Assessment of exposure to pesticides: residues in 24 h duplicate diets versus their metabolites in 24 h urine using suspect screening and target analysis.

Human biomonitoring can add value to chemical risk assessment by reducing the assumptions regarding consumption rates, residue occurrence, and processing effects and by integrating exposures from different sources (diet, household use, environmental). However, the relationship between exposure and concentration in human matrices is unknown for most pesticides. Therefore, we conducted a pilot study to gain more insight into the qualitative and quantitative relationship between dietary intake of pesticides (external exposure) and urinary excretion (reflecting internal exposure). In this cross-sectional observational study, 35 healthy consumers aged 18-65 years from the region of Wageningen, Netherlands, collected an exact duplicate portion of their diets during 24 h. On the same day, they also collected all their urine. The duplicate diets were analyzed using target screening by GC- and LC-HRMS; each duplicate diet contained at least five, up to 21, pesticide residues. The 24 h urine samples were analyzed using LC-HRMS in a suspect screening workflow. Metabolites were tentatively detected in all 24 h urine samples, ranging from six metabolites corresponding to four pesticides up to 40 metabolites originating from 16 pesticides in a single urine sample. In total, 65 metabolites originating from 28 pesticides were tentatively detected. After prioritization and additional confirmation experiments, 28 metabolites originating from 10 pesticides were identified with confidence level 1 or 2b. Next, quantitative analysis was performed for a selection of pesticides in duplicate diets and their metabolites in 24 h urine to assess quantitative relationships. In the quantitative comparisons between duplicate diet and 24 h urine, it was found that some metabolites were already present in the duplicate diet, which may give an overestimation of exposure to the parent pesticide based on measurement of the metabolites in urine. Additionally, the quantitative comparisons suggest a background exposure through other exposure routes. We conclude that suspect screening of 24 h urine samples can disclose exposure to mixtures of pesticide on the same day in the general population. However, more research is needed to obtain quantitative relationships between dietary intake and exposure.

Follicular fluid steroid profile in sows: relationship to follicle size and oocyte quality†.

Identification of reliable characteristics of follicle quality and developmental competence has been pursued in numerous studies, but with inconsistent outcomes. Here, we aimed to identify these characteristics by analysis of the follicular fluid (FF) steroid profile in relation to cumulus-oocyte complex (COC) morphology and follicle size, followed by molecular substantiation. Multiparous sows at weaning were used to facilitate analysis at the start of the follicular phase of the oestrus cycle. Sows with a higher average follicle size (≥5 mm vs. < 5 mm) had a higher follicular fluid ß-estradiol concentration, but did not differ in other measured steroids. Sows with high compared to low percentage high-quality COCs (<70% vs. ≥70% high-quality) had follicular fluid with a higher concentration of ß-estradiol, 19-norandrostenedione, progesterone, and α-testosterone, while the concentration of cortisol was lower. Transcriptome analysis of granulosa cells of healthy follicles of sows with a high percentage high-quality COCs showed higher abundance of transcripts involved in ovarian steroidogenesis (e.g., CYP19A2 and 3, POR, VEGFA) and growth (IGF1) and differential abundance of transcripts involved in granulosa cell apoptosis (e.g., GADD45A, INHBB). Differences in aromatase transcript abundance (CYP19A1, 2 and 3) were confirmed at the protein level. In addition, sows with a high percentage high-quality COCs lost less weight during lactation and had higher plasma IGF1 concentration at weaning, which may have affected COC quality. To the best of our knowledge, this study is also the first to report the relation between FF steroid profile and COC quality.

Consequences of negative energy balance on follicular development and oocyte quality in primiparous sows†.

