Matthew Sharman

Control of Strobilurin Fungicides in Wheat Using Direct Analysis in Real Time Accurate Time-of-Flight and Desorption Electrospray Ionization Linear Ion Trap Mass Spectrometry

Ambient mass spectrometry has been used for the analysis of strobilurin residues in wheat. The use of this novel, challenging technique, employing a direct analysis in a real time (DART) ion-source coupled with a time-of-flight mass spectrometer (TOF MS) and a desorption electrospray ionization (DESI) source coupled with a linear ion trap tandem MS (LIT MS(n)), permitted a direct screen of the occurrence of target fungicides in treated grains in less than 1 min. For quantification purpose by DART-TOF MS, an ethyl acetate extract had to be prepared. With the use of a prochloraz as an internal standard, the performance characteristics obtained by repeated analyses of extract, spiked at 50 microg kg(-1) with six strobilurins (azoxystrobin, picoxystrobin, dimoxystrobin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin), were in the following range: recoveries 78-92%, repeatability (RSD) 8-15%, linearity (R(2)) 0.9900-0.9978. The analysis of wheat with incurred strobilurin residues demonstrated good trueness of data generated by the DART-TOF MS method; the results were in a good agreement with those obtained by the conventional approach, i.e., by the QuEChERS sample handling procedure followed by identification/quantification employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry using DESI-LIT MS(n) provided a sufficient number of product ions for confirmation of the identity of azoxystrobin and pyraclostrobin in incurred wheat samples.

Meeting maximum residue limits: an improved screening technique for the rapid detection of antimicrobial residues in animal food products

A rapid, high-throughput antimicrobial screening assay was developed using either a physical fluid extraction or a solvent extraction technique coupled to the commercially available Premi®Test. The solvent extraction approach was fully validated for a wide range of tissues and the fluid extraction approach partially validated for porcine muscle. Both procedures can detect a wide range of antimicrobial compounds at or below maximum residue limit concentrations. The use of a solvent extraction provides an enhanced test capable of detecting a wider range of drugs than the fluid extraction approach at or below half maximum residue limit levels in a variety of matrices. Biochemical methods for the class-specific identification of β-lactams and sulphonamides following initial screening were developed and validated. The approach is a significant improvement on existing methodologies as a tool for residues monitoring in surveillance programmes.

A generic method for the quantitative analysis of aminoglycosides (and spectinomycin) in animal tissue using methylated internal standards and liquid chromatography tandem mass spectrometry

Aminoglycosides (AGs) are a large and diverse group of antibiotics. Although AGs may cause side effects of nephrotoxicity and ototoxicity, they are still occasionally being used for the treatment of serious infections. In this study the development of a method is described for the quantitative determination and confirmation of seven aminoglycosides (and relevant isomers) and spectinomycin in animal tissues. The extraction was based on an extraction followed by a concentration and clean-up step using weak cation exchange solid phase extraction. The separation was performed by ion-pair liquid chromatography on a C(18) column followed by mass spectrometric detection. The method was validated according to the EU requirements for a quantitative confirmatory method. Permethylated aminoglycosides (in-house synthesised internal standards) were used for accurate quantification. The accuracy of the analyses of AGs in kidney ranged from 94 to 111%, intra-day precision ranged between 2.5 and 7.4% (R.S.D.(r)) and inter-day precision ranged between 2.2 and 17.3% (R.S.D.(RL), n=21, MRL level). Accuracy (muscle tissue) varied from 83 to 128% with an intra-day precision between 2.2 and 17.3% (R.S.D.(r), n=7, MRL level). From the results it was concluded that the method was able to monitor MRL levels which ranged from 750 to 20,000 microgkg(-1) for kidney and from 50 to 10,000 microgkg(-1) for muscle tissue.

Toggled RNA Aptamers Against Aminoglycosides Allowing Facile Detection of Antibiotics Using Gold Nanoparticle Assays

We have used systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers against aminoglycoside antibiotics. The SELEX rounds were toggled against four pairs of aminoglycosides with the goal of isolating reagents that recognize conserved structural features. The resulting aptamers bind both of their selection targets with nanomolar affinities. They also bind the less structurally related targets, although they show clear specificity for this class of antibiotics. We show that this lack of aminoglycoside specificity is a common property of aptamers previously selected against single compounds and described as “specific”. Broad target specificity aptamers would be ideal for sensors detecting the entire class of aminoglycosides. We have used ligand-induced aggregation of gold-nanoparticles coated with our aptamers as a rapid and sensitive assay for these compounds. In contrast to DNA aptamers, unmodified RNA aptamers cannot be used as the recognition ligand in this assay, whereas 2′-fluoro-pyrimidine derivatives work reliably. We discuss the possible application of these reagents as sensors for drug residues and the challenges for understanding the structural basis of aminoglycoside-aptamer recognition highlighted by the SELEX results.

