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Analytica Chimica Acta (v.700, #1-2)
Development, validation and data quality assurance of screening methods: A case study by Roberta Galarini; Roberta Buratti; Laura Fioroni; Lidia Contiero; Francesca Lega (pp. 2-10).
Display Omitted► Some confusion exists about the validation of qualitative screening methods according to Commission Decision 2002/657/EC. ► The state of art is outlined reviewing the published papers about this subject. ► The main approaches with their advantages and drawbacks are summarised. ► A case of study is reported describing in practice the possible steps in the validation of a screening method.Despite the growing importance of qualitative screening tests in routine laboratories involved in the EU official control, their validation is not as deeply explained in Commission Decision 2002/657/EC as the validation of quantitative confirmatory methods. At the same time, the issue of quality assurance of screening assays defining internal quality control (IQC) procedures as required by accreditation bodies is undoubtedly less developed in this analytical field. As an example the present study describes the development, the validation and the IQC implemented for a commercial enzyme linked immunosorbent assay (ELISA) able to detect 17-α-19-nortestosterone (α-NT) and 17-β-19-nortestosterone (β-NT) isomers in bullock urine. In order to select a suitable sample treatment, two SPE purification protocols were preliminary compared. The chosen method was therefore fully validated determining the mandatory parameters required by Commission Decision 2002/657/EC: specificity, detection capability and robustness. An in-depth discussion was carried out illustrating the possible validation approaches and their implications especially in the assessment of the key performance characteristic: detection capability. Finally, the control charts implemented for continuous method verification during analyses of real samples were reported.
Keywords: Screening test; Enzyme linked immunosorbent assay; Detection capability; Validation; Commission Decision 2002/657/EC
Validation study on avermectine residues in foodstuffs by L. Giannetti; A. Giorgi; F. Necci; G. Ferretti; F. Buiarelli; B. Neri (pp. 11-15).
Avermectines are antiparasitic agents widely used as veterinary drugs for food producing animals. The European Community, due to their side effects, limited the use of these molecules establishing maximum residue limits (MRLs) in some foods.A validated qualitative and quantitative high performance liquid chromatography method with fluorescence detection (HPLC-FL) is presented for the simultaneous determination of ivermectin (IVM), abemectin (ABA), moxidectin (MOX), eprinomectin (EPR), doramectin (DOR) and emamectin (EMA) in foodstuffs (muscle, eggs and milk). Samples were extracted with acetonitrile, purified with liquid-liquid extraction (LLE), and analysed by HLPC-FL previous derivatization with trifluoroacetic anhydride (TFAA) in presence of 1-methyl-imidazole (MI) and acetic acid.To date, the presented method is the first validated for the matrix eggs, and in accordance with the requirements set by Commission Decision 2002/657/EC. Recoveries of the methods, calculated spiking the samples in the range 5.0–100.0μgkg−1, were 64–83% for muscle, 65–89% for milk and 63–84% for eggs. The precision (CV) ranged between 9.2 and 17.1% for muscle, 9.9 and 16.6% for milk and from 9.4 to 17.4% for eggs. Linearity for the six analytes was calculated from 5.0 to 200.0μgkg−1.The main advantages of the presented method are its rapidity, the specificity, the good precision and recovery that make it very suitable to the detection and determination of avermectines.
Keywords: Avermectines; Residue; Validation; Liquid chromatography; Fluorescence
Metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry to screen for the illegal use of estradiol and progesterone in cattle by Patricia Regal; Sébastien Anizan; Jean-Philippe Antignac; Bruno Le Bizec; Alberto Cepeda; Cristina Fente (pp. 16-25).
Administration of hormonal compounds as growth promoters in livestock farming was banned by Council Directive 93/22/EC, however, this kind of substances are sometimes reported within the framework of European monitoring residue plans. Various analytical methods have been previously developed to screen for their misuse, and they are now especially efficient for monitoring the illegal administration of synthetic and semi-synthetic hormones. Nevertheless, proving an exogenous administration of hormones from natural origin (i.e. estradiol-17β or progesterone) still remains a challenging task for European authorities. As a result of their origin, these target compounds are indeed always present in the analytical matrix, and because the concentration levels of natural steroids are extremely variable from one animal to another, the establishment of reference thresholds appears very difficult. During this preliminary study, metabolomic data was acquired on a high performance liquid chromatography system coupled to high resolution mass spectrometer (HPLC–LTQ-Orbitrap). Serum samples were collected from dairy cows treated or not with sex steroid hormones commonly employed in animal husbandry: estradiol-17β (or its ester estradiol benzoate) and progesterone. After appropriate data processing and multivariate statistical analyses (OPLS-DA), it was possible to highlight significant metabolic modifications in serum consecutively to the administration of estradiol and/or progesterone. Those differences were used to build predictive models able to suspect illegal administration of these hormones in cattle. Potential biomarker candidates of estradiol and/or progesterone were pointed out, that remains to be structurally elucidated.
