Simultaneous determination of five metabolites of nitrofurans including the metabolite nifursol in shrimp and fish by UPLC- MS/MS: in-house method validation according to commission implementing regulation (EU) 2021/808.

For the simultaneous identification and quantification of five nitrofurans metabolites in farmed shrimp and fish, 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoine (AHD), semicarbazide (SEM), and 3,5-dinitrosalicylic acid hydrazide (DNSH), an accurate, precise, and specific method was developed. The mixture of water and methanol (60/40; v/v) was found to be the final optimised solvent for injection. The analytical run duration was 7 min, and the mobile phase included 2 mM methanol and ammonium formate. The new reference point for action (RPA) of 0.50 µg kg-1 as per EC/1871/2019 was taken into consideration and evaluated for the performance characteristics as per the CIR (EC)/2021/808 criteria. Specificity, relative retention time (≤0.25%) relative ion ratio (≤40%), linearity (0.25 to 2.0 µg kg-1), trueness (between 82.8 and 118.1%), repeatability (RSDr ≤14%), within lab reproducibility (RSDwr ≤16.9%), CCα (0.32-0.36 µg kg-1), ruggedness and relative matrix effect (≤14.26%) achieved acceptable values.

Discrimination between the exogenous and endogenous origin of thiouracil in farm animals, the final chapter?

Thiouracil (2-thiouracil) is a thyreostatic compound that can be used as an illegal growth promoter. In bovine, porcine and other farm animals, low concentrations of thiouracil are detected in urine. There is much debate on which concentrations can be considered to originate from feed ('natural') and which concentrations are caused by the illegal administration of thiouracil for growth-promoting purposes. Currently, a threshold value of 10 µg/L in urine is applied. The threshold value is based on epidemiological data. Data on thiouracil from animals treated with thiouracil is scarce. We conducted a study whereby animals were fed with rapeseed, rapeseed with thiouracil, or regular feed with thiouracil (low and high concentration). It was determined that administration of thiouracil leads to concentrations higher than the current 10 µg/L threshold of thiouracil and its metabolites in urine during treatment. Animals fed with rapeseed showed higher thiouracil concentrations than the control group, mostly above 10 µg/L and in some cases above 30 µg/L. In the discovery study, several biomarkers for thiouracil treatment were tentatively identified and confirmed with reference standards. One metabolite was identified as indicative for thiouracil abuse, namely 6-methyl-thiouracil. Another metabolite, 4-thiouracil, was indicative for endogenous formation and did not increase during 2-thiouracil treatment. 6-Methyl-thiouracil was not found in urine samples from the Dutch routine control programmes that contained (endogenous) 2-thiouracil above the threshold value. However, 4-thiouracil was found at high concentrations in the same samples when 2-thiouracil was present. This study's overall conclusion is that the threshold value for thiouracil in bovine urine samples should be set at 10 µg/L and for porcine urine samples at 30 µg/L. Also, confirmation of 6-methyl-thiouracil and 4-thiouracil should be used as indicators for exogenous or endogenous origin in routine control monitoring programmes.

USB-Powered Coated Blade Spray Ion Source for On-Site Testing Using Transportable Mass Spectrometry.

On-site testing in food analysis using mass spectrometry (MS) requires miniaturization of vacuum systems, mass analyzers, sample cleanup, and ionization sources. In this study, a simple coated blade spray (CBS) ion source was developed that enables high voltage generation on the blade by ubiquitous certified (micro-)USB On-The-Go devices like smartphones, tablets, and power banks. CBS is capable of performing both analyte enrichment by solid-phase microextraction (SPME) material coated on the metal substrate and direct-spray ionization. The USB-CBS device was used on two different MS systems, a transportable single-quadrupole and a benchtop triple-quadrupole tandem MS. Various characteristics of the USB-CBS device, including high voltage generation and angular positioning, were studied. The potential of the newly developed device for food safety applications is demonstrated by banned and regulated veterinary drugs such as ß-agonists and sulfonamide antibiotics, covering a wide range of molecular weights and polarities. The results highlight the potential of the developed, simplified, inexpensive (less than 10 USD), and universal vendor-independent USB-powered CBS ion source coupled with MS(/MS) systems for semiquantitative applications, in laboratories, and in future on-site food quality and safety testing. Apart from that, most likely on-site environmental, biomedical, and forensic testing will also benefit from this USB-CBS instrumental development that is compatible with any atmospheric inlet MS system.

Enhanced UHPLC-MS/MS screening of selective androgen receptor modulators following urine hydrolysis.

Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (ß-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacophores in urine from a range of species (i.e. equine, bovine, human, canine and rodent). The newly presented semi-quantitative UHPLC-MS/MS-based assay is shown to be fit-for-purpose, being rapid and offering high-throughput, with validation findings fulfilling criteria stipulated within relevant doping and food control legislation.•CCß values determined at 1 ng mL-1 for majority of analytes.•Deconjugation step included in the method led to significantly increased relative abundance of ostarine in analysed incurred urine samples demonstrating the requirement for hydrolysis to detect a total form of emerging SARMs.•Assay amenable for use within routine testing to ensure fair play in animal and human sports and that animal-derived food is free from contamination with SARM residues.

Development and validation of a semi-quantitative ultra-high performance liquid chromatography-tandem mass spectrometry method for screening of selective androgen receptor modulators in urine.

A semi-quantitative method was developed to monitor the misuse of 15 SARM compounds belonging to nine different families, in urine matrices from a range of species (equine, canine, human, bovine and murine). SARM residues were extracted from urine (200 µL) with tert-butyl methyl ether (TBME) without further clean-up and analysed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). A 12 min gradient separation was carried out on a Luna Omega Polar C18 column, employing water and methanol, both containing 0.1% acetic acid (v/v), as mobile phases. The mass spectrometer was operated both in positive and negative electrospray ionisation modes (ESI±), with acquisition in selected reaction monitoring (SRM) mode. Validation was performed according to the EU Commission Decision 2002/657/EC criteria and European Union Reference Laboratories for Residues (EU-RLs) guidelines with CCß values determined at 1 ng mL-1, excluding andarine (2 ng mL-1) and BMS-564929 (5 ng mL-1), in all species. This rapid, simple and cost effective assay was employed for screening of bovine, equine, canine and human urine to determine the potential level of SARMs abuse in stock farming, competition animals as well as amateur and elite athletes, ensuring consumer safety and fair play in animal and human performance sports.

Determination and confirmation of selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors in bovine and porcine urine using UHPLC-MS/MS.

Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial - the potential of enantiomeric separation for doping control analysis.

The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes' claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed.

Sex hormone-binding globulin regulation of androgen bioactivity in vivo: validation of the free hormone hypothesis.

Sex hormone-binding globulin (SHBG) is the high-affinity binding protein for androgens and estrogens. According to the free hormone hypothesis, SHBG modulates the bioactivity of sex steroids by limiting their diffusion into target tissues. Still, the in vivo physiological role of circulating SHBG remains unclear, especially since mice and rats lack circulating SHBG post-natally. To test the free hormone hypothesis in vivo, we examined total and free sex steroid concentrations and bioactivity on target organs in mice expressing a human SHBG transgene. SHBG increased total androgen and estrogen concentrations via hypothalamic-pituitary feedback regulation and prolonged ligand half-life. Despite markedly raised total sex steroid concentrations, free testosterone was unaffected while sex steroid bioactivity on male and female reproductive organs was attenuated. This occurred via a ligand-dependent, genotype-independent mechanism according to in vitro seminal vesicle organ cultures. These results provide compelling support for the determination of free or bioavailable sex steroid concentrations in medicine, and clarify important comparative differences between translational mouse models and human endocrinology.

A view on the analytical design of future risk based residue control.

The current laboratory network system in support of residue monitoring programmes within the EU formally started in the early 1990s. Since then, it has undergone a gentle evolution incorporating new techniques and methods for quality assurance and, in parallel to the extension of the European Union itself, was further extended. However, a paradigm shift from production-based to risk-based control now is foreseen. This will have a serious impact on the type of methodologies used and subsequently on the specific roles of EU reference laboratories also. Here, we present our view on the changes that will inevitably take place in the years to come. Copyright © 2016 John Wiley & Sons, Ltd.

Toward a new European threshold to discriminate illegally administered from naturally occurring thiouracil in livestock.

Thiouracil is a thyrostat inhibiting the thyroid function, resulting in fraudulent weight gain if applied in the fattening of livestock. The latter abuse is strictly forbidden and monitored in the European Union. Recently, endogenous sources of thiouracil were identified after frequently monitoring low-level thiouracil positive urine samples and a "recommend concentration" (RC) of 10 µg/L was suggested by the EURL to facilitate decision-making. However, the systematic occurrence of urine samples exceeding the RC led to demands for international surveys defining an epidemiologic threshold. Therefore, six European member states (France, Poland, The Netherlands, United Kingdom, Norway, and Belgium) have shared their official thiouracil data (2010-2012) collected from bovines, porcines, and small livestock with 95 and 99% percentiles of 8.1 and 18.2 µg/L for bovines (n = 3894); 7.4 and 13.5 µg/L for porcines (n = 654); and 7.4 µg/L (95% only) for small livestock (n = 85), respectively. Bovine percentiles decreased with the animal age (nonadults had significantly higher levels for bovines), and higher levels were observed in male bovines compared to female bovines.