Metabolic demands of modern hybrid sows have increased over the years, which increases the chance that sows enter a substantial negative energy balance (NEB) during lactation. This NEB can influence the development of follicles and oocytes that will give rise to the next litter. To study effects of a lactational NEB on follicular development, we used 36 primiparous sows of which 18 were subjected to feed restriction (3.25 kg/day) and 18 were full-fed (6.5 kg/day) during the last 2 weeks of a 24.1 ± 0.3 day lactation. Feed restriction resulted in a 70% larger lactational body weight loss and 76% higher longissimus dorsi depth loss, but similar amounts of backfat loss compared to the full fed sows. These changes were accompanied by lower plasma insulin-like growth factor 1 (IGF1) and higher plasma creatinine levels in the restricted sows from the last week of lactation onward. Ovaries were collected 48 h after weaning. Restricted sows had a lower average size of the 15 largest follicles (-26%) and cumulus-oocyte complexes showed less expansion after 22 h in vitro maturation (-26%). Less zygotes of restricted sows reached the metaphase stage 24 h after in vitro fertilization and showed a higher incidence of polyspermy (+89%). This shows that feed restriction had severe consequences on oocyte developmental competence. Follicular fluid of restricted sows had lower IGF1 (-56%) and steroid levels (e.g., ß-estradiol, progestins, and androgens), which indicated that follicles of restricted sows were less competent to produce steroids and growth factors needed for oocytes to obtain full developmental competence.

Combining Melphalan Percutaneous Hepatic Perfusion with Ipilimumab Plus Nivolumab in Advanced Uveal Melanoma: First Safety and Efficacy Data from the Phase Ib Part of the Chopin Trial.

PURPOSE: To define a safe treatment dose of ipilimumab (IPI) and nivolumab (NIVO) when applied in combination with percutaneous hepatic perfusion with melphalan (M-PHP) in metastatic uveal melanoma (mUM) patients (NCT04283890), primary objective was defining a safe treatment dose of IPI/NIVO plus M-PHP. Toxicity was assessed according to Common Terminology Criteria for Adverse Events version 4.03 (CTCAEv4.03). Secondary objective was response rate, PFS and OS. MATERIALS AND METHODS: Patients between 18-75 years with confirmed measurable hepatic mUM according to RECIST 1.1 and WHO performance score 0-1 were included. Intravenous IPI was applied at 1 mg/kg while NIVO dose was increased from 1 mg/kg in cohort 1 to 3 mg/kg in cohort 2. Transarterial melphalan dose for M-PHP was 3 mg/kg (maximum of 220 mg) in both cohorts. Treatment duration was 12 weeks, consisting of four 3-weekly courses IPI/NIVO and two 6-weekly M-PHPs. RESULTS: Seven patients were included with a median age of 63.6 years (range 50-74). Both dose levels were well tolerated without dose-limiting toxicities or deaths. Grade III/IV adverse events (AE) were observed in 2/3 patients in cohort 1 and in 3/4 patients in cohort 2, including Systemic Inflammatory Response Syndrome (SIRS), febrile neutropenia and cholecystitis. Grade I/II immune-related AEs occurred in all patients, including myositis, hypothyroidism, hepatitis and dermatitis. There were no dose-limiting toxicities. The safe IPI/NIVO dose was defined as IPI 1 mg/kg and NIVO 3 mg/kg. There was 1 complete response, 5 partial responses and 1 stable disease (3 ongoing responses with a median FU of 29.1 months). CONCLUSION: Combining M-PHP with IPI/NIVO was safe in this small cohort of patients with mUM at a dose of IPI 1 mg/kg and NIVO 3 mg/kg.

Thyreostatic drugs, stability in bovine and porcine urine.

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981. For monitoring their illegal use, sensitive and specific analytical methods are required. In this context, the knowledge of the stability in a matrix is of primary importance. This study aimed at evaluating the effects of preservation, number of freeze-thaw cycles, and matrix-related variables on the stability of thyreostatic drugs in the urine of livestock. Finally, the developed conservation approach was applied on incurred urine samples, which displayed traces of the thyreostat thiouracil below the recommended concentration of 10 µg L(-1). The stability study confirmed the negative influence of preservation (8 h) at room temperature and at -70 °C, decreases in concentration of more than 78.0% were observed for all thyreostats, except for 1-methyl-2-mercaptoimidazole and 2-mercaptobenzimidazole. Additionally, investigation of matrix-related variables indicated significant impacts of the presence of copper (p = 0.001) and the pH (p = 0.002). Next, an optimised pre-treatment (pH 1 and 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate) significantly differing from the original conservation approach (p < 0.05) was developed, which proved capable of delaying the decrease in concentration and improved the detection in time for both spiked as well as incurred urine samples. In the future, it seems highly advisable to apply the developed pre-treatment on incurred urines upon sampling, before thyreostat analysis. Additionally, it is recommendable to limit preservation of urine samples at room temperature, but also in the freezer prior to thyreostat analysis.

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment.