Keywords: Estradiol-17β; Progesterone; Serum; Cattle; Metabolomic; OPLS
Determination of eleven coccidiostats in animal feed by liquid chromatography–tandem mass spectrometry at cross contamination levels by Mark Cronly; P. Behan; B. Foley; E. Malone; P. Shearan; L. Regan (pp. 26-33).
A confirmatory multi-residue method has been developed to allow for the detection, confirmation and quantification of eleven coccidiostats in animal feed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The method can be used to determine halofuginone, robenidine, nicarbazin, diclazuril, decoquinate, semduramicin, lasalocid, monensin, salinomycin, narasin, maduramicin at levels relating to unavoidable carry over as stated in Regulation 2009/8/EC. Feed samples are extracted with water and acetonitrile with the addition of anhydrous magnesium sulphate and sodium chloride. The extract then undergoes a freezing out step before being diluted and injected onto the LC–MS/MS system. The LC–MS/MS system is run in MRM mode with both positive and negative electrospray ionisation and can confirm all eleven analytes in a run time of 19min. The sensitivity of the method allows quantification and confirmation for all coccidiostats at a 0.5% carry over level. The method was validated over three days in accordance with of European legislation; Commission Decision 2002/657/EC. Validation criteria of accuracy, precision, decision limit (CCα), and detection capability (CCβ) along with measurement uncertainty are calculated for all analytes. The method was then successfully used to analyse a number of feed samples that contained various coccidiostat substances.
Keywords: Coccidiostats; Regulation 2009/8/EC; LC–MS/MS; Animal feed; Validation
The application of reporter gene assays for the detection of endocrine disruptors in sport supplements by Monika Plotan; Christopher T. Elliott; Marie Louise Scippo; Marc Muller; Jean-Philippe Antignac; Edward Malone; Toine F.H. Bovee; Samuel Mitchell; Lisa Connolly (pp. 34-40).
The increasing availability and use of sports supplements is of concern as highlighted by a number of studies reporting endocrine disruptor contamination in such products. The health food supplement market, including sport supplements, is growing across the Developed World. Therefore, the need to ensure the quality and safety of sport supplements for the consumer is essential.The development and validation of two reporter gene assays coupled with solid phase sample preparation enabling the detection of estrogenic and androgenic constituents in sport supplements is reported. Both assays were shown to be of high sensitivity with the estrogen and androgen reporter gene assays having an EC50 of 0.01ngmL−1 and 0.16ngmL−1 respectively.The developed assays were applied in a survey of 63 sport supplements samples obtained across the Island of Ireland with an additional seven reference samples previously investigated using LC–MS/MS. Androgen and estrogen bio-activity was found in 71% of the investigated samples. Bio-activity profiling was further broken down into agonists, partial agonists and antagonists. Supplements (13) with the strongest estrogenic bio-activity were chosen for further investigation. LC–MS/MS analysis of these samples determined the presence of phytoestrogens in seven of them. Supplements (38) with androgen bio-activity were also selected for further investigation. Androgen agonist bio-activity was detected in 12 supplements, antagonistic bio-activity was detected in 16 and partial antagonistic bio-activity was detected in 10. A further group of supplements (7) did not present androgenic bio-activity when tested alone but enhanced the androgenic agonist bio-activity of dihydrotestosterone when combined.The developed assays offer advantages in detection of known, unknown and low-level mixtures of endocrine disruptors over existing analytical screening techniques. For the detection and identification of constituent hormonally active compounds the combination of biological and physio-chemical techniques is optimal.
Keywords: Endocrine disruptor; Estrogen; Androgen; Bioassay; Health food supplement; Food safety
Detection of benzimidazole carbamates and amino metabolites in liver by surface plasmon resonance-biosensor by Jemma Keegan; Richard O’Kennedy; Steven Crooks; Christopher Elliott; David Brandon; Martin Danaher (pp. 41-48).
Two surface plasmon resonance (SPR) biosensor screening assays were developed and validated to detect 11 benzimidazole carbamate (BZT) and four amino-benzimidazole veterinary drug residues in liver tissue. The assays used polyclonal antibodies, raised in sheep, to detect BZTs and amino-benzimidazoles. A modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction method was developed to isolate benzimidazole carbamate residues. Liver samples were extracted using an acetonitrile extraction method. BZTs were purified by dispersive solid phase extraction (d-SPE) using C18 sorbent. Residues of amino-benzimidazoles were effectively cleaned-up using a simple cyclohexane defatting step. The assays were validated in accordance with the performance criteria described in 2002/657/EC. The BZT assay limit of detection was calculated to be 32μgkg−1, the detection capability (CCβ) was determined to be 50μgkg−1 and the mean recovery of analytes was in the range 77–132%. The amino-benzimidazole assay limit of detection was determined to be 41μgkg−1, the CCβ was determined to be 75μgkg−1 and analyte recovery was in the range 103–116%. Biosensor assay performance was tested by analysing liver tissue from animals treated with benzimidazole drugs and comparing the results with an ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) confirmatory method. All non-compliant samples were identified using the biosensor assays.
