-cutaneous ear implant and urine collected for 24

  Biddle et al. reported a quantitative oestradiol metabolism study in the bovine in 2003. Three steers and three heifers were treated with a 43.9 mg oestradiol sub-cutaneous ear implant and urine collected for 24 days. The concentrations of epioestradiol were quantified throughout the excretion study. In steers, time of peak epioestradiol concentration varied between day 0 and day 3 post-dose. Two of the three animals produced peak concentrations of 14'452.0 and 22'450.8 pg/mL, but in the third animal 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

  , an extensive population study of natural steroid concentrations in bovine urine samples obtained in the UK has been conducted and strategies for screening and confirming the abuse of boldenone, nandrolone, testosterone, progesterone and oestradiol in the bovine have been suggested

Figure 1 -

 1 Figure 1 -structures of the metabolites studied (name in brackets indicates the parent steroid).

Figure 2 -

 2 Figure 2 -plots of creatinine (µmol/L) versus steroid concentration (pg/mL) for the different analytes quantified.

Figure 3 -

 3 Figure 3 -histogram of ABA-pregnanediol concentrations (pg/mL) in A) heifer and B) steer urine.

Figure 4 -

 4 Figure 4 -histogram of BAB-androstanediol concentrations (pg/mL) in A) heifer and B) steer urine.

Figure 5 -

 5 Figure 5 -histogram of ABA-estranediol concentrations (pg/mL) in A) heifer and B) steer urine.

Figure 6 -

 6 Figure 6 -histogram of 19-noretiocholanolone concentrations (pg/mL) in A) heifer and B) steer urine.

Figure 7 -

 7 Figure 7 -histogram of epinandrolone concentrations (pg/mL) in A) heifer and B) steer urine.

Figure 8 -

 8 Figure 8 -histogram of epiboldenone concentrations (pg/mL) in A) heifer and B) steer urine.

Figure 9 -

 9 Figure 9 -histogram of 17β-hydroxy-5β-androst-1-ene-3-one concentrations (pg/mL) in heifer and steer urine (combined population).

Figure 10 -

 10 Figure 10 -histogram of epioestradiol concentrations (pg/mL) in A) heifer and B) steer urine.

Table 8 .

 8 The only exception to the 1 in

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

  Pinel, G., Rambaud, L., Monteau, F., Elliot, C., & Le Bizec, B. Estranediols profiling in calves' urine after 17β-nandrolone laureate ester administration. (2010). Journal of Steroid Biochemistry and Molecular Biology (in press).

	Plusquellec, P., & Bouissou, M. -. (2001). Behavioural characteristics of two
	dairy breeds of cows selected (Hérens) or not (brune des alpes) for fighting
	and dominance ability. Applied Animal Behaviour Science, 72(1), 1-21.
	F o r F o r F o r Prévost, S., Buisson, C., Monteau, F., Andre, F. and Le Bizec, B. (2004). Is GC-C-IRMS a possible analytical approach to clear up misuse situations for forbidden natural substances in edible tissues? Euroresidue 5. 777-782. F o r
	administration of one or two different steroids. Specifically relating to nandrolone, either 5α-estrane-3β,17β-diol (ABB-estranediol) or 5β-estrane-3α,17β-diol (BAB-estranediol) should also be detected qualitatively in pregnant or injured animals whose samples exceed one of the specified P e e r P e e r P e e r P Scarth, J., Akre, C., Van Ginkel, L., Le Bizec, B., De Brabander, H., Korth, W., e Points, J., Teale, P. and Kay, J. (2009a). The presence and metabolism of endogenous androgenic-anabolic steroid hormones in meat producing e r
	nandrolone thresholds before the result is considered non-compliant. R R R animals. A review. Food Additives and Contaminants: part A. Vol. 26(5), 640-671. R
	e e e e
	v i e w v i e w v i e w Scarth, J., Clarke, A., Hands, J., Teale, P. and Kay, J. (2009b). Validation of a quantitative multi-residue urinary assay for the detection of androgen, v i e estrogen and progestagen abuse in the bovine. Submitted to Chromatographia in September 2009. w
	O n l O n l O n l O n l
	y y y y

  *Table1-limits of detection and quantification for the steroid metabolites. The limits of detection were calculated during the validation(Scarth et al. 2009b).

	ABA-pregnanediol	12.2	1'168.5
	BAB-androstanediol	94.1	449.3
	ABA-estranediol	10.9	178.7
	Epinandrolone	19.8	81.7
	19-noretiocholanolone	32.4	82.7
	Epiboldenone	10.5	55.9
	17β-hydroxy-5β-androst-1-ene-3-one	160.7	250
	Epioestradiol	14.8	475.1

*

Table 2 -

 2 relative standard deviations (RSDs) of steroid concentrations with and without adjustment for creatinine concentration.

	% RSD of steroid only	% RSD of steroid adjusted for creatinine concentration
	ABA-pregnanediol	108	165
	BAB-androstanediol	444	590
	ABA-estranediol	126	160
	Epinandrolone	160	243
	19-noretiocholanolone	125	178
	Epiboldenone	153	270
	17B-OH-androst-1-en-3-one	239	382
	o Epioestradiol	736	792
	r
	P
	e e r
	R e v i e w
		O n
			l y

Table 8 -

 8 suggested strategy for confirming the abuse of natural steroids in the urine of non-pregnant animals (not including bulls) where confirmation is based on urinary concentration thresholds. Unless stated otherwise, all thresholds are derived at 1 in 10'000 probabilities.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

This study was funded by the Department for Environment, Food and Rural Affairs.

* Indicates that the concentration of epioestradiol in two of the six pregnant animal urines were determined to be above the validated calibration range and are therefore omitted from the results in the above table. It was not possible to reanalyse these samples with dilution because the remaining volume of urine was required for other assays. The true mean concentration for this steroid may therefore be significantly higher than that shown.

Suggested GC-C-IRMS confirmatory analyte: Epioestradiol

Table 7 -suggested strategy for screening for the abuse of natural steroids in the urine of non-pregnant animals (not including bulls) where confirmation is based on GC-C-IRMS, an on-farm visit and/or another non-threshold method. Unless stated otherwise, 'one sample' thresholds are derived at 1 in 1'000 probabilities and 'two of two' thresholds at 1 in 10'000 probabilities.

* In addition to the concentration of an individual steroid, the overall steroid profile of the animal should be 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 would not be expected following the administration of one or two different steroids.