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10-12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCbeta levels (5-20 microg/kg) after 5-7 weeks. When treatment is repeated two times, the CCbeta levels are reached after 9-11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but--in contrast with the pour-on application--after i.m. injection, significant increase of 17beta-testosterone and 17beta-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.

Multiclass screening in urine by comprehensive two-dimensional liquid chromatography time of flight mass spectrometry for residues of sulphonamides, beta-agonists and steroids.

Nowadays routine residue monitoring involves the analysis of many compounds from different classes, mainly in urine. In the past two decades, developments heavily focused on the use of mass spectrometers (MS) and faster and more sensitive MS detectors have reached the market. However, chromatographic separation (CS) was rather ignored and the cognate developments in CS were not in line. As a result, residue analysis did not improve to the extent anticipated. CS by LC x LC is a promising technique and will enable a further increase in the range of compounds and compound classes that can be detected in a single run. In the present study, a self-built LC x LC system, using a 10 port valve, was connected to a single quadrupole MS with electrospray interface. Standards containing a mixture of sulphonamides, ß-agonists and (steroid) hormones, 53 compounds, in total, were analysed. Results demonstrated that these compounds were well separated and could be detected at low levels in urine, i.e. limit of detection (LOD) from 1 µg L-1 for most ß-agonists to 10 µg L-1 for some sulphonamides and most hormones. To enhance the sensitivity, optimisation was performed on an advanced commercial LC x LC system connected to a full scan accurate MS. This ultimately resulted in a fast high throughput untargeted method, including a simple sample clean-up in a 96-well format, for the analysis of urine samples.

Applicability of an innovative steroid-profiling method to determine synthetic growth promoter abuse in cattle.

A robust LC-MS/MS method was developed to quantify a large number of phase I and phase II steroids in urine. The decision limit is for most compounds lower than 1ngml-1 with a measurement uncertainty smaller than 30%. The method is fully validated and was applied to assess the influence of administered synthetic steroids and beta-agonists on the steroidogenesis. From three animal experiments, clenbuterol, diethylstilbestrol and stanozolol, the steroid profiles in urine of bovine animals were compared before and after treatment. It was demonstrated that the steroid profiles were altered due to these treatments. A predictive multivariate model was built to identify deviations from normal population steroid profiles. The abuse of synthetic steroids can be detected in urine samples from bovine animals using this model. The samples from the animal experiments were randomly analysed using this method and predictive model. It was shown that these samples were predicted correctly in the exogenous steroids group.

Workshop 2 (synthesis): principles for management of urban water services.

The main conclusion of the workshop was that there is a widespread need for reform in the water sector, with clear separation of policy, regulatory and service delivery functions in the management of urban water services.

Endogenous steroid profiling by gas chromatography-tandem mass spectrometry and multivariate statistics for the detection of natural hormone abuse in cattle.

For years it has been suspected that natural hormones are illegally used as growth promoters in cattle in the European Union. Unfortunately there is a lack of methods and criteria that can be used to detect the abuse of natural hormones and distinguish treated from non-treated animals. Pattern recognition of steroid profiles is a promising approach for tracing/detecting the abuse of natural hormones administered to cattle. Traditionally steroids are analysed in urine as free steroid after deconjugation of the glucuronide (and sulphate) conjugates. The disadvantage of this deconjugation is that valuable information about the steroid profile in the sample is lost. In this study we develop a method to analyse steroids at very low concentration levels (ng l(-1)) for the free steroid, glucuronide and sulphate conjugates in urine samples. This method was used to determine concentrations of natural (pro)hormones in a large population (n = 620) of samples from male and female bovine animals and from bovine animals treated with testosterone-cypionate, estradiol-benzoate, dihydroepiandrosterone and pregnenolone. The data acquired were used to build a statistical model applying the multivariate technique 'Soft Independent Modeling of Class Analogy' (SIMCA). It is demonstrated that by using this model the results of the urine analysis can indicate which animal may have had illegal treatment with natural (pro)hormones.

Human Papillomavirus and Oropharyngeal Squamous Cell Carcinoma: A Case-Control Study regarding Tobacco and Alcohol Consumption.