Keywords: SPR biosensor; Benzimidazoles; Liver; Screening assay
Development and validation of a liquid chromatography–tandem mass spectrometry method for the simultaneous determination of nine corticosteroid residues in bovine liver samples by Guglielmo Dusi; Mara Gasparini; Michele Curatolo; Walter Assini; Eros Bozzoni; Nadia Tognoli; Enrica Ferretti (pp. 49-57).
A liquid chromatography tandem mass spectrometry (LC–MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography–negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness.All the corticosteroids can be detected at a concentration around 1μgkg−1; the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.
Keywords: Corticosteroids; Liver; Mass spectrometry; Decision 2002/657/EC
Preparation and characterisation of in-house reference material of tylosin in honey and results of a proficiency test by Detlef A. Bohm; Carolin S. Stachel; Rudolf Hackenberg; Petra Gowik (pp. 58-62).
The analysis of incurred material from animals treated with pharmacologically active substances is an efficient way to check the accuracy of a method. Tylosin A was chosen for the preparation of that material because it is highly effective in controlling active infections of American Foulbrood (AFB), a global threat to apiculture, but residues in honey are not allowed according to European legislation. For this reason an in-house reference material of honey containing the macrolide tylosin A and its degradation product desmycosin (tylosin B) was prepared. After the treatment of a beehive with the appropriate macrolide tylosin A, the honey samples were collected. The incurred honey material was diluted by mixing with blank honey. Concentrations of 25.81μgkg−1 for tylosin A and of 19.28μgkg−1 for its degradation product desmycosin (tylosin B) were reached. The homogeneity was checked by analysing 12 bottles in duplicate. The stability was tested at different defined temperatures and storage conditions. The reference material described above was homogeneous and stable.Samples of this in-house reference material were used for the realisation of a proficiency test with international participation. All participants accomplished satisfying results with the exception of one laboratory.
Keywords: In-house reference material; Honey; Tylosin A; Desmycosin (tylosin B); Stability; Proficiency test
Feasibility of desorption electrospray ionization mass spectrometry for rapid screening of anabolic steroid esters in hair by M.W.F. Nielen; A.W.J.M. Nijrolder; H. Hooijerink; A.A.M. Stolker (pp. 63-69).
Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionization (DESI) mass spectrometry (MS). In this work the feasibility of DESI application for the rapid screening of intact esters of anabolic steroids in bovine hair has been studied. Using a linear ion trap both full scan and data-dependent collision induced dissociation MS n spectra were acquired in minutes for testosterone cypionate, testosterone decanoate and estradiol benzoate standard solutions deposited on a glass or PTFE surface. However direct analysis of incurred hair failed due to inefficient desorption ionization and the minute quantities of steroid esters present. Therefore a simplified ultrasonic liquid extraction procedure was developed, allowing rapid DESI analysis of a few microliters of the concentrate and a total analysis time of 2–4h per batch instead of 3 days. The potential of this DESI approach is clearly demonstrated by MS3 data from hair samples incurred with high levels (300–800μgkg−1) of steroid esters, levels which do occur in samples from controlled- and illegally treated animals. For much lower levels state-of-the-art ultra high performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) screening methods remain the method of choice and might benefit from the proposed simplified extraction as well.
Keywords: Mass spectrometry; Desorption electrospray ionization; DESI; Anabolic steroid; Hair; Veterinary control; Forensic
Ultra-high performance liquid chromatography–tandem mass spectrometry in high-throughput confirmation and quantification of 34 anabolic steroids in bovine muscle by Lynn Vanhaecke; Julie Vanden Bussche; Klaas Wille; Karen Bekaert; Hubert F. De Brabander (pp. 70-77).
An ultra-high performance liquid chromatography tandem mass spectrometry multi-residue method for the determination of 34 anabolic steroids (10 estrogens including stilbenes, 14 androgens and 10 gestagens) in meat of bovine origin is reported. The extraction and clean-up procedure involved homogenization with methanol, defatting with hexane, liquid/liquid extraction with diethylether and finally SPE clean-up with coupled Si and NH2 cartridges. The analytes were separated on a 1.9μm Hypersil Gold column (100×2.1mm) and quantified on a triple quadrupole mass spectrometer (TSQ Vantage) operating simultaneously in both positive and negative atmospheric pressure chemical ionisation (APCI) modes. This analytical procedure was subsequently validated according to EU criteria (CD 2002/657/EC), resulting in decision limits and detection capabilities ranging between 0.04 and 0.88μgkg−1 and 0.12 and 1.9μgkg−1, respectively. The method obtained for all, natural and synthetic steroids, adequate precisions and intra-laboratory reproducibilities (relative standard deviation below 20%), and the linearity ranged between 0.991 and 0.999. The performance characteristics fulfill the recommended concentrations fixed by the Community Reference Laboratories. The developed analysis is sensitive, and robust and therefore useful for confirmation and quantification of anabolic steroids for research purposes and residue control programs.