** The 'two of two' thresholds for epioestradiol are not derived from 1 in 10'000 probabilities since these may lead to an unacceptably high level of false compliances. A pragmatic approach based on setting the threshold at the highest level that is predicted to detect abuse based on knowledge of epioestradiol post-administration concentrations was therefore adopted (see more details in the text).


Kevin Mark Cooper

Michelle Whelan

Martin Danaher

Dr David Glenn Kennedy

Stability during cooking of anthelmintic veterinary drug residues in beef

Keywords: anthelmintics, veterinary drug residues, QuEChERS, stability, cooking, beef, ProSafeBeef

come

Introduction

A wide range of veterinary drugs are used in prophylaxis and treatment of endoparasitic infections and ectoparasitic infestations in domestic livestock. Anthelmintic drugs used to control nematode (roundworm), cestode (tapeworm) and trematode (fluke) infections include various benzimidazole compounds (including fenbendazole, triclabendazole, mebendazole and albendazole), imidazothiazoles (particularly levamisole), macrocyclic lactones (including ivermectin) and a range of flukicidal compounds (including oxyclozanide, rafoxanide, closantel, nitroxynil and clorsulon).

Concerns over the toxicity of some of these drugs [START_REF] Mckellar | The benzimidazole anthelmintic agents -a review[END_REF][START_REF] Lankas | Induction of reversible urothelial cell hyperplasia in rats by clorsulon, a flukicide with weak carbonic-anhydrase inhibitory activity[END_REF] led the European Union to establish Maximum Residue Limits (MRLs) for their residues in tissues of food-producing species (collated in European Commission 2010). Drugs, other than those listed in this legislation, are not licensed for use in food-producing animals and therefore their residues ought not to be present in food. The occurrence and determination of benzimidazole and macrocyclic lactone residues in biological matrices have recently been reviewed by [START_REF] Danaher | Review of methodology for the determination of macrocyclic lactone residues in biological matrices[END_REF]2007). Food safety and the exposure of the consumer to chemical residues in food are of continuing concern and there is an ongoing need for new analytical techniques and information on the occurrence of potentially harmful residues of veterinary drugs in our food. ProSafeBeef (www.prosafebeef.eu) is a EU Sixth Framework Integrated Project involving 41 leading research and industrial organisations working in 18 different countries, aiming to advance beef safety and the diversification of the European beef sector. Under this project, a novel analytical method, based on state-of-the-art ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), was developed simultaneously to detect 38 anthelmintic drug residues in bovine tissues [START_REF] Kinsella | A dual validation approach to detect anthelmintic residues in bovine liver over an extended concentration range[END_REF][START_REF] Whelan | Determination of anthelmintic drug residues in milk using ultra high performance liquid chromatography-tandem mass spectrometry with rapid polarity switching[END_REF]. This technique was subsequently applied in a survey of retail beef across Europe to determine the prevalence of anthelmintic drug residues in meat available to the European consumer, and in a study of the stability of anthelmintic drugs in solution, tissue extracts and stored tissue to assist the analyst in accurate quantification of residues of these drugs in food. Results of both these studies will be published elsewhere. Studies of the attitudes of consumers towards the safety of the meat they consume were also a major pillar of ProSafeBeef research [START_REF] Van Wezemael | European consumers and beef safety: Perceptions, expectations and uncertainty reduction strategies[END_REF]. Should unacceptable residues of anthelmintic drugs find their way into our foods, legislators, analysts and consumers alike will wish to know if such residues are destroyed when that food is cooked.

Between 1995 and 2005, Rose and co-workers in the UK Central Science Laboratory demonstrated that residues of a range of veterinary drugs exhibit varying degrees of stability during cooking and, therefore, that cooking influences the level of risk posed by such residues [START_REF] Rose | The effect of cooking on veterinary drug residues in food: 4. oxytetracycline[END_REF]. This compound-dependent stability during cooking is supported by studies of nitrofuran residues by [START_REF] Cooper | Stability studies of the metabolites of nitrofuran antibiotics during storage and cooking[END_REF] and malachite green residues by [START_REF] Mitrowska | The effects of cooking on residues of malachite green and leucomalachite green in cup muscles[END_REF]. However, to date, the assessment of the stability of anthelmintic drug residues in foods has been piecemeal and limited to the effects of pasteurisation on the macrocyclic lactones, including ivermectin, in milk [START_REF] Imperiale | Thermal stability of antiparasitic macrocyclic lactones milk residues during industrial processing[END_REF][START_REF] Cerkvenik | Thermal and long-term freezing stability of ivermectin residues in sheep milk[END_REF], and the effects of various cooking procedures on ivermectin in porcine liver and bovine, porcine and salmon muscle [START_REF] Rose | The effect of cooking on veterinary drug residues in food: 7. ivermectin[END_REF][START_REF] Slanina | Ivermectin residues in the edible tissues of swine and cattle: effect of cooking and toxicological evaluation[END_REF], levamisole in bovine and porcine muscle (Rose et al. 1995a;[START_REF] Hsu | Influence of cooking processing conditions on levamisole residues in swine muscle tissues[END_REF] and oxfendazole in bovine liver [START_REF] Rose | The effect of cooking on veterinary drug residues in food: 5. oxfendazole[END_REF].

To complement the outcomes of the ProSafeBeef European retail beef survey and aid relevant authorities and the consumer in assessing their exposure to anthelmintic drug residues, the current study set out to determine the stability during cooking, by frying and roasting, of a wide range of anthelmintic residues in meat and liver of cattle treated with commercially available veterinary parasiticide preparations.

Materials and methods

Materials and instrumentation

Reference standards and internal standards analysed in this study are listed in Table 1 along heated to 60°C. A binary gradient mobile phase was applied at a flow rate of 0.6 mL min -1 , phase A being 0.01% acetic acid in acetonitrile:water (10:90, v/v), phase B being 5 mM ammonium formate in methanol:acetonitrile (75:25, v/v). The gradient profile was: (1) 0-0.5 min, 100% A, (2) 5 min, 50% A, (3) 7 min, 10% A, (4) 8.5 min, 10% A, (5) 8.51 min, 0% A, (6) 9.0 min, 0% A, (7) 9.01 min, 100% A, (8) 11 min 100% A. The UPLC weak wash was methanol:water (20:80, v/v) and strong wash was methanol:propan-2-ol:water (80:10:10, v/v).

A Testo 915-1 Universal probe thermometer was obtained from Testo AG (Lenzkirch, Germany).

Tissues were minced in a domestic food processor before homogenisation in a SL2 laboratory homogeniser (Silverson Machines Ltd., Chesham, UK). QuEChERS dispersive clean-up tubes (1.5 g MgSO 4 and 0.5 g C 18 in 50 mL centrifuge tubes) were obtained from UCT (Bristol, PA, USA).

