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Hoppner S.,German Sport University Cologne | Delahaut P.,CER Groupe | Schanzer W.,German Sport University Cologne | Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents
Journal of Pharmaceutical and Biomedical Analysis | Year: 2014

The NAD+ depending enzyme SIRT1 regulates the mitochondrial biogenesis, fat and glucose metabolism through catalyzing the deacetylation of several metabolism-related protein-substrates. Recently, synthetic activators of SIRT1 referred to as STACs (Sirtuin activating compounds, e.g. SRT2104) were identified and tested in clinical studies for the treatment of aging-related diseases such as type 2 diabetes, Alzheimer's and obesity. Although the mechanism of SIRT1 activation by small molecules has caused considerable controversy, STACs demonstrated a significant performance enhancement in mice experiments including an improvement of endurance, muscle strength, and locomotor behavior. Due to their potential to increase exercise tolerance in healthy individuals, SIRT1 activators are currently being monitored by anti-doping authorities. In the present study, the in vivo metabolic clearance of three SIRT1 activators was investigated in rats by the collection of urine, DBS (dried blood spots) and plasma samples following a single oral administration. The resulting metabolic products were studied by positive electrospray ionization - (tandem) mass spectrometry and confirmed by the comparison with in vitro generated metabolites using human and rat liver microsomal preparations. Subsequently, a screening procedure for five SIRT1 activators and the metabolite M1-SRT1720 in DBS specimens was developed. Liquid-liquid-extraction and liquid chromatography/tandem mass spectrometry was employed based on diagnostic ion transitions recorded in multiple reaction monitoring mode and two deuterated internal standards namely d8-SRT1720 and d8-M1-SRT1720 were utilized. The doping control assay was characterized with regard to specificity, limit of detection (10-50ng/ml), recovery (65-83%) and imprecision (7-20%) and ion suppression/enhancement effects (<10%), demonstrating its fitness-for-purpose for sports drug testing applications. © 2013 Elsevier B.V.


Fodey T.L.,Agri Food and Biosciences Institute of Northern Ireland | George S.E.,Queen's University of Belfast | Traynor I.M.,Agri Food and Biosciences Institute of Northern Ireland | Delahaut P.,CER Groupe | And 3 more authors.
Journal of Immunological Methods | Year: 2013

Thiamphenicol and florfenicol are antibacterial agents permitted for use as veterinary drugs in animals used for food production. However, as the EU has established maximum residue limits for both and the metabolite florfenicol amine, there is a requirement to monitor animal food products for their residues. In this study antisera were generated which can simultaneously detect thiamphenicol, florfenicol and florfenicol amine in an immunoassay. Details of the various coupling techniques employed to prepare immunogens and enzyme labels are provided and the antibodies produced have been assessed, in homologous and heterologous ELISA formats, with respect to sensitivity and specificity. It was found that while the antisera raised to thiamphenicol and florfenicol generally performed better in a heterologous set up, those raised to florfenicol amine were not only less affected by the assay format but also produced the most sensitive antibodies to all three target analytes. Antisera matched previous sensitivity (IC50<1ngmL-1) but had improved cross-reactivity (>100%) to thiamphenicol and florfenicol. © 2013.


Robert C.,CER Groupe | Gillard N.,CER Groupe | Brasseur P.-Y.,CER Groupe | Pierret G.,CER Groupe | And 3 more authors.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2013

Multi-class UHPLC-MS/MS was developed for the analysis of more than 160 regulated or banned compounds of various classes: anthelmintics including benzimidazoles, avermectins and others; antibiotics including amphenicols, beta-lactams, macrolides, pyrimidines, quinolones, sulphonamides and tetracyclines; beta-agonists; corticosteroids; ionophores; nitroimidazoles; non-steroidal anti-inflammatory agents; steroids; and tranquillisers. Samples were extracted with acetonitrile, without any additional purification step, and analysed by using UHPLC-MS/MS. Validation was done in accordance with the guidelines laid down by European Commission Decision 2002/657/EC for qualitative screening methods. This simple method proved applicable to routine screening for residues in egg, honey, milk and muscle samples at half the maximum concentration permitted by the European Union for each drug. In most cases, the target value was set at 5 μg kg-1 for unauthorised compounds. © 2013 Copyright Taylor and Francis Group, LLC.


De Clercq N.,Ghent University | Vanden Bussche J.,Ghent University | Croubels S.,Ghent University | Delahaut P.,CER Groupe | Vanhaecke L.,Ghent University
Journal of Chromatography A | Year: 2014

Faecal glucocorticoid analysis is a powerful non-invasive tool for the study of the animal endocrine status and stress physiology, which is mainly carried out by immunoassays, characterised by some limitations. In this study, an ultra high-performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry (U-HPLC-HRMS) method was developed to confirm the presence of glucocorticoids in bovine faeces during a long-term stability study. Because of the complex nature of faeces, an appropriate extraction and purification procedure was developed. To this extent, a Plackett-Burman experimental design was successfully applied to determine the key conditions for optimal extraction of glucocorticoids from faeces. The targeted analysis, including natural and synthetic glucocorticoids, was successfully validated according to CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone, methylprednisolone and the metabolites 20α-dihydroprednisolone and 20β-dihydroprednisolone ranged, respectively, from 0.15 to 2.95 μg kg-1 and from 0.40 to 5.20 μg kg-1. Limits of detection and limits of quantification for the natural glucocorticoids dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.55 to 2.10 μg kg-1 and from 0.70 to 5.00 μg kg-1.The stability study of glucocorticoids in faecal matrix demonstrated that lyophilising the faeces, storage at -80°C, and aerobic conditions were optimal for preservation and able to significantly (p < 0.05) limit degradation up to 10 weeks. © 2014 Elsevier B.V.