Keywords: U-HPLC–MS/MS; 2002/657/EC; Hormones; Validation; CC; α; CC; β
Discrimination of eight chloramphenicol isomers by liquid chromatography tandem mass spectrometry in order to investigate the natural occurrence of chloramphenicol by Bjorn J.A. Berendsen; Tina Zuidema; Jacob de Jong; Linda (A)A.M. Stolker; Michel W.F. Nielen (pp. 78-85).
This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450μgkg−1. It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para-configuration and in the meta-configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta-stereoisomers and four para-stereoisomers. It is known that only RR- p-CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR- p-CAP and SS- p-CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.
Keywords: Chloramphenicol; Dextramycin; Chiral separation; Mass spectrometry; Natural occurrence; Feed
Development of an improved high resolution mass spectrometry based multi-residue method for veterinary drugs in various food matrices by A. Kaufmann; P. Butcher; K. Maden; S. Walker; M. Widmer (pp. 86-94).
Multi-residue methods for veterinary drugs or pesticides in food are increasingly often based on ultra performance liquid chromatography (UPLC) coupled to high resolution mass spectrometry (HRMS). Previous available time of flight (TOF) technologies, showing resolutions up to 15,000 full width at half maximum (FWHM), were not sufficiently selective for monitoring low residue concentrations in difficult matrices (e.g. hormones in tissue or antibiotics in honey). The approach proposed in this paper is based on a single stage Orbitrap mass spectrometer operated at 50,000FWHM. Extracts (liver and kidney) which were produced according to a validated multi-residue method (time of flight detection based) could not be analyzed by Orbitrap because of extensive signal suppression. This required the improvement of established extraction and clean-up procedures. The introduced, more extensive deproteination steps and dedicated instrumental settings successfully eliminated these detrimental suppression effects. The reported method, covering more than 100 different veterinary dugs, was validated according to the EU Commission Decision 2002/657/EEC. Validated matrices include muscle, kidney, liver, fish and honey. Significantly better performance parameters (e.g. linearity, reproducibility and detection limits) were obtained when comparing the new method with the older, TOF based method. These improvements are attributed to the higher resolution (50,000 versus 12,000FWHM) and the superior mass stability of the of the Orbitrap over the previously utilized TOF instrument.
Keywords: High resolution mass spectrometry; Orbitrap; Veterinary drugs; Multi-residue methods; Validation
Effects of low-dose dexamethasone and prednisolone long term administration in beef calf: Chemical and morphological investigation by Francesca Tiziana Cannizzo; Pierluigi Capra; Sara Divari; Valentina Ciccotelli; Bartolomeo Biolatti; Marco Vincenti (pp. 95-104).
An analytical, pharmacokinetic and histopathologic investigation was conducted by two experimental trials on beef cattle in order to determine fate and effects of dexamethasone and prednisolone, administered to distinct cattle groups at low dosage for long periods of time. In trial 1, eighteen Charolaise beef cattle, male, 17–22-months-old, were divided in three groups: to group A ( n=6) dexamethasone-21-sodium-phosphate 0.7mgday−1 per os for 40 days was administered; group B ( n=6) was orally treated with prednisolone 15mgday−1 for 30 days, while group C ( n=6) served as negative control. Urine was collected at days 0, 7, 15, 25 and 47 from groups A and C, and at days 0, 8, 18 and 42 from group B. In trial 2, sixteen Friesian cattle, male, 10–17-months-old, were randomly divided into two groups: group D ( n=8) was administered prednisolone 30mgday−1 per os for 35 days, while group K ( n=8) served as control. In both trials, the animals were slaughtered after a 6-days drug withdrawal and thymus and livers were collected and properly stored until the analysis was performed.Quantitative determinations of dexamethasone, prednisolone and its main metabolite, prednisone, in urine and liver samples were conducted by HPLC–MS/MS, after the analytical procedure was optimized and fully validated. The method validation included the assessment of specificity, linearity, precision, trueness, robustness, CCα and CCβ values.By a morphological point of view, severe atrophy of thymus parenchyma was observed in group A, together with a significant ( P<0.005) reduction of the mean thymus weight (217±94g), while group B (646±215g) presented normal thymus features and weights (group C, 415±116g). Accordingly, no differences were found in trial 2 for groups D (727±275g) and K (642±173g).Average dexamethasone concentrations in group A urine samples ranged from 1.4 to 3.0μgL−1 during the treatment, while no residue was detected in the urine samples collected 6–7 days after the end of the treatment. Low amounts of dexamethasone (<1μgL−1) were detected in liver samples of group A.All average prednisolone concentrations in group B urine samples (sum of conjugate and free form) turned out to be below 1.0μgL−1 during the treatment, despite the much higher concentration administered (15–30mgday−1) with respect to dexamethasone in group A (0.7mgday−1). No prednisolone residues were found in the urine and liver samples taken at the slaughterhouse.The absence of any prednisolone residue in the urine samples of control group animals supports the theory that the origin of this molecule is fundamentally exogenous, at least for this cattle category maintained under unstressing conditions. Remarkable findings are represented by the absence of thymus atrophy in the prednisolone treated animals and the extremely low residue concentrations found in urine during the treatment. Both findings reveal that the detection of illegal growth-promoting treatments with this drug is difficult.