We aimed to determine the role of HPV in the pathogenesis and outcome of oropharyngeal squamous cell carcinoma (OSCC) in lifelong nonsmoking and nondrinking patients. A case-case analysis was performed to compare the presence of HPV-DNA in tumor cells of 16 nonsmoking and nondrinking with 16 matched smoking and drinking patients (matching criteria: age at incidence, gender, tumor sublocation, tumor stage). HPV was detected using 2 PCR tests, FISH analysis, and p16(INK4A) immunostaining. Nonsmoking and nondrinking patients had more HPV-positive tumors than smoking and drinking patients (n = 12; 75% versus n = 2; 13%; P < 0.001). All HPV-positive tumors showed p16(INK4A) overexpression, and 1 HPV-negative tumor had p16(INK4A) overexpression, (P < 0.001). Overall survival and disease-specific survival were higher for HPV-positive compared to HPV-negative cases (P = 0.027, P = 0.039, resp.). In conclusion, HPV is strongly associated with OSCC of nonsmoking and nondrinking patients. Specific diagnostic and therapeutic actions should be considered for these patients to achieve a better prognosis.

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS.

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.

Development of a method which discriminates between endogenous and exogenous beta-boldenone.

One potential explanation for the presence of beta-boldenone in calf urine is contamination of the sample with feces containing beta-boldenone. It has been demonstrated that after oral and intramuscular administration of beta-boldenone esters, several metabolites are formed and excreted in urine. One of the (minor) metabolites is 6beta-hydroxy-17alpha-boldenone. This paper describes an analytical method that can discriminate between unconjugated boldenone, its glucuronide- and sulphate-conjugates, 6beta-hydroxy-17alpha/beta-boldenone and coprostanol, a marker for fecal contamination. The method was applied to all samples suspected to contain boldenone within the Dutch National Residue Control Plan. Approximately 10,000 samples of urine were screened (LC-MS) in 2004-2005 by VWA-East, one of the official Dutch control laboratories, from which 261 samples were suspected to contain boldenone. These samples were all analyzed for their conjugation state, 6beta-hydroxy-17alpha/beta-boldenone and for the presence of coprostanol. Alfa-boldenone, the major metabolite in bovine urine after boldenone-ester administration, was found in a large number of these samples. The presence of alpha-boldenone was proven also to be a result of fecal contamination. None of the samples tested contained residues of the metabolite 6beta-hydroxy-17alpha/beta-boldenone. Not finding this metabolite indicates that the origin of alpha-boldenone-conjugates is endogenous. The results confirm that the presence of unconjugated beta-boldenone and alpha-boldenone conjugates next to alpha-boldenone are no indicators for illegal administration of boldenone-esters. No indications were obtained that conjugated beta-boldenone can be of endogenous origin.

Determination of resorcylic acid lactones in biological samples by GC-MS. Discrimination between illegal use and contamination with fusarium toxins.

An EU project, FAIR5-CT-1997-3443, has been undertaken to distinguish illegal use of zeranol from consumption of food contaminated with Fusarium spp. toxin. One of the tasks was development of screening and confirmatory methods of analysis. This paper describes a new method based on two-step clean-up and GC-MS analysis. The first clean-up step is matrix-dependant; the second is applicable to both urine and meat. The MS is operated in negative chemical ionisation mode. The method is quantitative for zeranol and taleranol, alpha- and beta-zearalenol, and zearalenone and qualitative for zearalanone. Validation was performed according to the latest EU performance criteria (Commission Decision 2002/657). For analysis of urine CC(alpha) and CC(beta) for the method (microg L(-1)) were 0.06-0.11 for zeranol, 0.07-0.12 for taleranol, 0.07-0.11 for alpha-zearalenol, 0.21-0.36 for beta-zearalenol, 0.35-0.60 for zearalenone, and 0.19-0.33 zearalanone. Within-laboratory reproducibility was 16.2, 11.2, 31.9, 30.1, 26.6, and 54.2% for zeranol, taleranol, alpha-zearalenol, beta-zearalenol, zearalenone, and zearalanone, respectively. It was found that all the compounds are stable in urine at -20 degrees C for at least a year. Part of the validation program was organisation of a small proficiency study (ringtest) and a correlation study with an LC-MS-MS method developed by the Veterinary Science Division (VSD; Belfast, UK-NI). This study showed there was good correlation between results from both laboratories. The method can be used for quantitative analysis discriminating illegal use of zeranol from consumption of zearalenone-contaminated food.

Nortestosterone: endogenous in urine of goats, sheep and mares?