QuEChERS extraction salts, anhydrous MgSO 4 (4 g) and NaCl (1 g), were obtained from Sigma-Aldrich and pre-weighed in-house.

[INSERT TABLE 1 ABOUT HERE]

Production of incurred bovine tissues

Seven pasture-fed beef cattle (male, 16-17 months of age, approximately 450 kg body weight) were treated with commercially available parasiticide preparations (comprising 11 active ingredients corresponding to 21 marker residues) at the manufacturers' recommended doses and routes (subcutaneous injection or oral drench) split half-and-half over two consecutive days (Table 2).

Administration was staggered over two days to encourage the formation of both parent and metabolite drug residues in the tissues. Short withdrawal periods of one or two days were observed after the second dose before cattle were slaughtered and Longissimus dorsi muscle (sirloin/filet steak) and liver were sampled. Tissues were stored at -20°C prior to analysis.

Cooking of incurred beef and liver

One portion of L. dorsi muscle and one portion of liver were selected from each medicated cow.

After thawing to room temperature, adjoining pieces of muscle were sliced for frying (mean 185 g) and roasting (mean 300 g). A third adjoining slice was retained for analysis without cooking.

Single slices of liver (mean 170 g) were taken for frying and adjoining uncooked slices retained for analysis without cooking. Excess juices and blood were removed from each sample using absorbent tissue before weighing immediately prior to cooking (or before mincing of uncooked samples). Cooked samples were re-weighed after cooling to room temperature and removal of excess juices. Cooked and raw samples were then individually minced in a domestic food processor and re-frozen at -20°C prior to analysis by LC-MS/MS. Cooking conditions were as follows.

Roasting. Muscle was roasted for 40 min at 190°C on the middle shelf of a pre-heated fan-assisted domestic oven, while wrapped in aluminium foil in an uncovered tray. All juices expressed during roasting were collected and weighed.

Frying. Muscle was fried for 4-6 min each side (depending on sample size, to give medium cooked meat) on a high heat setting on a domestic ceramic hob, in a minimal volume of sunflower oil (one teaspoon) to prevent burning. Small volumes of expressed juices were collected following frying of the majority of muscle samples. Liver was fried in approximately 10 mL sunflower oil, searing for 2 min each side on a high heat setting, then frying on medium heat for a further 10-15 min, depending on sample size, to give well done cooked liver in the centre of the portion. Significant amounts of juices were not expressed during frying of liver.

The maximum internal temperature of each sample was measured on completion of cooking by inserting a digital penetration probe thermometer into the centre of the sample.

LC-MS/MS analysis

Each sample, raw and cooked, was analysed in triplicate as described below. Juices were analysed in duplicate. Different sample types were stored under identical conditions at all times and analysed in the same analytical batch to avoid time-dependent (stability) differences in residue concentrations. This analytical method is based on the QuEChERS-UPLC-MS/MS protocol developed by [START_REF] Kinsella | New method for the analysis of flukicide and other anthelmintic residues in bovine milk and liver using liquid chromatography-tandem mass spectrometry[END_REF]2010) under the ProSafeBeef project.

Briefly, samples (10 g) were extracted by homogenisation in acetonitrile (12 mL). Phase separation was achieved by shaking homogenised samples with anhydrous MgSO 4 (4 g) and NaCl (1 g).

Following centrifugation, the supernatant was cleaned-up using dispersive SPE by vortex mixing with anhydrous MgSO 4 (1.5 g) and C 18 sorbent (0.5 g). Following centrifugation, a portion of the extract (1-3 mL) was mixed with dimethylsulphoxide (DMSO, 0.5-1 mL) and the acetonitrile evaporated under nitrogen at 50°C. The volumes of extract and DMSO employed were varied for a given analytical batch to keep the MS/MS signal within a linear range. Furthermore, highly concentrated samples were diluted with L. dorsi muscle or liver from untreated cattle, up to a total of 10 g, prior to homogenisation. Extracted matrix calibration standard curves were prepared by fortifying 10 g of L. dorsi muscle or liver from untreated cattle with standard mixtures prior to homogenisation and extraction. Final extracts in DMSO were injected (4 µL) onto the Acquity-Premier XE UPLC-MS/MS system described above. Ionisation polarities and internal standards applied are listed in Table 1. Calibration standards (n=5 to 7 depending on range of linearity) were injected in duplicate with acceptable regression coefficients (r 2 ) above 0.98 for analytes employing isotopic internal standards and 0.95 for those employing non-isotopic internal standards (Table 1).

Analyte concentrations in samples were calculated by comparing the ratio of the response of the analyte's base peak to the internal standard response with the same ratio in calibration curve standards, followed by a correction for any sample dilution applied prior to extraction.

Results and discussion

Changes in anthelmintic drug residue concentrations during cooking are shown in Tables 345. Two cattle received medication containing levamisole -only data from that which received Levafas Diamond are presented. The concentrations detected in raw (uncooked) tissues were adjusted for sample weight changes resulting from the cooking procedures to give concentrations "expected after cooking". These expected concentrations were then compared with the concentrations detected after cooking and a two-tailed, unpaired Student's t-test applied to the triplicate analyses. For those samples from which juices were expressed, mean residues detected in cooked tissue and juices were combined in absolute terms (µg residue in total cooked sample + µg residue in total cooked juices) to permit comparison with µg of residue in total uncooked sample (Tables 45).

Replicate data (n=3) from tissues and juices cannot be paired to give replicate concentration data for combined cooked samples, therefore a t-test cannot be applied Maximum internal sample temperatures during cooking ranged from 55 to 96°C (Tables 345). In keeping with the differing sample sizes and cooking times, higher internal temperatures were achieved during roasting of muscle (mean 88°C) than frying (mean 65°C). Liver, fried thoroughly for up to 19 min, reached the highest temperatures (mean 91°C).

Major residue instability was evident only for nitroxynil which dropped by 96% in roast muscle and by 78% in fried muscle (Tables 4 and5). Nitroxynil was not detected in uncooked liver. This disparity between nitroxynil concentrations in uncooked bovine muscle (531 µg kg -1 ) and liver (<10 µg kg -1 ) is in keeping with a previous report indicating that up to 98% of nitroxynil was metabolised in the liver of treated cattle (EMEA 1999). Our observed loss of nitroxynil in muscle is in agreement with the limited data of [START_REF] Ekström | Determination and health-risk evaluation of nitroxynil residues in the edible tissues of cattle[END_REF] who saw the complete destruction of nitroxynil in beef boiled for 2 h and a loss of up to 55% when fried.