Keywords: Cattle; Dexamethasone; Prednisolone; Thymus; High performance liquid chromatography–tandem mass spectrometry; Urine
Targeted phase II metabolites profiling as new screening strategy to investigate natural steroid abuse in animal breeding by Sebastien Anizan; Domenica Di Nardo; Emmanuelle Bichon; Fabrice Monteau; Nora Cesbron; Jean-Philippe Antignac; Bruno Le Bizec (pp. 105-113).
The use of steroid hormones as growth promoters in cattle is banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. While efficient targeted confirmatory methods have been implemented in control laboratories for many years, fast and reliable screening methods are still required, especially in the case of natural hormones abuse, but more globally for new “fishing” strategies allowing to reveal the use of even unknown anabolic agents. The development of focused profiling or untargeted metabolomic approaches is thus emerging in this context. The present study was a focused profiling study using steroids phase II metabolites, with the aim to get a better understanding of the steroid metabolism disruptions after exogenous administration of androstenedione and finally reveal potential biomarkers signing its administration. A sample preparation procedure was first developed, based on a separation of 31 glucuronide and sulphate conjugate compounds using an anion exchange SPE system. Each fraction was then analysed by UPLC–MS/MS in MRM mode showing a rapid (between 4h and 4 days after treatment) and huge excretion of several direct metabolites of androstenedione such as etiocholanolone-glucuronide or epiandrosterone-sulphate.
Keywords: Mass spectrometry; Chemical food safety; Phase II steroid hormones; Natural steroid metabolism; UPLC–MS/MS; Androstenedione
Influence of natural organic matter on the screening of pharmaceuticals in water by using liquid chromatography with full scan mass spectrometry by Zahira Herrera Rivera; Efraim Oosterink; Luuk Rietveld; Frans Schoutsen; Linda Stolker (pp. 114-125).
The influence of natural organic matter on the screening of pharmaceuticals in water was determined by using high resolution liquid chromatography (HRLC) combined with full scan mass spectrometry (MS) techniques like time of flight (ToF) or Orbitrap MS. Water samples containing different amount of natural organic matter (NOM) and residues of a set of 11 pharmaceuticals were analyzed by using Exactive Orbitrap™ LC-MS. The samples were screened for residues of pharmaceuticals belonging to different classes like benzimidazoles, macrolides, penicillins, quinolones, sulfonamides, tetracyclines, tranquillizers, non-steroidal anti-inflammatory drugs (NSAIDs), anti-epileptics and lipid regulators. The method characteristics were established over a concentration range of 0.1–500μgL−1. The 11 pharmaceuticals were added to two effluent and two influent water samples. The NOM concentration within the samples ranged from 2 to 8mgL−1 of dissolved organic carbon. The HRLC-Exactive Orbitrap™ LC-MS system was set at a resolution of 50,000 (FWHM) and this selection was found sufficient for the detection of the list of pharmaceuticals. With this resolution setting, accurate mass measurements with errors below 2ppm were found, despite of the NOM concentration of the different types of water samples. The linearities were acceptable with correlation coefficients greater than 0.95 for 30 of the 51 measured linearities. The limit of detection varies between 0.1μgL−1and 100μgL−1. It was demonstrated that sensitivity could be affected by matrix constituents in both directions of signal reduction or enhancement. Finally it was concluded that with direct shoot method used (no sample pretreatment) all compounds, were detected but LODs depend on matrix-analyte-concentration combination. No direct relation was observed between NOM concentration and method characteristics. For accurate quantification the use of internal standards and/or sample clean-up is necessary. The direct shoot method is only applicable for qualitative screening purposes.The use of full scan MS makes it possible to search for unknown contaminants. With the use of adequate software and a database containing more than 50,000 entries a tool is available to search for unknowns.
Keywords: Accurate mass measurements; Dissolved natural organic matter; Non-target compounds; High resolution; Pharmaceuticals; Drinking water treatment
Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control by Yves Moulard; Ludovic Bailly-Chouriberry; Sophie Boyer; Patrice Garcia; Marie-Agnès Popot; Yves Bonnaire (pp. 126-136).
