J. Vanden Bussche

Residue analysis: future trends from a historical perspective.

A residue is a trace (microg kg(-1), ng kg(-1)) of a substance, present in a matrix. Residue analysis is a relatively young discipline and a very broad area, including banned (A) substances as well as registered veterinary medicinal products (B substances). The objective of this manuscript is to review future trends in the analysis of residues of veterinary drugs in meat producing animals out of historical perspectives. The analysis of residues in meat producing animals has known a tremendous evolution during the past 35-40 years. In the future, it can be foreseen that this evolution will proceed in the direction of the use of more and more sophisticated and expensive machines. These apparatus, and the necessary human resources for their use, will only be affordable for laboratories with sufficient financial resources and having guarantee for a sufficient throughput of samples.

A validated analytical method for the determination of perfluorinated compounds in surface-, sea- and sewagewater using liquid chromatography coupled to time-of-flight mass spectrometry

Perfluorinated compounds (PFCs), which are extensively used in a wide variety of applications because of their specific surfactant properties, have recently appeared as an important new class of global environmental pollutants. Quantitative analysis of PFCs in aqueous matrices remains, however, a challenging task. During this study, a new analytical method for the determination of 14 PFCs in surface-, sewage- and seawater was developed and validated. The target analytes were extracted using solid-phase extraction followed by liquid chromatography coupled to a time-of-flight mass spectrometer (LC-ToF-MS). The use of very narrow mass tolerance windows (< 10 ppm) resulted in a highly selective MS-technique for the detection of PFCs in complex aqueous matrices. Validation of this analytical method in surface-, sewage- and seawater resulted in limits of quantification (LOQs) varying from 2 to 200 ng L⁻¹, satisfying recoveries (92-134%), and good linearity (R²=0.99 for most analytes). Analysis of samples of the North Sea, the Scheldt estuary, and three harbours of the Belgian coastal region led to the detection of four different PFCs. Perfluorooctane sulfonate (PFOS) was found to be the most abundant PFC in levels up to 38.9 ng L⁻¹.

Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). For monitoring their illegal use, sensitive and specific analytical methods are required. In this study an UHPLC-MS/MS method was described for quantitative analysis of eight thyreostatic drugs in urine, this without a derivatisation step. The sample pretreatment involved a reduction step with dithiothreitol under denaturating conditions at 65 degrees C, followed by liquid-liquid extraction with ethyl acetate. This analytical procedure was subsequently validated according to the EU criteria (2002/657/EC Decision), resulting in decision limits and detection capabilities ranging between 1.1 and 5.5 microg L(-1) and 1.7 and 7.5 microg L(-1), respectively. The method obtained for all, xenobiotic thyreostats, a precision (relative standard deviation) lower than 15.5%, and the linearity ranged between 0.982 and 0.999. The performance characteristics fulfill not only the requirements of the EU regarding the provisional minimum required performance limit (100 microg L(-1)), but also the recommended concentration fixed at 10 microg L(-1) in urine set by the Community of Reference Laboratories. Future experiments applying this method should provide the answer to the alleged endogenous status of thiouracil.

Analysis of thyreostats: A history of 35 years

Thyreostatic drugs (TS), illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). This paper reviews the trends in the analytical approaches for the determination of TS drugs in biological matrices. After a brief introduction on the different groups of compounds with a thyreostatic action, the most relevant legislation regarding the residue control of these compounds is presented. An overview of the analytical possibilities for the determination of TS in animal matrices, covering sample extraction, purification, separation techniques and detection methods is provided. Additionally, a brief outline of animal experiments is described that illustrates the excretion and distribution profiles of TS residues. Finally, the novel developments in TS analysis are highlighted. Also the possible semi-endogenous status of thiouracil is discussed.

A validated ultra-high performance liquid chromatography coupled to high resolution mass spectrometry analysis for the simultaneous quantification of the three known boar taint compounds.

Boar taint is an off-odour that can occur when meat or fat from entire male pigs is heated. Most of the currently available analytical methods are not capable of detecting the three known boar taint compounds (indole, skatole and androstenone) simultaneously, which renders their analysis often labour-intensive and time-consuming as separate analyses are required. In this study a validated U-HPLC-HR-Orbitrap-MS analysis method is described for the quantitative determination of the three boar taint compounds in fat. The sample pre-treatment involves a melting step followed by extraction with methanol and clean-up consisting of a freezing step and solid phase extraction (HLB cartridges). The analytes are then chromatographically separated and detected with an Exactive high-resolution mass spectrometer. Due to the absence of guidelines for the analysis of boar taint in fat, the Commission Decision 2002/657/EC [18] and ISO 17025 [19] guidelines were used as guideline for validation of the developed detection method. This resulted in limits of detection and limits of quantification between 2.5 and 7 μg kg(-1) and between 5 and 10 μg kg(-1) for the three compounds, respectively, which is far below the threshold values set at 100 μg L(-1) for indole, 200 μg L(-1) for skatole and 1000 μg L(-1) for androstenone in pig fat samples. The method obtained for the three compounds a repeatability (RSD) lower then 12.7% and a within-laboratory reproducibility (RSD) lower than 16.9%. The recovery of the three compounds ranged between 99 and 112 and an excellent linearity (R(2) ≥ 0.99) was found. In the future, this method may be extended with other compounds that turn out to be correlated with boar taint.

Epigenetic alterations and decreasing insecticide sensitivity of the Asian tiger mosquito Aedes albopictus.

