Mary Moloney

Current trends in sample preparation for growth promoter and veterinary drug residue analysis.

A comprehensive review is presented on the current trends in sample preparation for the isolation of veterinary drugs and growth promoters from foods. The objective of the review is to firstly give an overview of the sample preparation techniques that are applied in field. The review will focus on new techniques and technologies, which improve efficiency and coverage of residues. The underlying theme to the paper is the developments that have been made in multi-residue methods and particularly multi-class methods for residues of licensed animal health products, which have been developed in the last couple of years. The role of multi-class methods is discussed and how they can be accommodated in future residue surveillance.

Determination of 20 coccidiostats in egg and avian muscle tissue using ultra high performance liquid chromatography-tandem mass spectrometry.

A quantitative, comprehensive multiresidue method which includes 20 coccidiostat residues has been developed. The method described uses a simple one-step liquid extraction with acetonitrile to isolate analytes from both the polyether ionophore and chemical classes of coccidiostats. Subsequent to a further concentration step, samples were analysed via UHPLC-MS/MS. The method was validated according to the Commission Decision 2002/657/EEC in egg and avian muscle. The method permitted quantitative confirmation for 13 compounds below target concentrations, and screening for a further 7 compounds. Within-laboratory repeatability gave accuracy values in the range of 68-129%, while reproducibility ranged between 75 and 123%. Calibration ranges were typically 1-50 μg kg⁻¹, although higher ranges were used for dinitrocarbanilide, imidocarb and toltrazuril residues. A regression coefficient (R²) value of greater than 0.98 was obtained for all analytes. Precision results ranged from 2.3 to 19.7% CV for egg and from 2.6 to 23.6% CV in muscle. CCα was in the range from 1.13 μg kg⁻¹ (clopidol) to 179 μg kg⁻¹ (lasalocid) in egg. In muscle, CCα ranged from 2.25 μg kg⁻¹ (aprinocid) to 4579 μg kg⁻¹ (dinitrocarbanilide). CCβ was from 1.29 μg kg⁻¹ (clopidol) to 209 μg kg⁻¹ (lasalocid) in egg, and 2.58 μg kg⁻¹ (arprinocid) to 6060 μg kg⁻¹ (dinitrocarbanilide) in muscle. Limits of quantification were 1 μg kg⁻¹ for all compounds, except imidocarb and dinitrocarbanilide (10 μg kg⁻¹), and toltrazuril and metabolites (50 μg kg⁻¹).

A review of coccidiostats and the analysis of their residues in meat and other food

Coccidiostats are used in the control of protozoan infections in different food producing animals. They are most widely used as feed additives in intensively reared species such as pigs and poultry to maintain animal health and in some cases enhance feed conversion. However, a number of these drugs are used in the control of infections in beef and lamb production. Coccidiostat residues have been frequently reported in meat and eggs in a number of countries since the late 1990s. This has prompted increased research and surveillance of coccidiostat residues in food. This paper reviews the various coccidiostat agents used in animal production, including their chemical properties, mode of action and activity. Legislation concerning coccidiostats, limits for residues in food, monitoring and occurrence of residues in food is discussed. Methods for residue determination in food, including screening and physicochemical methods are discussed in depth. The paper concludes with a synopsis of the current state of coccidiostat residue analysis and future perspectives.

Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements.

A surface plasmon resonance (SPR) immunobiosensor assay was developed and validated to detect microcystin toxins in Spirulina and Aphanizomenon flos-aquae blue-green algae (BGA) food supplements. A competitive inhibition SPR-biosensor was developed using a monoclonal antibody to detect microcystin (MC) toxins. Powdered BGA samples were extracted with an aqueous methanolic solution, centrifuged and diluted in HBS-EP buffer prior to analysis. The assay was validated in accordance with the performance criteria outlined in EU legislation 2002/657/EC. The limit of detection (LOD) of the assay was calculated from the analysis of 20 known negative BGA samples to be 0.561 mg kg(-1). The detection capability (CCβ) of the assay was determined to be ≤ 0.85 mg kg(-1) for MC-LR. The biosensor assay was successfully applied to detect MC-LR toxins in BGA samples purchased on the Irish retail market. MC-LR was detected in samples at levels ranging from <0.5 to 2.21 mg kg(-1). The biosensor results were in good agreement with an established LC-MS/MS assay. The assay is advantageous because it employs a simple clean-up procedure compared to chemical assays and allows automated unattended analysis of samples unlike ELISA.