Liquid chromatography–mass spectrometry (LC–MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity.In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50000) and high mass accuracy (<5ppm) in full scan MS mode for more than 235 target analytes screened from an initial volume of 5mL of urine.The sample preparation was classically founded on solid phase extraction by means of reverse phase C18 cartridges. LC–MS analyses were carried out on a Shimadzu binary HPLC pumps linked to a C18 Sunfire column associated with the high resolution exactive benchtop orbitrap mass spectrometer. This screening was performed alternatively in positive–negative ionization mode during the same run. Thus, the identification of compounds of interest was made using their exact mass in positive–negative ionization mode at their expected retention time. All data obtained were processed by ToxID software (ThermoFisherScientific) which is able to identify a molecule by theoretical mass and retention time.In order to illustrate this innovative technology applied in our laboratory, sample preparation, validation data performed on 20 target compounds from 16 different horse urine samples, chromatograms and spectra will be discussed in this paper.
Keywords: Screening; Equine; Urine; Mass spectrometry; LC–MS; High resolution; Mass accuracy
Determination of MRL regulated corticosteroids in liver from various species using ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC) by Yoann Deceuninck; Emmanuelle Bichon; Fabrice Monteau; Jean-Philippe Antignac; Bruno Le Bizec (pp. 137-143).
Dexamethasone, betamethasone, prednisolone and methylprednisolone are corticosteroids widely used in animal husbandry. These compounds are licensed for therapy in veterinary practices while their use for growth promoting practices, mainly in combination with other growth promoters, is prohibited within the European Union. In order to protect the consumer, maximum residue limits (MRLs) have been set by the European Community in liver to 2.0μgkg−1 (dexamethasone and betamethasone) and 10.0μgkg−1 (prednisolone and methylprednisolone) for different species. The major challenges in the analysis of dexamethasone and betamethasone consist in performing an efficient separation of both isomers and in detecting and identifying all the molecules according to the regulatory requirements fixed in Commission Decision 2002/657/EC. In this context, an UHPLC–MS/MS method with a short runtime (7min) and using the SRM acquisition mode was developed and validated. An efficient selectivity of the sample preparation combined with a high sensitivity of the measurement system allowed identifying and quantifying the four corticosteroids of interest in this complex biological matrix. Signals obtained were found very repeatable, even at very low concentration levels with an unambiguous identification of the compounds. The performance limits of the method have been validated according to the regulatory requirements and the method has been successfully applied to the confirmation of incurred liver samples.
Keywords: Corticosteroids; Control; Veterinary drugs; UHPLC; Mass spectrometry; Maximum residue limit (MRL); Liver; Validation
Assessment of two complementary liquid chromatography coupled to high resolution mass spectrometry metabolomics strategies for the screening of anabolic steroid treatment in calves by Gaud Dervilly-Pinel; Stefan Weigel; Arjen Lommen; Sylvain Chereau; Lauriane Rambaud; Martien Essers; Jean-Philippe Antignac; Michel W.F. Nielen; Bruno Le Bizec (pp. 144-154).
Anabolic steroids are banned in food producing livestock in Europe. Efficient methods based on mass spectrometry detection have been developed to ensure the control of such veterinary drug residues. Nevertheless, the use of “cocktails” composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention. New analytical tools able to detect such abuse are today mandatory. In this context, metabolomics may represent new emerging strategies for investigating the global physiological effects associated to a family of substances and therefore, to suspect the administration of steroids. The purpose of the present study was to set up, assess and compare two complementary mass spectrometry-based metabolomic strategies as new tools to screen for steroid abuse in cattle and demonstrate the feasibility of such approaches. The protocols were developed in two European laboratories in charge of residues analysis in the field of food safety. Apart from sample preparation, the global process was different in both laboratories from LC-HRMS fingerprinting to multivariate data analysis through data processing and involved both LC-Orbitrap-XCMS and UPLC-ToF-MS-MetAlign strategies. The reproducibility of both sample preparation and MS measurements were assessed in order to guarantee that any differences in the acquired fingerprints were not caused by analytical variability but reflect metabolome modifications upon steroids administration. The protocols were then applied to urine samples collected on a large group of animals consisting of 12 control calves and 12 calves administrated with a mixture of 17β-estradiol 3-benzoate and 17β-nandrolone laureate esters according to a protocol reflecting likely illegal practices. The modifications in urine profiles as indicators of steroid administration have been evaluated in this context and proved the suitability of the approach for discriminating anabolic treated animals from control ones. Such an approach may therefore open a new way for the screening of anabolic steroid administration through targeted monitoring of relevant biomarkers highlighted as a result of the metabolomics study.