Evaluation of the discriminative potential of a novel biomarker for estradiol treatments in bovine animals.

The endogenous occurrence of natural hormones obstructs the application of classical targeted methods as confirmatory options. In the case of estradiol, the ultimate confirmation of its exogenous administration relies on gas chromatography coupled to combustion/isotope ratio mass spectrometry (GC-C/IRMS). A serum dipeptide composed of pyroglutamic acid and phenylalanine was identified as a potential biomarker of estradiol treatments in adult cows. To evaluate its potential to pinpoint suspicious samples, samples from prepubertal females under different estrogenic treatments have been analyzed. The results confirmed the up-regulation of the dipeptide in adult bovines. The 2-week-old females exhibited short-lasting responses only in a few animals. The 6-month-old female showed a delayed but clear increase on the biomarker level. The composition of the anabolic preparations, the dose, and/or the administration route are possible additional reasons for the reduced response in young animals. A comparison to previous results reported by various researchers is included.

Ultratrace LC-MS/MS analysis of segmented calf hair for retrospective assessment of time of clenbuterol administration in Agriforensics.

In agriforensics, time of administration is often debated when illegal drug residues, such as clenbuterol, are found in frequently traded cattle. In this proof-of-concept work, the feasibility of obtaining retrospective timeline information from segmented calf tail hair analyses has been studied. First, an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) hair analysis method was adapted to accommodate smaller sample sizes and in-house validated. Then, longitudinal 1 cm segments of calf tail hair were analyzed to obtain clenbuterol concentration profiles. The profiles found were in good agreement with calculated, theoretical positions of the clenbuterol residues along the hair. Following assessment of the average growth rate of calf tail hair, time of clenbuterol administration could be retrospectively determined from segmented hair analysis data. The data from the initial animal treatment study (n = 2) suggest that time of treatment can be retrospectively estimated with an error of 3-17 days.

Investigation of the presence of prednisolone in bovine urine.

Over the past two years low levels of prednisolone have been reported in bovine urine by a number of laboratories in European Union member states. Concentrations vary, but are reported to be below approximately 3 µg l(-1). Forty per cent of bovine urine samples from the Dutch national control plan had concentrations of prednisolone between 0.11 and 2.04 µg l(-1). In this study the mechanism of formation of prednisolone was investigated. In vitro conversion of cortisol by bacteria from faeces and soil, bovine liver enzymes and stability at elevated temperatures were studied. In vitro bovine liver S9 incubation experiments showed a significant 20% decrease of cortisol within 6 h, and formation of prednisolone was observed from 0.2 g l(-1) at t = 0 to 0.5 g l(-1) at t = 6. Under the influence of faeces, the stability of cortisol in urine is reduced and cortisol breaks down within 50 h. Prednisolone is formed up to 4 µg l(-1) at 70°C after 15 h. However, this decreases again to zero after 50 h. With soil bacteria, a slower decrease of cortisol was observed, but slightly higher overall formation of prednisolone, up to 7 µg l(-1) at 20°C. As opposed to incurred urine, in fortified urine incubated with faeces or soil bacteria no prednisolone was detected. This difference may be explained by the presence of natural corticosteroids in the incurred sample. With UPLC-QToF-MS experiments, in urine and water samples incubated with faeces, metabolites known from the literature could be (tentatively) identified as 20ß-hydroxy-prednisolone, cortisol-21-sulfate, oxydianiline, tetrahydrocortisone-3-glucuronide and cortexolone, but for all compounds except 20ß-hydroxy-prednisolone no standards were available for confirmation. Based on the results of this study and literature data, for regulatory purposes a threshold of 5 µg l(-1) for prednisolone in bovine urine is proposed. Findings of prednisolone in concentrations up to 5 µg l(-1) in bovine urine can, most likely, originate from other sources than illegal treatment with growth promoters.

Selective androgen receptor modulators: in vitro and in vivo metabolism and analysis.