Multiresidue determination of triarylmethane and phenothiazine dyes in fish tissues by LC-MS/MS.

The occurrence of residues of malachite green and its leuco-metabolite in tissues of farmed fish for human consumption have long been of concern and there is extensive literature on methods of analysis and surveillance for these compounds. Recently, concern has been expressed that the use of other related compounds in place of malachite green may go undetected. This paper describes a new method for extending the range of triarylmethane and related phenothiazine dyes that can be detected in fish. In this procedure 13 parent compounds are monitored, with any potential leuco-forms being oxidized back to the parent prior to determination. The method utilizes a buffer-acetonitrile extraction followed by liquid-liquid extraction. Oxidant is added and the extracts further purified by cation exchange chromatography. Final determination is carried out using LC-MS/MS. The method has been validated to the standards of Commission Decision 2002/657/EC.

Effects of European foulbrood treatment regime on oxytetracycline levels in honey extracted from treated honeybee (Apis mellifera) colonies and toxicity to brood

This study aimed to assess oxytetracycline (OTC) residue levels in honey up to 12 weeks after treatment of honeybee colonies with two methods of application (in liquid sucrose and in powdered icing sugar). Significantly greater mortality was seen in the all stages of brood development for the treated colonies when compared with the controls. Samples of honey were extracted up to 12 weeks after treatment and analysed by HPLC following metal chelation with a limit of quantitation of 0.05 mg/kg. These data showed that the current method of application of Terramycin in liquid form results in very high residue levels in honey with residues of 3.7 mg/kg eight weeks after application. Further work is required to determine whether the levels can be further reduced by changes in the method of application whilst ensuring efficacy and minimizing the effects on brood.

A rapid microbial inhibition-based screening strategy for fluoroquinolone and quinolone residues in foods of animal origin

A rapid, high-throughput antimicrobial screening assay for the detection of fluoroquinolone and 4-quinolone residues in foods of animal origin has been developed in ampoule format. The assay employs a single Escherichia coli species sensitive to those Gram-negative inhibitiory antimicrobial compounds and is presented in a comparable format to the existing commercially available Premi Test and Delvotest ampoule-based microbial inhibition tests (DSM, Delft, The Netherlands). In the novel E. coli assay the microorganism, in vegetative state, is inoculated into a nutrient agar pellet containing a pH sensitive acid-base indicator dye. A simple extraction protocol that is selective for fluoroquinolone and quinolone compounds was developed to recover, cleanup and concentrate the target analyte(s) from a variety of tissue types and matrices prior to screening analysis. The method detected 16 target compounds at concentrations equal to or below the maximum residue limits (where applicable). The method has been validated using the prototype assay in accordance with the 2002/657/EC guidelines for the validation of qualitative screening assays. False positive and false negative responses rates for the procedure have been determined as less than 5%. The stability of a selection of representative target analytes has been demonstrated for a 20-week period under a variety of storage conditions both in tissue and in extract.

Multi-residue determination of phenolic and salicylanilide anthelmintics and related compounds in bovine kidney by liquid chromatography-tandem mass spectrometry

This paper describes an analytical method for four phenolic and salicylanilide anthelmintics authorised for use within the EU (nitroxinil, oxyclozanide, rafoxanide and closantel) in bovine kidney, and the extension of this procedure to include a number of related compounds; ioxynil, niclosamide, salicylanide and 3-trifluoromethyl-4-nitrophenol (TFM). The method comprises a solvent extraction with 1% acetic acid in acetone and clean-up using a mixed-mode anion-exchange solid phase extraction column. Determination is by reversed phase LC-MS/MS. The method was validated to the latest EU requirements (Commission Decision 2002/657/EC) using both spiked and incurred tissues and was subject to second laboratory evaluation.