Keywords: Metabolomics; Steroids; Nandrolone; Estradiol; Biomarkers; LC-HRMS; Screening
Liquid chromatography tandem mass spectrometry with ion trap and triple quadrupole analyzers for determination of thyreostatic drugs in urine and muscle tissue by Barbara Wozniak; Iwona Matraszek Zuchowska; Jan Zmudzki; Piotr Jedziniak; Beata Korycinska; Katarzyna Sielska; Sebastian Witek; Alicja Klopot (pp. 155-166).
Liquid chromatography tandem mass spectrometry methods were developed and validated to screen for and confirm residues of the thyreostatic drugs: tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil in bovine and porcine urine and muscle tissues using dimethylthiouracil as internal standard. Thyreostats were extracted from urine samples with diethyl ether after derivatisation with 3-iodobenzylbromide in basic medium (pH 8.0) and analyzed by gradient elution on a Nucleosil C18 column with ion trap mass spectrometry detection using an electrospray source and triple quadrupole MS detection with turbo spray source. Thyreostats were extracted from muscle tissue with methanol, the denaturation of matrix protein was performed and then the same steps as for the urine samples were carried out. The methods were validated in accordance with the Commission Decision 2002/657/EC. Good thyreostats recoveries were obtained (from 82% to 117%) as well as acceptable within-lab reproducibility. The values of the decision limit CCα and the detection capability CCβ of five thyreostatic drugs are found to be below the recommended concentration set at 10μgL−1 (kg−1). The results of the validation demonstrate that liquid chromatography mass spectrometry with ion trap detection does not meet the criteria for confirmation for some thyreostats and therefore was applied for screening purpose only.
Keywords: Thyreostatic drugs; Residue analysis; Liquid chromatography; Mass spectrometry
Development and validation of a multi-residue liquid chromatography–tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs by Roberta Galarini; Laura Fioroni; Simone Moretti; Laura Pettinacci; Guglielmo Dusi (pp. 167-176).
A confirmatory method for the determination of residues of eleven coccidiostats including ionophore antibiotics: lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin and chemical coccidiostats: decoquinate, diclazuril, halofuginone, nicarbazin and robenidine in poultry eggs was developed and validated. The sample was extracted with acetonitrile, defatted with hexane and cleaned-up on a silica SPE cartridge. The analytes were identified and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The method performance characteristics required by Commission Decision 2002/657/EC were estimated adopting a more flexible and simple validation design. In this alternative approach the experimental study focuses on a larger dynamic range with progressively increasing validation levels. Each of them presents equal concentrations of all the analytes. On the contrary the classical Decision plan investigates a restricted concentration range necessarily different for each analyte being determined by the individual permitted limit (0.5–1.5 times the permitted limit). As a consequence each validation level involves the simultaneous fortification with complex mixtures containing different concentrations of each analyte. Adopting this alternative strategy the validation of several substances with significantly different permitted limits is considerably simplified and a deeper knowledge of the method is achieved. The results proved that the method enables the confirmation of regulated coccidiostats in eggs at the levels required in the official control of residues (CCα in the range of 2.2–174μgkg−1, depending on the coccidiostat). The repeatability (CVr in the range of 1.1–19%) and within-laboratory reproducibility (CVRw in the range of 1.8–30%) are also acceptable. The procedure was successfully verified in the proficiency test and implemented in the national residue control plan.
Keywords: Coccidiostats; Eggs; Liquid chromatography–tandem mass spectrometry; 2002/657/EC
Screening method for the detection of a range of nitrofurans in avian eyes by optical biosensor by Colin S. Thompson; Imelda M. Traynor; Terence L. Fodey; Steven R.H. Crooks; D. Glenn Kennedy (pp. 177-182).
An immunobiosensor assay was developed for the multi-residue screening of a range of nitrofuran compounds in avian eyes. A polyclonal antibody which binds at least 5 of the major parent nitrofurans was raised in a rabbit after inoculation with a nitrofuran mimic-protein conjugate. Sample homogenates were extracted into 0.1M hydrochloric acid and subjected to clean-up by solid phase extraction and micro-centrifugation prior to biosensor analysis. Validation data obtained from the analysis of 21 fortified samples has shown that the method has a detection capability (CCβ) of less than 1ngeye−1 for nitrofurazone (NFZ). In addition, cross-reactivity data and the analysis of a smaller number of fortified samples have shown that the method will also detect a range of other major parent nitrofurans including furazolidone (FZD), furaltadone (FTD), nitrofurantoin (NFA) and nifursol (NFS). Intra-assay variation ( n=10) was calculated at 12.9% and 10.1% at concentrations of 1ngeye−1 and 2ngeye−1 NFZ respectively. Inter-assay variation ( n=3) was determined to be 10.8% and 4.7% at the same NFZ concentrations respectively. The cross-reactivity profile and validation data for the detection of these nitrofurans are presented together with the results obtained following the analysis of a small number of incurred samples using the developed method.