Smaller, but significant, losses of levamisole (42% in fried liver, 11% in fried muscle), rafoxanide (17% in fried muscle, 18% in roast muscle) and triclabendazole (23% in fried liver, 47% in roast muscle) were also seen in the current study.

A small loss of levamisole from fried bovine muscle (11%) was observed. Rose et al. (1995a) saw no loss of levamisole during frying of incurred porcine muscle or in boiling water at 100°C.

Roasting bovine muscle in the current study led to a 17% loss of levamisole in the meat but this was wholly accounted for by the residue detected in the expressed juices (Table 5). Rose et al. (1995a) also demonstrated a loss of levamisole during roasting of porcine muscle but expressed juices were not analysed in that instance. [START_REF] Hsu | Influence of cooking processing conditions on levamisole residues in swine muscle tissues[END_REF] total levamisole residues post-cooking were detected in the juices; Table 5), it is conceivable that this 42% loss may be accounted for, at least in part, by juices expressed during frying which then evaporated in the pan and were thus unavailable for analysis. Levamisole residues remaining in the pan following evaporation of expressed juices may still be ingested if the consumer chooses to prepare sauces or gravy in the same pan.

Rafoxanide (Tables 345) was seen to decrease by 17-18% in cooked muscle and by 12% in fried liver (although this was not statistically significant). [START_REF] Caldow | Multi-residue determination of phenolic and salicylanilide anthelmintics and related compounds in bovine kidney by liquid chromatography-tandem mass spectrometry[END_REF] demonstrated that rafoxanide was generally stable during storage, although degradation in fortified tissue and low concentration solvent solution was evident after one week storage at room temperature in daylight.

The significance of a minor reduction (less than one fifth) in residues of a drug to which relatively low EU Maximum Residue Limits have been assigned (30 µg kg -1 bovine muscle and 10 µg kg -1 bovine liver; Table 2) is debatable. Consumers want their meat to be residue-free on the supermarket shelf, and on their plate, and will view such partial degradation of residues during cooking as no safeguard to their health or the perceived quality of their food. Furthermore, it cannot be discounted that residues of any compound lost during cooking are in fact being converted to unidentified products with equal or greater toxicity.

Triclabendazole was the only benzimidazole compound which exhibited instability during cooking when expressed as the sum of its metabolites as defined by Commission Regulation 37/2010 (European Commission 2010). Total triclabendazole fell by 23% in fried liver and by 47% in roast muscle. This two-fold difference in stability (cooking temperatures were comparable) may be due in part to the different starting metabolite concentrations in the uncooked tissues. TCB-SO 2 is the predominant metabolite in muscle, followed by TCB, while the reverse is the case in liver (Tables 3 and4). However, in both tissues TCB-SO 2 exhibited higher percentage losses than TCB during cooking, leading to a substantially greater reduction in total triclabendazole residues in muscle than in liver. Total triclabendazole did not fall significantly when muscle was fried -the treatment Minor statistically significant reductions (6%) in total albendazole and total mebendazole residues were evident in roast muscle, but these can be considered negligible in the context of food safety.

Analysis of the metabolites of albendazole suggests a shift towards higher ABZ-NH 2 -SO 2 concentrations when liver and muscle are fried, but this was not evident when muscle was roasted (Tables 345). Analysis of the metabolites of mebendazole suggested no shift in their relative concentrations during cooking with the exception of NH 2 -MBZ which doubled its concentration in liver during frying, leading to a considerable 86% increase in total mebendazole residues. This change was not replicated in cooked muscle in which the starting concentration of mebendazole residues was 10 fold lower than in liver. The source of this elevated NH 2 -MBZ in fried liver was not transformation of MBZ or OH-MBZ, and must therefore be attributed to other unidentified hepatic metabolites of mebendazole. [START_REF] Rose | The effect of cooking on veterinary drug residues in food: 5. oxfendazole[END_REF] postulated the formation of an amine of oxfendazole (FBZ-SO) when cooking incurred bovine liver and concluded that, while cooking did not destroy the residues, it may affect the equilibrium between FBZ-SO, FBZ-SO 2 , FBZ and other unidentified fenbendazole metabolites in incurred tissue. The current study supports this assertion. FBZ-SO is a very minor metabolite in muscle and no significant change in total fenbendazole residues or shift in the relative concentrations of its metabolites was evident following cooking. Similarly, no significant drop in total fenbendazole residues was seen following frying of incurred liver although the concentration of FBZ-SO 2 did fall by 80%. [START_REF] Slanina | Ivermectin residues in the edible tissues of swine and cattle: effect of cooking and toxicological evaluation[END_REF] observed ivermectin losses of up to 50% when bovine muscle was minced before boiling or frying. In the former study, the majority of the missing residue was detected in the cooking fluids which the latter study did not analyse.

F

In addition to ivermectin, the flukicides oxyclozanide, closantel and clorsulon exhibited no instability under cooking in the present study. However, statistically significant increases (27-29%)

were apparent for oxyclozanide and clorsulon after frying of muscle (Table 4). As postulated above for NH 2 -MBZ, these increases may arise from transformation of other unidentified metabolites during cooking, although it is difficult to explain why they were not replicated in roast muscle or fried liver.

Migration of drug residues into expressed juices during cooking is one aspect of studies such as this which is difficult to control and to assess accurately. The availability of expressed juices is sample and operator-dependent, being affected by many factors including the fat and water contents of the tissue, cooking temperature, the physical treatment of the tissue (slicing, squeezing) and resting time post-cooking. Furthermore, the fate of any residues which do migrate into cooking juice is dependent upon consumer choice -the choice to discard juices or use them in sauces or gravy or to baste the meat. Culinary practices do not always fit neatly with analytical laboratory practices.

Nevertheless, the evidence of the current study suggests that migration of anthelmintic drug residues into juices during roasting or shallow frying of beef or bovine liver is not a major consideration. With the exception of levamisole (17% from roast muscle), residues in expressed juices constituted only 8% or less of the total residues remaining after cooking (Tables 4 and5).

The extent of drug residue migration into cooking juices is known to be dependent upon the compound (<2% for nicarbazin; [START_REF] Tarbin | The effect of cooking on veterinary drug residues in food: Nicarbazin (dinitrocarbanilide component)[END_REF]) and method of cooking (20% for sulphamethazine from microwaved pig meat, but 54% when pressure cooked; Rose et al. 1995b).