Detection of banned nitrofuran metabolites in animal plasma samples using UHPLC–MS/MS

The use of nitrofurans as veterinary drugs in food-producing animals has been banned in the EU since the 1990s. Monitoring programs in the EU are based on the detection of protein-bound metabolites after slaughter. An UHPLC-MS/MS method was developed and validated for pre slaughter determination of four nitrofuran metabolites (AHD, AOZ, SEM, AMOZ) in animal plasma (bovine, ovine, equine and porcine). This method is proposed as an alternative method for on-farm surveillance. Plasma samples were derivatised with 2-nitrobenzaldehyde and subsequently extracted with organic solvent. Extracts were concentrated and then analysed by UHPLC-MS/MS. The method was validated according to Commission Decision 2002/657/EC. Inter-species recovery for AHD, AOZ, SEM and AMOZ was 72, 74, 57 and 71%, respectively. Decision limits (CCα) were calculated from within laboratory reproducibility experiments to be 0.070, 0.059, 0.071 and 0.054 μg kg(-1), respectively. In addition, the assay was applied to incurred plasma samples taken from pigs treated with furazolidone.

Design and implementation of an imprinted material for the extraction of the endocrine disruptor bisphenol A from milk.

This paper describes the determination of bisphenol A (BPA) in milk samples, using a novel molecularly imprinted polymer. The imprinted polymer was developed using a rational design approach, and pre-polymerization interactions were investigated using molecular dynamics simulations and X-ray crystallography. A hydroquinone-imprinted polymer was used for solid phase extraction (SPE) clean-up of samples. BPA was quantified by high performance liquid chromatography (HPLC) and fluorescence (FLD) detection. Following validation, the method described was capable of determining bisphenol A in milk down to a limit of detection of 1.32μgkg(-1). The method was applied to a survey (n=27) of commercial milk products; BPA was detected in one of the samples, at a level of 176μgkg(-1). Test results were confirmed by a parallel UHPLC-MS/MS analytical method. This demonstrates the utility of the hydroquinone-imprinted polymer for application to selective sample clean-up and analysis of bisphenol A in milk, avoiding possible detrimental affects associated with template bleeding and without the need for expensive or difficult-to-obtain template.

Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip screening assay

A chemiluminescence-based biochip array sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplex approach, metabolites of the four main nitrofuran antibiotics could be simultaneously detected. Individual antibodies specific towards the metabolites were spotted onto biochips. A competitive assay format, with chemiluminescent response, was employed. The method was validated in accordance with EU legislation (2002/657/EC, 2002), and assessed by comparison with UHPLC-MS/MS testing of 134 honey samples of worldwide origin. A similar extraction method, based on extraction of the analytes on Oasis™ SPE cartridges, followed by derivatisation with nitrobenzaldehyde and partition into ethyl acetate, was used for both screening and LC-MS/MS methods. The biochip array method was capable of detecting all four metabolites below the reference point for action of 1 μg kg(-1). The detection capability was below 0.5 μg kg(-1) for the metabolites AHD, AOZ and AMOZ; it was below 0.9 μg kg(-1) for SEM. IC(50) values ranged from 0.14 μg kg(-1) (AMOZ) to 2.19 μg kg(-1) (SEM). This biosensor method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety technology.

Determination of 20 coccidiostats in milk, duck muscle and non-avian muscle tissue using UHPLC-MS/MS