Keywords: Screening; Detection; Nitrofurans; Optical biosensor
Bioassay based screening of steroid derivatives in animal feed and supplements by Jeroen C.W. Rijk; Helen Ashwin; Sandra J.A. van Kuijk; Maria J. Groot; Henri H. Heskamp; Toine F.H. Bovee; Michel W.F. Nielen (pp. 183-188).
Receptor binding transcription activation bioassays are valuable tools for the screening of steroid hormones in animal feed and supplements. However, steroid derivatives often lack affinity for their cognate receptor and do not show any direct hormonal activity by themselves. These compounds are thus not detected by these kinds of bioassays and need a bioactivation step in order to become active, both in vivo and in vitro. In this study a comparison was made between different in vitro activation methods for hormone esters and hormone glycosides. Testosterone acetate and testosterone decanoate were chosen as model compounds for the hormone esters, representing the broad range of steroid esters of varying polarities, while genistin was used as a substitute model for the steroid-glycosides. Concerning bioactivation of the steroids esters, the efficiency for alkaline hydrolysis was 90–100% and much better as compared to enzymatic deconjugation by esterase. As a result 1μg testosterone ester per gram of animal feed could easily be detected by a yeast androgen bioassay. When comparing different enzyme fractions for deglycosilation, genistin was shown to be deconjugated most efficiently by β-glucuronidase/aryl sulfatase from Helix pomatia, resulting in a significant increase of estrogenic activity as determined by a yeast estrogen bioassay. In conclusion, chemical and enzymatic deconjugation procedures for ester and glycoside conjugates respectively, resulted in a significant increase in hormonal activity as shown by the bioassay readouts and allowed effective screening of these derivatives in animal feed and feed supplements.
Keywords: Bioassay; Steroid ester; Steroid glycoside; Hormone abuse; Cattle
Development and validation of an enzyme-linked immunosorbent assay for the detection of circulating antibodies raised against growth hormone as a consequence of rbST treatment in cows by Sandrine Rochereau-Roulet; Isabelle Gaudin; Sylvain Chéreau; Stéphanie Prévost; Geneviève André-Fontaine; Gaud Pinel; Bruno Le Bizec (pp. 189-193).
The recombinant bovine growth hormone (rbST) is used to increase lactating performances of dairy cows. Administration of rbST is banned in the European Union; nevertheless, its use is probable. Until now, efficient analytical strategies to detect such practice are based on the direct detection by mass spectrometry of the presence of rbST in biological fluids, which suits for confirmatory purposes. Current screening strategies do not offer satisfactory performances; therefore, alternative screening strategies are required.The aim of the present work is to develop and validate an ELISA to measure the production of specific antibodies upon rbST in bovine sera. In this immunoassay, rbST is absorbed onto microtiter plate. After specific purification of the antibodies in serum, samples are analysed and the presence of antibodies anti-rbST is detected by Protein G peroxidase conjugate and 2-2′-azino di-ethyl benz-thiazoline-6-sulphonic acid (ABTS). The mean reproducibility of the OD ( λ=405nm) measurement was calculated with a CV of 13%. The intra- and inter-assay CVs ranged from 0.79% to 7.91% and from 2.69% to 20% respectively. The test presents cross-reaction with other growth hormones such as the recombinant equine (reST) and porcine (pST) (100% and 80% respectively). The specificity of the test toward rbST anabolic treatment was confirmed through the analysis of sera samples collected on animals administered with other anabolic compounds (steroids).The performances of the present anti-rbST ELISA proves its efficiency as a new screening tool to highlight illegal administration of rbST in cattle up to at least 3 weeks after treatment.
Keywords: Bovine somatotropin; Growth hormone; Serum; Dairy cow; ELISA; Protein G; Kinetic immune response
Screening method for the analysis of antiviral drugs in poultry tissues using zwitterionic hydrophilic interaction liquid chromatography/tandem mass spectrometry by D. Chan; J. Tarbin; M. Sharman; M. Carson; M. Smith; S. Smith (pp. 194-200).
A screening method for the analysis of seven anti-viral drugs in poultry tissue has been developed. These include anti-influenza drugs (amantadine, rimantadine, zanamivir and oseltamivir and its carboxylate metabolite), anti-herpes drugs (acyclovir and ganciclovir) and an immunomodulator (imiquimod). Poultry tissue was extracted in acetonitrile:water:acetic acid. After sample purification, using a strong cation exchange column, the eluate was split into two fractions. The first portion was dissolved in methanol:water and the second in acetonitrile:methanol:water. Both fractions were analysed on a zwitterionic hydrophilic interaction liquid chromatography column coupled to a triple quadrupole mass spectrometer. The screening method was successfully validated according to Commission Decision 2002/657/EC in three different laboratories with CCβ concentrations of ≤10μgkg−1.
Keywords: Antiviral; Amantadine; Acyclovir; Ganciclovir; Imiquimod; Oseltamivir; Oseltamivir carboxylate; Rimantadine; Poultry; Zanamivir