Study of the depletion of tylosin residues in honey extracted from treated honeybee (Apis mellifera) colonies and the effect of the shook swarm procedure

Bee colonies were dosed with tylosin tartrate 1.1 g per hive (single dose in sucrose solution) and samples of honey were then collected at intervals over a 20-week period. The samples were analysed for tylosin A and desmycosin (tylosin B) using LC-MS/MS. The mean concentration of tylosin A in the honey (pooled results) 3 days after dosing was 17 μg/g, declining to 0.9 μg/g after 140 days. The mean concentration of desmycosin was 2.3 μg/g, 3 days after dosing declining to 1.1 μg/g after 140 days. The shook swarm procedure was investigated and resulted in a tylosin A concentration in brood honey of 10 μg/g, 3 days after dosing declining to 0.02 μg/g, 140 days after dosing. A corresponding decrease in the mean concentrations of desmycosin in brood honey, 1.1 fxg/g, 3 days after dosing to 0.03 [μg/g, 140 days after dosing also was observed. Tylosin A depletes to desmycosin in honey and can still be detected 238 days after dosing. Thus a more accurate residue definition is the sum of tylosin A and desmycosin.ZusammenfassungTylosin wurde kürzlich in den USA für die Bekämpfung der Amerikanischen Faulbrut in Bienenvölkern zugelassen und stellt somit ein alternatives Antibiotikum zu Oxytetracyclin dar. Allerdings sind nach EU-Bestimmungen Tylosinrückstände in Honig nicht erlaubt und Honige aus den USA mit Tylosinrückständen wären auf dem EU-Markt nicht verkehrsfähig. Daher wurde hier die Beziehung zwischen Tylosin A und dem Abbauprodukt Desmykosin untersucht. Damit sollte eine Markersubstanz etabliert werden, um den Abbau von Tylosin im Bienenvolk zu erfassen und die Verwendung dieses Wirkstoffes in der Imkerei nachzuweisen.Bienenvölkern wurde eine Dosis von 1,1g Tylosintartrat pro Volk in Form einer einmaligen Zuckerlösung gegeben. Die Proben wurden vor der Futtergabe und danach über 20 Wochen in regelmäßigen Abständen und schließlich nach der Überwinterung gezogen. Die Proben wurden über HPLC-MS auf Tylosin A und Desmycosin analysiert.Die Konzentration an Tylosin A im Honig nahm im Zeitraum der Probennahmen kontinuierlich ab: Von 17 [μg/g 3 Tage nach Applikation über 3,3 μg/kg 56 Tage danach bis auf 0,9 μg/kg 140 Tage danach. Die Konzentration von Desmycosin nahm lediglich von 2,3 μg/kg 3 Tage nach Applikation auf 1,1 μg/kg 140 Tage danach ab (Abb. 1, Tab. III und IV). Es gibt einen raschen Abbau an Tylosin A während der ersten 28 Tage nach Applikation gefolgt von einer geringeren Abbaurate danach. Trotz der Abnahme an Tylosin bleibt die Konzentration an Desmycosin weitgehend konstant, vermutlich wegen einer kontinuierlichen Umwandlung von Tylosin A zu Desmycosin. Die Konzentrationen von Desmycosin und Tylosin A gleichen sich 84 Tage nach der Applikation an (Abb. 2).Die Kunstschwarmbildung auf neues Wabenwerk 7 Tage nach der Applikation reduzierte die Rückstandskonzentrationen von Tylosin A und Desmycosin um den Faktor 30 zum Ende der Probennahme (140 Tage nach Applikation). Tab. V zeigt vergleichend die Abnahme der Rückstandskonzentrationen für behandelte Bienenvölker mit und ohne Kunstschwarmbildung. Nach Applikation von Tylosin können Rückstände auch 238 Tage danach noch nachgewiesen werden selbst wenn zwischenzeitlich die Kunstschwarmmethode angewendet wird. Tylosin A ist eine geeignete Markersubstanz um den Gebrauch bzw. Missbrauch von Tylosin nachzuweisen. Eine exakte Bestimmung von Tylosinrückständen sollte allerdings über die Summe von Tylosin A und Desmycosin erfolgen.

Investigation into the Occurrence in Food of Veterinary Medicines, Pharmaceuticals, and Chemicals Used in Personal Care Products

Human exposure to emerging contaminants by indirect routes is of increasing interest. This study assessed the contamination of food by chemicals used in human pharmaceuticals (HPs), veterinary medicines (VMs), and personal care products (PCPs). A prioritization study was undertaken to identify the chemicals and food-producing scenarios most likely to result in contamination of food. Around 400 samples of mushrooms, vegetables, aquaculture products, and animal tissues were collected from sites in the United Kingdom, along with aquaculture products imported from Southeast Asia. A number of multianalyte methods were developed and validated for the analysis of the prioritized compounds in these samples. The analysis of all sample-method combinations required approximately 18000 determinations. Around 325 individual residues, including parabens, musk compounds, and antibiotics, were detected in 118 individual samples, but mostly at low nanograms per gram concentrations. Results suggest that the limited contamination of target chemicals occurred in the realistic food-producing scenarios investigated.