For future targeted screening in National Residue Control Programmes, the metabolism of seven SARMs, from the arylpropionamide and the quinolinone classes, was studied in vitro using S9 bovine liver enzymes. Metabolites were detected and identified with ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry (ToF-MS) and triple quadrupole mass spectrometry (QqQ-MS). Several metabolites were identified and results were compared with literature data on metabolism using a human cell line. Monohydroxylation, nitro-reduction, dephenylation and demethylation were the main S9 in vitro metabolic routes established. Next, an in vivo study was performed by oral administration of the arylpropionamide ostarine to a male calf and urine samples were analysed with UPLC-QToF-MS. Apart from two metabolites resulting from hydroxylation and dephenylation that were also observed in the in vitro study, the bovine in vivo metabolites of ostarine resulted in glucuronidation, sulfation and carboxylation, combined with either a hydroxylation or a dephenylation step. As the intact mother compounds of all SARMs tested are the main compounds present after in vitro incubations, and ostarine is still clearly present in the urine after the in vivo metabolism study in veal calves, the intact mother molecules were selected as the indicator to reveal treatment. The analytical UPLC-QqQ-MS/MS procedure was validated for three commercially available arylpropionamides according to European Union criteria (Commission Decision 2002/657/EC), and resulted in decision limits ranging from 0.025 to 0.05 µg l⁻¹ and a detection capability of 0.025 µg l⁻¹ in all cases. Adequate precision and intra-laboratory reproducibility (relative standard deviation below 20%) were obtained for all SARMs and the linearity was 0.999 for all compounds. This newly developed method is sensitive and robust, and therefore useful for confirmation and quantification of SARMs in bovine urine samples for residue control programmes and research purposes.

Confirmation and 3D profiling of anabolic steroid esters in injection sites using imaging desorption electrospray ionisation (DESI) mass spectrometry.

In this study, desorption electrospray ionisation (DESI) linear ion trap tandem mass spectrometry (MS(n)) was applied for the confirmation and three-dimensional profiling of anabolic steroid esters in an injection site of bovine muscle. The spatial resolution of the DESI-MS(n) was demonstrated by scanning hormone esters and marker ink lines drawn at various distances on a microscopic slide at set distances, using an x-scanner with manual y and z adjustment. Tissue slices of bovine muscle injected with a hormone cocktail were analysed. All anabolic steroid esters could be directly detected in the sample and confirmed on the basis of identification points awarded for selected MS/MS transitions according to the performance criteria given in Commission Decision 2002/657/EC. Moreover, the injection site could be mapped by two-dimensional and three-dimensional imaging MS, showing a horizontal and vertical distribution through the muscle tissue. This DESI approach offers potential for analysis of injection sites of steroid esters from illegally treated animals; moreover, direct analysis by ambient imaging DESI-MS still allows conventional extraction and analysis of the whole tissue for further confirmatory or contra-analysis afterwards.

Adverse analytical findings with clenbuterol among U-17 soccer players attributed to food contamination issues.

The illicit use of growth promoters in animal husbandry has frequently been reported in the past. Among the drugs misused to illegally increase the benefit of stock farming, clenbuterol has held a unique position due to the substance's composition, mechanism of action, metabolism, and disposition. Particularly clenbuterol's disposition in animals' edible tissues destined for food production can cause considerable issues on consumption by elite athletes registered in national and international doping control systems as demonstrated in this case-related study. Triggered by five adverse analytical findings with clenbuterol among the Mexican national soccer team in out-of-competition controls in May 2011, the Fédération Internationale de Football Association (FIFA) initiated an inquest into a potential food contamination (and thus sports drug testing) problem in Mexico, the host country of the FIFA U-17 World Cup 2011. Besides 208 regular doping control samples, which were subjected to highly sensitive mass spectrometric test methods for anabolic agents, 47 meat samples were collected in team hotels during the period of the tournament and forwarded to Institute of Food Safety, RIKILT. In 14 out of 47 meat samples (30%), clenbuterol was detected at concentrations between 0.06 and 11 µg/kg. A total of 109 urine samples out of 208 doping control specimens (52%) yielded clenbuterol findings at concentrations ranging from 1-1556 pg/ml, and only 5 out of 24 teams provided urine samples that did not contain clenbuterol. At least one of these teams was on a strict 'no-meat' diet reportedly due to the known issue of clenbuterol contamination in Mexico. Eventually, owing to the extensive evidence indicating meat contamination as the most plausible reason for the extraordinary high prevalence of clenbuterol findings, none of the soccer players were sanctioned. However, elite athletes have to face severe consequences when testing positive for a prohibited anabolic agent and sufficient supporting information corroborating the scenario of inadvertent ingestion are required to be acquitted from anti-doping rule violations. Hence, governmental contribution is urgently needed to combat the illegal use of clenbuterol in stock breading.

Hormone analysis in food products.

The administration of growth-promoting compounds, to food producing animals is banned within the European Union. We developed several methods whereby the cleanup is based on LLE and SPE and detection based on GC-MSMS or LC-MSMS to identify and confirm the identity of different growth promoting agents in several food products. This chapter describes methods to isolate and identify these growth promoting agents.

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays.

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical-analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical-analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical-analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.