In summary, with the exception of nitroxynil, residues of anthelmintic drugs in beef and bovine liver were generally resistant to degradation during roasting and frying. Apparent increases in residue concentrations during cooking may be due to conversion of unidentified metabolites into the specified marker residues. Consequently, conventional domestic cooking cannot be considered a safeguard against ingestion of residues of commonly used anthelmintic veterinary drugs in beef.

How such findings impact upon the consumer of beef and their perception of beef quality is another aspect of the research conducted under the ProSafeBeef project. [START_REF] Verbeke | Why consumers behave as they do with respect to food safety and risk information[END_REF] described how consumers of food do not differentiate significantly between different types of risk within a particular food group. For example, in the mind of the consumer dioxins in poultry, hormones in beef and antibiotics in pork were conflated and considered to be equivalent. Furthermore, risks in one food group can be erroneously attributed to another -for example, the belief that bovine spongiform encephalopathy (BSE) was a cause for concern in poultry [START_REF] Verbeke | Beliefs, attitude and behaviour towards fresh meat revisited after the Belgian dioxin crisis[END_REF]. A recent ProSafeBeef project study of European consumer perceptions of beef safety demonstrated that, rather than fearing the separate risks of contamination of their food by either "bugs" (microbiological risks) or "drugs" (chemical risks), consumers applied an overarching criterion such that safe beef was perceived as beef that is not bad for consumers' health (''It shouldn't be making me ill"; [START_REF] Van Wezemael | European consumers and beef safety: Perceptions, expectations and uncertainty reduction strategies[END_REF]. Consumers may thus apply the well-understood public advice on bacterial risks (store the meat properly and cook it thoroughly) believing that their vigilance will also afford a degree of protection against the health risks of chemical contaminants. The present study and much of the scientific literature demonstrate that such action affords the consumer little or no such protection against veterinary drug residues. This study adds weight to the general principle that veterinary drug residues in food are resistant to degradation under conventional

  with their sources and abbreviated names. Standards were obtained from Sigma-Aldrich (Poole, UK), Witega Laboratorien Berlin-Adlershof (Berlin, Germany), Janssen Animal Health (Beerse, Belgium), Pfizer Animal Health (Sandwich, UK) and QuChem (formerly of Belfast, UK). Unless stated, all other chemicals were obtained from Sigma-Aldrich. Commercially available parasiticide preparations were purchased from the following manufacturers: Levafas Diamond (Norbrook Laboratories Ltd.; Newry, UK), Curafluke 10% (Univet Ltd.; Cootehill, Ireland), Supaverm™ Oral Suspension (Janssen Animal Health; High Wycombe, UK), Valbazen® 10% (Pfizer Animal Health; Ringaskiddy, Ireland), Fasinex Super® 19.5% (Novartis Animal Health Ireland Ltd.; Waterford, Ireland), Deldrax® 34% (Intervet Ireland Ltd.; Dublin, Ireland) and Ivomec Super® Injection for Cattle (Merial Animal Health; Harlow, UK). An Acquity UPLC® binary pump and sample management system (Waters Corporation, Manchester, UK) coupled to a Quattro Premier™ XE tandem mass spectrometer (Waters Corporation), both operating under MassLynx™ software, were used for sample analysis. The mass spectrometer operated under Electrospray Ionisation (ESI) with rapid polarity switching. Data acquisition was in Multiple Reaction Monitoring mode (MRM) with a total run time of 11 min.Data analysis was performed using Waters TargetLynx™ software. MS source settings were as follows: capillary voltage 3.0 kV (ESI+) and 2.2 kV (ESI-), source temperature 150°C, desolvation temperature 400°C, cone nitrogen gas flow 150 L h -1 , desolvation nitrogen gas flow 800 L h -1 . The UPLC system was equipped with an Acquity HSS T3 UPLC analytical column (100 × 2.1 mm) packed with HSS C 18 (1.8 µm) and an in-line filter unit (0.2 µm, 2.1 mm) (Waters Corporation),

  Concentrations of the benzimidazoles are expressed as the sums of their parent compounds and/or metabolites as defined by Commission Regulation 37/2010 (European Commission 2010) which recently replaced Commission Regulation 2377/90 as an alphabetical listing of Maximum Residue Limits and marker residues for allowed pharmacologically active substances in foodstuffs of animal origin. Calculations of the concentrations of designated benzimidazole marker residues were as follows: Albendazole: ABZ = (0.943×ABZ-SO) + (0.892×ABZ-SO 2 ) + (1.109×ABZ-NH 2 -SO 2 ) Fenbendazole: FBZ-SO 2 = FBZ-SO 2 + (1.107×FBZ) + (1.051×FBZ-SO) Mebendazole: MBZ = MBZ + (1.244×NH 2 -MBZ) + (1.014×OH-MBZ) Triclabendazole: KetoTCB = KetoTCB + (0.916×TCB) + (0.878×TCB-SO) + (0.842×TCB-SO 2 )

  shortest time and lowest temperatures. In this case the predominant TCB-SO 2 metabolite concentration fell slightly but was off-set by an increase in TCB concentration. Of the four triclabendazole metabolites analysed, TCB-SO exhibited the largest percentage reductions during cooking, but it comprised only a small proportion (approximately 2%) of total triclabendazole residues.

  the approach of minimising and controlling the use of veterinary medicines at source to safeguard both the health of the consumer and the profitability of food producers.

  The finding that ivermectin is stable under normal cooking conditions is in agreement with[START_REF] Rose | The effect of cooking on veterinary drug residues in food: 7. ivermectin[END_REF] who fried, microwaved and boiled medicated cattle and pig tissues, and[START_REF] Cerkvenik | Thermal and long-term freezing stability of ivermectin residues in sheep milk[END_REF] who saw no degradation of ivermectin in milk pasteurised at 80°C or boiled at 100°C.[START_REF] Rose | The effect of cooking on veterinary drug residues in food: 7. ivermectin[END_REF]

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Table 1 .