For a number of species it is known that nortestosterone, either the alpha- or beta-epimer, can be of endogenous origin. For goats and mares similar results have not yet been published. As a follow-up on the experiments with cattle, a large number of urine samples per animal were collected from pregnant goats, sheep and mares. These samples were analysed for the presence of alpha- and beta-nortestosterone and alpha-estradiol using GC-MS. The results show that in the goats and mares studied alpha-nortestosterone is present during pregnancy. In this study no alpha-nortestosterone could be demonstrated in sheep. From our study and recently published data, however, it is proven that alpha-nortestosterone can occur endogenously.

Confirmatory assay for zeranol, taleranol and the Fusarium spp. toxins in bovine urine using liquid chromatography-tandem mass spectrometry.

A method is described for the quantitative determination of the veterinary drug zeranol, its epimer taleranol and the mycotoxins zearalenone, alpha-zearalenol and beta-zearalenol in bovine urine. The method is based on liquid chromatography coupled to negative-ion electrospray mass spectrometry-mass spectrometry of urine extracts prepared by solid-phase extraction with C(18) columns. Two transition ions at m/z 277 and 91 are monitored for zeranol and taleranol along with the transition ion at m/z 281 for their respective deuterated (d(4)) internal standards. Similarly, two transitions are monitored for each of the three mycotoxins along with a transition ion for each of their corresponding internal standards. The method has been validated according to the new European Union criteria for analysis of veterinary drug residues, and is suitable for monitoring urine samples taken under National Surveillance Schemes. The method has been validated at 1, 1.5 and 2 ng ml(-1) for zeranol and taleranol and at 5, 10 and 15 ng ml(-1) for each of the three mycotoxins. Correlation between the described method and a routine method, based on gas chromatography-mass spectrometry, was assessed using a range of naturally incurred samples.

Characterization of two cell lines, derived from two malignant fibrous histiocytomas.

We have investigated the phenotype and ultrastructure of tumour cells from two cell lines each derived from a malignant fibrous histiocytoma (MFH) as a means of studying the histogenesis of this group of tumours. The first MFH (MFH-I) was of the pleomorphic subtype, with a predominantly histiocytic appearance, the second was of the pleomorphic subtype associated with myxoid and storiform areas (MFH-II). In vitro tumour cells from both neoplasms showed aberrant growth properties. Xenografts in nude mice from both neoplasms showed a similar histology to that of the original tumour. Both tumours showed hyaluronidase sensitive alcian blue staining. Phenotypic studies of the two cell lines and of the tumour tissues demonstrated that the cells differed in the presence of collagen types I and III. They did not show evidence of histiocytic, endothelial, leiomyoblastic, rhabdomyoblastic, lipoblastic of schwannian origin. Ultrastructurally, the two cell lines were found to be different. In vitro and in xenografts the cell type of MFH-I resembled a primitive mesenchymal cell. Whereas that of MFH-II resembled a fibroblast-like cell. We concluded that the group of MFH is heterogeneous and is probably derived from more than one progenitor cell.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits.

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1). The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Prevalence of zeranol, taleranol and Fusarium spp. toxins in urine: implications for the control of zeranol abuse in the European Union.

There is currently little information concerning the prevalence of zeranol and taleranol in animal urine following metabolism of the naturally occurring Fusarium spp. toxins. An epidemiological study is described which involves four European Union control laboratories in which 8008 urine samples were screened for the presence of zeranol using a time-resolved fluoroimmunoassay (TR-FIA). Of these samples, 93.6% screened negative for zeranol. All samples testing positive for zeranol were then analysed with a confirmatory method. Based on the confirmatory results, the TR-FIA-positive samples were then categorized as false-positive, true-positive or 'equivocal' (zeranol/taleranol and the Fusarium spp. toxins detected). The true-positive samples represented only 0.05% of the total number of samples (n = 4). After statistical analysis, 170 of 174 equivocal samples proved to belong to a 'normal' population in which the amount of zeranol/taleranol could be related to the total amount of Fusarium spp. toxins through a linear regression with a 99% prediction interval. This suggested that the presence of zeranol in these samples might be due to in vivo metabolism of the Fusarium spp. toxins. The presence of zeranol in the four remaining 'outliers' might be attributable to zeranol abuse rather than to natural contamination. The results are of interest for control laboratories as they might provide an analytical tool to help distinguish between abuse and natural contamination in zeranol testing.