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

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

Towards the Development of (Certified) Reference Materials for Effective Veterinary Drug Residue Testing in Food-producing Animals

Reliable analysis of veterinary drug residues in animal-derived foodstuffs represents an important measure to ensure consumer protection. European Legislation addresses this issue, for instance by defining maximum residue level, specifying sampling and monitoring plans, and stipulating performance and validation criteria for analytical methods. Certified reference materials (CRMs) constitute an important tool for method validation and method performance verification. This article addresses the current status of legislation, describes characteristics of analytical methods in veterinary drug residue testing and then focuses on the challenges and considerations towards the development of new (certified) reference materials in this fieldJRC.D.2-Reference material

Food and feed safety : Cases and approaches to identify the responsible toxins and toxicants

There are food and feed safety monitoring programs to protect consumers. These programs however, are strongly focused on known and regulated substances. New or unexpected substances that might be of risk for consumers will thus escape routine controls. These risks are therefore mainly discovered by human or animal intoxications. All kind of analytical chemical methods, in-vitro bioassays, tracking, and chain analysis are then used to reveal the substance(s) responsible for the intoxication. Only in a few occasions (new) risks were revealed in time by analytical chemical methods or cell based in-vitro bioassays. This paper describes some relevant food and feed safety cases and how the causative substances were identified. This overview strongly indicates that more intense monitoring, including the use of cell based effect bioassays, can reduce the number of intoxications. Moreover, registration and follow-up actions should be arranged in a better way, for example by sharing information within the scientific communities or by establishing a national contact point. In addition, a strategy based on broad screening and bioassay directed identification with liquid chromatography high resolution mass spectrometry is proposed to prevent intoxications and identify toxin and toxicants relevant for food and feed safety

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

Food and feed safety : Cases and approaches to identify the responsible toxins and toxicants

There are food and feed safety monitoring programs to protect consumers. These programs however, are strongly focused on known and regulated substances. New or unexpected substances that might be of risk for consumers will thus escape routine controls. These risks are therefore mainly discovered by human or animal intoxications. All kind of analytical chemical methods, in-vitro bioassays, tracking, and chain analysis are then used to reveal the substance(s) responsible for the intoxication. Only in a few occasions (new) risks were revealed in time by analytical chemical methods or cell based in-vitro bioassays. This paper describes some relevant food and feed safety cases and how the causative substances were identified. This overview strongly indicates that more intense monitoring, including the use of cell based effect bioassays, can reduce the number of intoxications. Moreover, registration and follow-up actions should be arranged in a better way, for example by sharing information within the scientific communities or by establishing a national contact point. In addition, a strategy based on broad screening and bioassay directed identification with liquid chromatography high resolution mass spectrometry is proposed to prevent intoxications and identify toxin and toxicants relevant for food and feed safety.</p

Microsphere Peptide-Based Immunoassay for the Detection of Recombinant Bovine Somatotropin in Injection Preparations

The use of peptides in immunoassays can be favored over the use of the full protein when more cost effective or less toxic approaches are needed, or when access to the full protein is lacking. Due to restricted access to recombinant bovine somatotropin (rbST), a protein enhancing growth and lactating performances of livestock, which use has been banned in the EU, Canada and Australia (amongst others), we developed a peptide-based biorecognition assay on an imaging planar array analyzer. For this, we identified the rbST epitope that is responsible for binding to the rbST-targeting monoclonal antibody 4H12 (MAb 4H12) to be115 DLEEGILALMR125 . This linear peptide was synthesized and coupled to microspheres, after which it was tested in a biorecognition competitive inhibition assay format. We observed IC50 values of approximately 0.11 µg mL−1, which are lower than observed for the full rbST protein (IC50 = 0.20 µg mL−1 ). Importantly, there was no binding with the scrambled peptide. Preliminary results of directly coupled peptides in a microsphere biorecognition assay for detection of rbST are presented. Real-life applicability for detection of somatotropins (STs) in injection preparations of bovine-, porcine-and equine ST are shown. This newly developed immunoassay strongly supports future developments of peptide-based immunoassays to circumvent the limited access to the full protein

Detection of methionine- and alanine-recombinant bovine somatotropins and their induced antibodies in serum and milk of cows suggests blood-milk barrier specificity for these compounds

The elimination of recombinant bovine somatotropin (rbST) and its induced antibodies through milk of 2 formulations is studied to propose a control strategy for its use or abuse. Two dairy cows were treated with alanine-rbST (Ala-rbST), which is identical to endogenous bovine somatotropin, and ten dairy cows were treated with methionine-rbST (Met-rbST), which differs by 1 amino acid from endogenous bovine somatotropin. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method able to measure rbST at a decision limit (CCα) of 0.8 ng/mL and 2.3 ng/mL for serum and milk, respectively. The results show that the administered Ala-rbST is transferred from blood to milk but that this is not the case for Met-rbST. This suggests a blood-milk barrier-related specificity for these compounds. In addition, rbST-induced antibodies were formed in animals treated with Ala-rbST and those treated with Met-rbST. In both treatments, the rbST-induced antibodies were transferred from blood to milk, showing no blood-milk barrier specificity for these antibodies. These elimination patterns show that, for enforcement purposes, the detection of rbST-induced antibodies in tank milk can serve to screen for rbST administration, and subsequent confirmatory serum analysis by LC-MS/MS is needed to identify whether Ala-rbST or Met-rbST has been used.</p

Dietary supplement for energy and reduced appetite containing the β-agonist isopropyloctopamine leads to heart problems and hospitalisations

In 2013 the Dutch authorities issued a warning against a dietary supplement that was linked to 11 reported adverse reactions, including heart problems and in one case even a cardiac arrest. In the UK a 20-year-old woman, said to have overdosed on this supplement, died. Since according to the label the product was a herbal mixture, initial LC-MS/MS analysis focused on the detection of plant toxins. Yohimbe alkaloids, which are not allowed to be present in herbal preparations according to Dutch legislation, were found at relatively high levels (400–900 mg kg–1). However, their presence did not explain the adverse health effects reported. Based on these effects the supplement was screened for the presence of a β-agonist, using three different biosensor assays, i.e. the validated competitive radioligand β2-adrenergic receptor binding assay, a validated β-agonists ELISA and a newly developed multiplex microsphere (bead)-based β-agonist assay with imaging detection (MAGPIX®). The high responses obtained in these three biosensors suggested strongly the presence of a β-agonist. Inspection of the label indicated the presence of N-isopropyloctopamine. A pure standard of this compound was bought and shown to have a strong activity in the three biosensor assays. Analysis by LC-full-scan high-resolution MS confirmed the presence of this ‘unknown known’ β3-agonist N-isopropyloctopamine, reported to lead to heart problems at high doses. A confirmatory quantitative analysis revealed that one dose of the preparation resulted in an intake of 40–60 mg, which is within the therapeutic range of this compound. The case shows the strength of combining bioassays with chemical analytical techniques for identification of illegal pharmacologically active substances in food supplements