 1 Reference and internal standard abbreviations, sources, ESI modes and applied internal standards.

		Food Additives and Contaminants		Page 20 of 24
	Standard	Abbreviation	Source	ESI mode Internal standard
	Clorsulon	CLORS	Sigma-Aldrich	-	DNC-D8
	Closantel	CLOSAN	Sigma-Aldrich	-	TBCP
	Nitroxynil	NITROX	Sigma-Aldrich	-	TBCP
	Oxyclozanide	OXYCLOZ	Sigma-Aldrich	-	TBCP
	Rafoxanide	RAFOX	Sigma-Aldrich	-	DNC-D8
	Levamisole	LEVAM	Sigma-Aldrich	+	LEVAM-D5
	Ivermectin B1a	IVER	Sigma-Aldrich	+	SELA
	Albendazole	ABZ	Sigma-Aldrich	+	ABZ-D3
	Albendazole Sulphoxide	ABZ-SO	Witega	+	ABZ-SO-D3
	Albendazole Sulphone	ABZ-SO 2	Witega	+	ABZ-SO 2 -D3
	Albendazole 2-Amino-Sulphone	ABZ-NH 2 -SO 2	Witega	+	ABZ-D3
	Fenbendazole	FBZ	Sigma-Aldrich	+	FBZ-D5
	Fenbendazole-Sulphoxide	FBZ-SO	Sigma-Aldrich	+	FBZ-SO-D5
	Fenbendazole-Sulphone	FBZ-SO 2	Witega	+	FBZ-SO 2 -D5
	Mebendazole	MBZ	Sigma-Aldrich	+	TBZ-D4
	Hydroxy-Mebendazole	OH-MBZ	Janssen Animal Health (gift)	+	OH-MBZ-D3
	Amino-Mebendazole	NH 2 -MBZ	Janssen Animal Health (gift)	+	TBZ-D4
	Triclabendazole	TCB	Sigma-Aldrich	+	TCB-D3
	Triclabendazole-Sulphoxide	TCB-SO	Witega	-	TBCP
	Triclabendazole-Sulphone	TCB-SO 2	Witega	-	TBCP
	Keto-Triclabendazole	Keto-TCB	Witega	-	SALI
	Levamisole-D5	LEVAM-D5	QuChem (custom synthesis)	+	*
	Albendazole-D3	ABZ-D3	Witega	+	*
	Albendazole Sulphoxide-D3	ABZ-SO-D3	Witega	+	*
	Albendazole Sulphone-D3	ABZ-SO 2 -D3	Witega	+	*
	Fenbendazole-D5	FBZ-D5	QuChem (custom synthesis)	+	*
	Fenbendazole-Sulphoxide-D5	FBZ-SO-D5	QuChem (custom synthesis)	+	*
	Fenbendazole-Sulphone-D5	FBZ-SO 2 -D5	QuChem (custom synthesis)	+	*
	Thiabendazole-D4	TBZ-D4	QuChem (custom synthesis)	+	*
	Triclabendazole-D3	TCB-D3	Witega	+	*
	Hydroxy-Mebendazole-D3	OH-MBZ-D3	Witega	+	*
	Selamectin	SELA	Pfizer Animal Health UK (gift)	+	*
	Salicylanilide	SALI	Sigma-Aldrich	-	*
	2,2-Thiobis(4-chlorophenol)	TBCP	Sigma-Aldrich	-	*
	Dinitrocarbanilide-D8	DNC-D8	Witega	-	*

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Table 2 .

 2 Parasiticidal preparations administered to beef cattle (~450 kg body weight) and EU Maximum Residue Limits (MRL). NA = none assigned.

	Page 21 of 24			Food Additives and Contaminants
	Brand name Manufacturer Active ingredients	Dosage (mg kg -1	Total dose	Route	Days before	MRL bovine	MRL bovine
			(% w/v)	body weight)	(mL)		slaughter	muscle (µg kg -1 )	liver (µg kg -1 )
	Fasinex Super®	Novartis	Triclabendazole 12%	12	45	Oral	2	225	250
	19.5%		Levamisole HCl 7.5%	7.5				10	100
	Supaverm™	Janssen	Mebendazole 7.5%	15	90	Oral	1	NA	NA
	Oral Suspension		Closantel 5%	10				1000	1000
	Ivomec Super®	Merial	Clorsulon 10%	2	9	Injection	2	35	100
			Ivermectin 1%	0. 2		(subcut.)		20 (action limit)	100
	Levafas	Norbrook	Oxyclozanide 6%	15	112.5	Oral	1	20	500
	Diamond		Levamisole HCl 3%	7.5				10	100
	Deldrax® 34%	Intervet	Nitroxynil N-	10	13.5	Injection	2	400	20
			ethylglucamine 34%			(subcut.)
	Curafluke 10%	Univet	Fenbendazole 10%	11.25	50.6	Oral	1	50	500
			Rafoxanide 10%	11.25				30	10
	Valbazen® 10%	Pfizer	Albendazole 10%	10	45	Oral	1	100	1000

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Table 3 .

 3 Effect of frying on concentration of anthelmintic drug residues in bovine liver. Concentration data are mean ± standard error (n=3). P value indicates a statistically significant difference between expected and observed concentrations after cooking. Expected concentrations are corrected for weight loss during cooking. ND = none detected (estimated assay Limit of Detection accounting for sample dilution).