A range of environmental factors, including chemicals, can affect epigenetic processes in organisms leading to variations in phenotype. Thus, epigenetics displays an important environmentally responsive element. The transgenerational impact of environmental stressors on DNA methylation and phenotype was the focus of this study. The influence of two known DNA methylation-changing agents, the phytoestrogen genistein and the fungicide vinclozolin, on the overall DNA methylation level in the Asian tiger mosquito Aedes albopictus was investigated. The experiment comprised four generations in a full life-cycle design with an exposed parental generation and three consecutive non-exposed offspring generations. Application of the methylation agents to the parental generation of the study led to an alteration of the global DNA methylation level of the exposed individuals and those in two subsequent generations. The phenotypic variability of the offspring generations was assessed by examining their insecticide sensitivity. Here, a significant decrease in sensitivity (p<0.01) towards the model insecticide imidacloprid revealed alterations of the mosquitos phenotype in two subsequent generations. Thus, the evaluation of A. albopictus from an epigenetic perspective can contribute important information to the study of the high adaptability of this invasive disease vector to new environments, and its underlying mechanisms.

An approach based on ultra-high pressure liquid chromatography-tandem mass spectrometry to quantify O6-methyl and O6-carboxymethylguanine DNA adducts in intestinal cell lines.

O⁶-methylguanine (O⁶-MeG) and O⁶-carboxymethylguanine (O⁶-CMG) are characteristic promutagenic and toxic DNA adducts formed by nitrosated glycine derivates and N-nitrosopeptides. Since endogenous nitrosation has been hypothesised as a plausible origin for the association between red and processed meat intake and colorectal cancer, a highly sensitive, fast and specific quantitative assay is needed to correlate the dose of individual DNA adducts with the effects of food consumption and individual digestive and metabolic processes. An ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for quantitation of O⁶-MeG and O⁶-CMG, using the deuterated analogues as internal standards (ISTD), was developed. Samples of calf thymus DNA containing O⁶-MeG and O⁶-CMG were purified by acid hydrolysis and solid phase extraction prior to quantification by UHPLC-MS/MS in the selected reaction monitoring mode. The method was successfully validated in terms of repeatability (RSD<10%), reproducibility (RSD<15%) and linearity (99.9%) by incubating 0.1mg calf thymus DNA with the known N-nitroso compound potassium diazoacetate (KDA). The limit of quantitation was 30 fmol mg⁻¹ DNA for O⁶-MeG or 1 adduct per 10⁸ nucleotides and 50 fmol mg⁻¹ DNA for O⁶-CMG or 1.7 adducts per 10⁸ nucleotides. Subsequently, the method was applied to human colon carcinoma cell lines, Caco-2 and HT-29, treated with KDA to illustrate its capability to quantify O⁶-MeG and O⁶-CMG DNA adducts using biological relevant models in vitro. This method will support further research to unravel the mechanistic basis of endogenous nitrosation processes upon consumption of red and processed meat products.

Endogenous boldenone-formation in cattle: Alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.

The effect of redox conditions and adaptation time on organic micropollutant removal during river bank filtration: A laboratory-scale column study

This study investigated the redox dependent removal and adaptive behaviour of a mixture of 15 organic micropollutants (OMPs) in laboratory-scale soil columns fed with river water. Three separate pilot systems were used consisting of: (1) two columns, (2) ten columns and (3) twenty two columns to create oxic, suboxic (partial nitrate removal) and anoxic (complete nitrate removal). The pilot set-up has some unique features--it can simulate fairly long residence times (e.g., 45 days using the 22 column system) and reduced conditions developed naturally within the system. Dimethoate, diuron, and metoprolol showed redox dependent removal behaviour with higher biodegradation rates in the oxic zone compared to the suboxic/anoxic zone. The redox dependent behaviour of these three OMPs could not be explained based on their physico-chemical properties (hydrophobicity, charge and molecular weight) or functional groups present in the molecular structure. OMPs that showed persistent behaviour in the oxic zone (atrazine, carbamazepine, hydrochlorothiazide and simazine) were also not removed under more reduced conditions. Adaptive behaviour was observed for five OMPs: dimethoate, chloridazon, lincomycin, sulfamethoxazole and phenazone. However, the adaptive behaviour could not be explained by the physico-chemical properties (hydrophobicity, charge and molecular weight) investigated in this study and only rough trends were observed with specific functional groups (e.g. ethers, sulphur, primary and secondary amines). Finally, the adaptive behaviour of OMPs was found to be an important factor that should be incorporated in predictive models for OMP removal during river bank filtration.

A laboratory-scale column study comparing organic micropollutant removal and microbial diversity for two soil types

UNLABELLED This study investigated sorption and biodegradation behaviour of 20 organic micropollutants (OMPs) in lab-scale columns filled with two types of soil (fed with the same water quality) simulating river bank filtration (RBF) under oxic conditions. Retardation factors and OMP biodegradation rates were similar for the two soils that were characterised by a different cationic exchange capacity, organic matter and sand/silt/clay content. This result was supported by the microbial community composition (richness, evenness) of the two soils that became more similar as a result of feeding both columns with the same water quality. This indicates that microbial community composition and thereby OMP removal in soils is primarily determined by the composition of the aqueous phase (organic matter quantity and quality, nutrients) rather than the soil phase. These results indicate that different RBF sites located along the same river may show similar OMP removal (in case of similar water quality and residence time). CAPSULE This study shows that the microbial community composition and thus OMP removal is primarily determined by the aqueous phase (water quality) rather than the soil phase.