De Clercq N.,Ghent University | Julie V.B.,Ghent University | Croubels S.,Ghent University | Delahaut P.,CER Groupe | Vanhaecke L.,Ghent University
Journal of Chromatography A | Year: 2013

Due to their growth-promoting effects, the use of synthetic glucocorticoids is strictly regulated in the European Union (Council Directive 2003/74/EC). In the frame of the national control plans, which should ensure the absence of residues in food products of animal origin, in recent years, a higher frequency of prednisolone positive bovine urines has been observed. This has raised questions with respect to the stability of natural corticoids in the respective urine samples and their potential to be transformed into synthetic analogs. In this study, a ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) methodology was developed to examine the stability of glucocorticoids in bovine urine under various storage conditions (up to 20 weeks) and to define suitable conditions for sample handling and storage, using an Orbitrap Exactive™. To this end, an extraction procedure was optimized using a Plackett-Burman experimental design to determine the key conditions for optimal extraction of glucocorticoids from urine. Next, the analytical method was successfully validated according to the guidelines of CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone and methylprednisolone ranged, respectively, from 0.1 to 0.5μgL-1 and from 0.3 to 0.8μgL-1. For the natural glucocorticoids limits of detection and limits of quantification for dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.1 to 0.2μgL-1 and from 0.3 to 0.8μgL-1. The stability study demonstrated that filter-sterilization of urine, storage at -80°C, and acidic conditions (pH 3) were optimal for preservation of glucocorticoids in urine and able to significantly limit degradation up to 20 weeks. © 2013 Elsevier B.V.


Thomas A.,German Sport University Cologne | Delahaut P.,CER Groupe | Krug O.,German Sport University Cologne | Schanzer W.,German Sport University Cologne | Thevis M.,German Sport University Cologne
Analytical Chemistry | Year: 2012

New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity. © 2012 American Chemical Society.


Boutier M.,University of Liège | Ronsmans M.,University of Liège | Ouyang P.,University of Liège | Ouyang P.,Sichuan Agricultural University | And 11 more authors.
PLoS Pathogens | Year: 2015

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin. © 2015 Rossmann et al.


Delahaut P.,CER Groupe | Pierret G.,CER Groupe | Ralet N.,CER Groupe | Dubois M.,CER Groupe | Gillard N.,CER Groupe
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment | Year: 2010

A multi-residue HPLC-ESI-MS/MS method has been developed for the simultaneous extraction, detection and confirmation of the 11 coccidiostats referenced by Regulation 2009/8/EC (lasalocid sodium, narasin, salinomycin sodium, monensin sodium, semduramicin sodium, maduramicin ammonium alpha, robenidine hydrochloride, decoquinate, halofuginone hydrobromide, nicarbazin, and diclazuril) in feedstuffs at carry-over level. The sensitivity of the method allows quantification and confirmation for all coccidiostats below target concentration. The method was in-house validated and meets all criteria of European legislation (2002/657/EC). The precision of the method was determined under repeatability and within-laboratory reproducibility conditions; RSDr and RSDR were below the maximum permitted values for every tested concentration. The specificity was checked by analysing representative blank samples and blank samples fortified with potentially interfering substances (benzimidazoles, corticosteroides, triphenylmethane dyes, quinolones, nitrofurans, nitroimidazoles, phenicols); no interference were found. Concerning quantification, a quadratic regression model was fitted to every calibration curve with a regression coefficient r2 above 0.99 on each data set. Finally, the expanded uncertainty U was calculated with data obtained within the laboratory while applying the method during validation and in routine tests. © 2010 Taylor & Francis.


Michel B.,University of Liège | Leroy B.,University of Mons | Raj V.S.,University of Liège | Lieffrig F.,CER Groupe | And 4 more authors.
Journal of General Virology | Year: 2010

Koi herpesvirus, also known as cyprinid herpesvirus 3 (CyHV-3), is the aetiological agent of an emerging and mortal disease in common and koi carp. CyHV-3 virions present the characteristic morphology of other members of the order Herpesvirales, being composed of an envelope, a capsid containing the genome and a tegument. This study identified CyHV-3 structural proteins and the corresponding encoding genes using liquid chromatography tandem mass spectrometry-based proteomic approaches. In addition, exponentially modified protein abundance index analyses were used to estimate the relative abundance of the identified proteins in CyHV-3 virions. These analyses resulted in the identification of 40 structural proteins, which were classified based on bioinformatic analyses as capsid (three), envelope (13), tegument (two) and unclassified (22) structural proteins. Finally, a search for host proteins in purified CyHV-3 virions indicated the potential incorporation of up to 18 distinct cellular proteins. The identification of the proteins incorporated into CyHV-3 virions and determination of the viral genes encoding these proteins are key milestones for further fundamental and applied research on this virus. © 2010 SGM.


Thomas A.,German Sport University Cologne | Kohler M.,German Sport University Cologne | Schanzer W.,German Sport University Cologne | Delahaut P.,Cer Groupe | Thevis M.,German Sport University Cologne
Analyst | Year: 2011

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R3-IGF-1 and longR3-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL -1. The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR3-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR3-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL-1), recovery (10-35%), precision (<20%), linearity, robustness and stability. © 2011 The Royal Society of Chemistry.

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