				Food Additives and Contaminants
	LIVER	Weight	Weight	Max. internal	µg kg -1	µg kg -1 expected	µg kg -1 observed after	Residues % change
	residue	before (g)	after (g)	temp. (°C)	uncooked	after frying	frying	during frying
	Levamisole	143.0	103.7	91.3	2928.6 ± 65.0	4038.5 ± 89.6	2332.6 ± 43.2 (P<0.0001)	-42
	Oxyclozanide	143.0	103.7	91.3	4014.7 ±243.1	5536.2 ± 335.2	5861.0 ± 90.4	+6
	Closantel	133.4	99.2	92.4	5652.8 ±881.3	7601.7 ± 1185.1	7654.9 ± 692.2	+1
	Nitroxynil	209.5	133.8	96.1	ND (<10)	ND (<10)	ND (<10)	*
	Clorsulon	187.5	144.9	88.7	1470.2 ± 19.3	1902.5 ± 24.9	1727.3 ± 105.5	-9
	Ivermectin	187.5	144.9	88.7	487.2 ± 35.2	630.4 ± 45.6	485.9 ± 61.6	-23
	Rafoxanide	165.5	113.6	88.0	3160.2 ±334.8	4604.0 ± 487.7	4056.3 ± 171.1	-12
	ABZ	154.2	108.0	93.7	1112.2 ± 20.0	1588.0 ± 28.6	1368.6 ± 39.8 (P<0.02)	-14
	ABZ-SO	154.2	108.0	93.7	290.2 ± 6.3	414.3 ± 8.9	256.6 ± 25.4 (P<0.005)	-38
	ABZ-SO 2	154.2	108.0	93.7	1403.9 ±108.8	2004.5 ± 155.4	1854.8 ± 89.4	-8
	ABZ-NH 2 -SO 2	154.2	108.0	93.7	720.3 ± 21.4	1028.4 ± 30.6	1211.9 ± 43.2 (P<0.03)	+18
	SUM ABZ	154.2	108.0	93.7	2324.8 ± 78.4	3319.3 ± 111.9	3240.4 ± 99.7	-2
	MBZ	133.4	99.2	92.4	22.4 ± 1.0	30.2 ± 1.4	33.2 ± 2.0	+10
	OH-MBZ	133.4	99.2	92.4	73.2 ± 2.1	98.4 ± 2.8	99.7 ± 7.3	+1
	NH 2 -MBZ	133.4	99.2	92.4	297.8 ± 25.4	400.4 ± 34.2	833.5 ± 142.8 (P<0.05)	+108
	SUM MBZ	133.4	99.2	92.4	467.0 ± 32.4	628.0 ± 43.6	1171.2 ± 174.3 (P<0.04)	+ 86
	FBZ	165.5	113.6	88.0	9243 ± 998	13466 ± 1454	13623 ± 251	+1
	FBZ-SO	165.5	113.6	88.0	1791.1 ± 90.8	2609.3 ± 132.3	525.6 ± 21.0 (P<0.0001)	-80
	FBZ-SO 2	165.5	113.6	88.0	1029.4 ± 76.0	1499.7 ± 110.7	1637.3 ± 111.9	+9
	SUM FBZ	165.5	113.6	88.0	13144 ± 1085	19149 ± 1581	17270 ± 387	-10
	TCB	186.3	138.0	89.7	2911.0 ± 43.9	3929.9 ± 59.3	3160.4 ± 183.2 (P<0.02)	-20
	TCB-SO	186.3	138.0	89.7	ND (<50)	ND (<50)	ND (<50)	*
	TCB-SO 2	186.3	138.0	89.7	1162.9 ± 85.5	1569.9 ± 115.4	988.6 ± 53.1 (P<0.02)	-37
	Keto-TCB	186.3	138.0	89.7	383.8 ± 34.2	518.2 ± 46.2	453.7 ± 133.0	-12
	SUM TCB	186.3	138.0	89.7	4029.5 ± 171.7	5439.8 ± 231.8	4181.0 ± 246.9 (P<0.03)	-23

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Table 4 .

 4 Effect of frying on concentration of anthelmintic drug residues in bovine muscle. Tissue concentration data are mean ± standard error (n=3). Juice concentration data are means (n=2). NA = not available. ND = none detected (estimated assay Limit of Detection accounting for sample dilution). P value indicates a statistically significant difference between expected and observed concentrations after cooking.

					Food Additives and Contaminants
	MUSCLE	Weight	Weight	Weight	Max. internal	µg kg -1	µg kg -1 expected	µg kg -1 observed after	µg kg -1	% of total fried	Residue % change
	residue	before (g)	after (g)	juices (g)	temp. (°C)	uncooked	after frying	frying	juices	residue in juices	during frying (juices
											included)
	Levamisole	189.6	151.2	0	57.0	108.1 ± 0.9	135.5 ± 1.2	121.3 ± 0.3 (P<0.0003)	NA	NA	-11
	Oxyclozanide	189.6	151.2	0	57.0	547.5 ± 14.9	686.5 ± 18.7	868.6 ± 22.1 (P<0.004)	NA	NA	+27
	Closantel	149.2	117.5	3.4	67.3	2089.8 ± 74.7	2653.6 ± 94.8	2499.9 ± 95.0	684.8	<1	-5
	Nitroxynil	248.1	196.0	4.0	66.9	531.4 ± 7.2	672.7 ± 9.2	142.9 ± 9.7 (P<0.0001)	238.5	3	-78
	Clorsulon	151.1	119.5	3.9	70.2	289.2 ± 10.3	365.7 ± 13.0	470.0 ± 28.3 (P<0.03)	ND(<150)	0	+29
	Ivermectin	151.1	119.5	3.9	70.2	14.4 ± 0.6	18.1 ± 0.7	15.6 ± 0.9	NA	NA	-14
	Rafoxanide	124.2	88.0	3.6	68.6	1813.3 ± 49.1	2559.2 ± 69.4	2106.8 ± 125.0 (P<0.04) 133.3	<1	-17
	ABZ	227.6	170.5	0	55.0	5.30 ± 0.09	7.07 ± 0.12	7.02 ± 0.12	NA	NA	-1
	ABZ-SO	227.6	170.5	0	55.0	1.58 ± 0.36	2.11 ± 0.48	ND (<1) (P<0.02)	NA	NA	-100
	ABZ-SO 2	227.6	170.5	0	55.0	42.4 ± 0.4	56.6 ± 0.6	47.2 ± 0.3 (P<0.0002)	NA	NA	-17
	ABZ-NH 2 -SO 2	227.6	170.5	0	55.0	106.0 ± 4.5	141.5 ± 6.0	164.9 ± 2.1 (P<0.03)	NA	NA	+17
	SUM ABZ	227.6	170.5	0	55.0	156.9 ± 5.2	209.4 ± 6.9	225.0 ± 2.6	NA	NA	+7
	MBZ	149.2	117.5	0	67.3	0.89 ± 0.05	1.13 ± 0.06	1.01 ± 0.06	NA	NA	-10
	OH-MBZ	149.2	117.5	0	67.3	8.75 ± 0.11	11.1 ± 0.1	8.34 ± 0.31 (P<0.002)	NA	NA	-25
	NH 2 -MBZ	149.2	117.5	0	67.3	23.1 ± 0.7	29.3 ± 0.9	30.0 ± 0.3	NA	NA	+2
	SUM MBZ	149.2	117.5	0	67.3	38.5 ± 0.9	48.9 ± 1.1	46.8 ± 0.1	NA	NA	-4
	FBZ	124.2	88.0	3.6	68.6	265.3 ± 7.8	374.5 ± 11.0	340.0 ± 4.0 (P<0.05)	7.75	<1	-9
	FBZ-SO	124.2	88.0	3.6	68.6	5.57 ± 0.19	7.86 ± 0.26	2.53 ± 0.09 (P<0.0001)	ND (<2)	0	-68
	FBZ-SO 2	124.2	88.0	3.6	68.6	154.6 ± 1.6	218.2 ± 2.3	209.5 ± 13.6	24.9	<1	-4
	SUM FBZ	124.2	88.0	3.6	68.6	454.2 ± 9.6	641.1 ± 13.6	588.5 ± 17.5	33.4	<1	-8
	TCB	205.5	158.5	3.9	72.0	292.4 ± 9.7	379.1 ± 12.6	617.0 ± 27.9 (P<0.002)	16.2	<1	+63
	TCB-SO	205.5	158.5	3.9	72.0	72.1 ± 4.0	93.5 ± 5.2	23.1 ± 1.6 (P<0.0003)	ND (<50)	0	-75
	TCB-SO 2	205.5	158.5	3.9	72.0	2674.4 ± 165.3	3467.4 ± 214.3	2953.6 ± 175.9	55.6	<1	-15
	Keto-TCB	205.5	158.5	3.9	72.0	52.6 ± 0.4	68.2 ± 0.5	96.7 ± 3.3 (P<0.001)	ND (<50)	0	+42
	SUM TCB	205.5	158.5	3.9	72.0	2635.5 ± 149.2	3417 ± 193.5	3169.1 ± 169.2	61.6	<1	-7

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Table 5 .

