Sara Stead

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.

Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish.

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BG) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BG, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BG which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes.

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.

New method for the rapid identification of tetracycline residues in foods of animal origin — using the Premi®Test in combination with a metal ion chelation assay

A post-screening classification assay for tetracycline compounds has been developed and integrated into the previously reported optimized Premi®Test methodology. The new post-antimicrobial screening assay is based on a metal ion chelation using calcium and sodium chloride and has been shown to be specific towards the tetracycline class. The assay is both cost-effective and complementary to the post-screening procedures that have previously been developed for the β-lactam and sulfonamide compounds. A validation study was conducted in accordance with 2002/657/EC (Commission Decision). The method is rugged and applicable to a range of tetracyclines of differing antimicrobial potencies over a wide concentration range. A blind trial was undertaken in which all antimicrobial residues in the unknown samples were successfully identified by the analyst following the integrated Premi®Test procedure for the classification of antimicrobial compounds.

Prediction of Collision Cross Section Values for Small Molecules: Application to Pesticide Residue Analysis

The use of collision cross-section (CCS) values obtained by ion mobility high-resolution mass spectrometry has added a third dimension (alongside retention time and exact mass) to aid in the identification of compounds. However, its utility is limited by the number of experimental CCS values currently available. This work demonstrates the potential of artificial neural networks (ANNs) for the prediction of CCS values of pesticides. The predictor, based on eight software-chosen molecular descriptors, was optimized using CCS values of 205 small molecules and validated using a set of 131 pesticides. The relative error was within 6% for 95% of all CCS values for protonated molecules, resulting in a median relative error less than 2%. In order to demonstrate the potential of CCS prediction, the strategy was applied to spinach samples. It notably improved the confidence in the tentative identification of suspect and nontarget pesticides.