 5 Effect of roasting on concentration of anthelmintic drug residues in bovine muscle. Tissue concentration data are mean ± standard error (n=3). Juice concentration data are means (n=2). NA = not available. ND = none detected (estimated assay Limit of Detection accounting for sample dilution). P value indicates a statistically significant difference between expected and observed concentrations after cooking.

	MUSCLE	Weight	Weight	Weight	Max.
	residue	before (g)	after (g)	juices (g)	internal
					temp. (

°C) µg kg -1 uncooked µg kg -1 expected after roasting µg kg -1 observed after roasting µg kg -1 juices % of total roasted residue in juices Residue % change during roasting (juices included)

	Levamisole	278.1	190.4	31.2	87.0	108.1 ± 0.9	157.8 ± 1.3	131.7 ± 1.5 (P<0.0003)	164.1	17	+1
	Oxyclozanide	278.1	190.4	31.2	87.0	547.5 ± 14.9	799.6 ± 21.8	728.2 ± 31.6	ND (< 25)	0	-9
	Closantel	314.2	206.5	49.7	92.9	2089.8 ± 74.7	3179.7 ± 113.6	3447.7 ± 117.5	20.0	<1	+9
	Nitroxynil	311.6	198.9	68.3	92.8	531.4 ± 7.2	832.6 ± 11.4	34.3 ± 0.9 (P<0.0001)	ND (<10)	0	-96
	Clorsulon	314.7	211.4	54.1	84.2	289.2 ± 10.3	430.6 ± 15.3	463.2 ± 41.8	117.5	6	+15
	Ivermectin	314.7	211.4	54.1	84.2	14.4 ± 0.6	21.4 ± 0.9	26.9 ± 2.9	ND (<5)	0	+26
	Rafoxanide	262.9	169.3	45.0	87.6	1813.3 ± 49.1	2815.8 ± 76.3	2313.6 ± 114.9 (P<0.03)	17.8	<1	-18
	ABZ	325.1	224.5	35.0	82.0	5.30 ± 0.09	7.67 ± 0.13	6.28 ± 0.05 (P<0.0006)	0.47	1	-17
	ABZ-SO	325.1	224.5	35.0	82.0	1.58 ± 0.36	2.29 ± 0.52	2.91 ± 0.35	1.37	7	+36
	ABZ-SO 2	325.1	224.5	35.0	82.0	42.4 ± 0.4	61.4 ± 0.6	54.8 ± 1.5 (P<0.02)	28.6	8	-3
	ABZ-NH 2 -SO 2	325.1	224.5	35.0	82.0	106.0 ± 4.5	153.5 ± 6.5	129.4 ± 1.9 (P<0.03)	77.4	9	-8
	SUM ABZ	325.1	224.5	35.0	82.0	156.9 ± 5.2	227.1 ± 7.5	195.1 ± 3.6 (P<0.02)	112.7	8	-6
	MBZ	314.2	206.5	49.7	92.9	0.89 ± 0.05	1.36 ± 0.07	0.93 ± 0.02 (P<0.004)	0.23	6	-28
	OH-MBZ	314.2	206.5	49.7	92.9	8.75 ± 0.11	13.3 ± 0.2	13.4 ± 0.5	3.36	6	+6
	NH 2 -MBZ	314.2	206.5	49.7	92.9	23.1 ± 0.7	35.1 ± 1.1	29.1 ± 0.8 (P<0.01)	11.8	9	-9
	SUM MBZ	314.2	206.5	49.7	92.9	38.5 ± 0.9	58.6 ± 1.3	50.6 ± 1.2 (P<0.02)	18.3	8	-6
	FBZ	262.9	169.3	45.0	87.6	265.3 ± 7.8	412.0 ± 12.1	386.9 ± 7.0	12.3	1	-5
	FBZ-SO	262.9	169.3	45.0	87.6	5.57 ± 0.19	8.64 ± 0.29	21.5 ± 1.4 (P<0.0009)	13.7	14	+190
	FBZ-SO 2	262.9	169.3	45.0	87.6	154.6 ± 1.6	240.1 ± 2.5	250.3 ± 3.7	39.4	4	+9
	SUM FBZ	262.9	169.3	45.0	87.6	454.2 ± 9.6	705.3 ± 14.9	701.2 ± 5.9	67.3	2	+2
	TCB	302.2	203.1	44.9	88.3	292.4 ± 9.7	435.0 ± 14.5	475.3 ± 46.8	2.87	<1	+9
	TCB-SO	302.2	203.1	44.9	88.3	72.1 ± 4.0	107.3 ± 6.0	8.50 ± 0.88 (P<0.0001)	ND (<2)	0	-92
	TCB-SO 2	302.2	203.1	44.9	88.3	2674.4 ± 165.3	3979.3 ± 246.0	1869.8 ± 65.5 (P<0.002)	18.5	<1	-53
	Keto-TCB	302.2	203.1	44.9	88.3	52.6 ± 0.4	78.2 ± 0.6	68.2 ± 3.5 (P<0.05)	ND (<20)	0	-13
	SUM TCB	302.2	203.1	44.9	88.3	2635.5 ± 149.2	3921.5 ± 222.0	2085.4 ± 94.3 (P<0.002)	18.2	<1	-47

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Acknowledgements

The authors acknowledge the financial support of the European Commission for the project FOOD-CT-2006-36241 'ProSafeBeef' which funded this work. Grateful thanks are expressed to the farm and post-mortem room staff of AFBI Veterinary Sciences Division, Belfast. We thank Janssen Animal Health and Pfizer Animal Health UK for donating standard materials and the EU Community Reference Laboratory (BVL, Berlin, Germany) for the formulae for benzimidazole metabolite calculations.

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Food Additives and Contaminants