In this paper, methods were developed to measure coccidiostats in bovine milk, duck muscle and non-avian species. The methods were validated to the maximum levels and MRLs laid down in European Union legislation. A simple sample preparation procedure was developed for the isolation of coccidiostat residues from bovine, ovine, equine, porcine and duck muscle tissue, based on solvent extraction with acetonitrile and concentration. An alternative method had to be developed for milk samples based on the QuEChERS sample preparation approach because of the high water content in this matrix. Milk samples were adjusted to basic pH with sodium hydroxide and extracted by using a slurry of acetonitrile, MgSO4 and NaCl. Purified sample extracts were subsequently analysed by using UHPLC-MS/MS in a 13.2-min chromatographic run. It was found that the use of rapid polarity switching enabled both negatively and positively charged ions to be analysed from a single injection. By using this approach, solvent usage was reduced significantly and sample throughput improved. The method was validated for the analysis of 20 coccidiostats (arprinocid, clopidol, decoquinate, diclazuril, diaveridine, ethopabate, halofuginone, laidlomycin, lasalocid, maduramicin, monensin, narasin, nequinate, nicarbazin, robenidine, salinomycin, semduramicin, toltrazuril, toltrazuril sulphoxide and toltrazuril sulphone) in muscle and milk. The method is quantitative for toltrazurils, but it cannot be used for confirmation because only the precursor ion is monitored. Accuracy values for muscle ranged from 80% to 125%, while CCα ranged from 2.2 µg kg−1 for clopidol to 122 µg kg−1 for toltrazuril sulphoxide. Bovine milk accuracy ranged from 84% to 120% for all analytes except maduramicin, semduramicin and salinomycin, for which the values were higher. CCα values achieved ranged from 1.1 µg kg−1 for arprinocid, nequinate and lasalocid to 27 µg kg−1 for toltrazuril.

Determination and Occurrence of Phenoxyacetic Acid Herbicides and Their Transformation Products in Groundwater Using Ultra High Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry

A sensitive method was developed and validated for ten phenoxyacetic acid herbicides, six of their main transformation products (TPs) and two benzonitrile TPs in groundwater. The parent compounds mecoprop, mecoprop-p, 2,4-D, dicamba, MCPA, triclopyr, fluroxypr, bromoxynil, bentazone, and 2,3,6-trichlorobenzoic acid (TBA) are included and a selection of their main TPs: phenoxyacetic acid (PAC), 2,4,5-trichloro-phenol (TCP), 4-chloro-2-methylphenol (4C2MP), 2,4-dichlorophenol (DCP), 3,5,6-trichloro-2-pyridinol (T2P), and 3,5-dibromo-4-hydroxybenzoic acid (BrAC), as well as the dichlobenil TPs 2,6-dichlorobenzamide (BAM) and 3,5-dichlorobenzoic acid (DBA) which have never before been determined in Irish groundwater. Water samples were analysed using an efficient ultra-high performance liquid chromatography (UHPLC) method in an 11.9 min separation time prior to detection by tandem mass spectrometry (MS/MS). The limit of detection (LOD) of the method ranged between 0.00008 and 0.0047 µg·L−1 for the 18 analytes. All compounds could be detected below the permitted limits of 0.1 µg·L−1 allowed in the European Union (EU) drinking water legislation [1]. The method was validated according to EU protocols laid out in SANCO/10232/2006 with recoveries ranging between 71% and 118% at the spiked concentration level of 0.06 µg·L−1. The method was successfully applied to 42 groundwater samples collected across several locations in Ireland in March 2012 to reveal that the TPs PAC and 4C2MP were detected just as often as their parent active ingredients (a.i.) in groundwater.

Maximum residue level validation of triclabendazole marker residues in bovine liver, muscle and milk matrices by ultra high pressure liquid chromatography tandem mass spectrometry.

Triclabendazole is the only anthelmintic drug, which is active against immature, mature and adult stages of fluke. The objective of this work was to develop an analytical method to quantify and confirm the presence of triclabendazole residues around the MRL. In this work, a new analytical method was developed, which extended dynamic range to 1-100 and 5-1000 μg kg(-1) for milk and tissue, respectively. This was achieved using a mobile phase containing trifluoroacetic acid (pK(a) of 0.3), which resulted in the formation of the protonated pseudomolecular ions, [M+H](+), of triclabendazole metabolites. Insufficient ionisation of common mobile phase additives due to low pK(a) values (<2) was identified as the cause of poor linearity. The new mobile phase conditions allowed the analysis of triclabendazole residues in liver, muscle and milk encompassing their EU maximum residue levels (MRL) (250, 225 and 10 μg kg(-1) respectively). Triclabendazole residues were extracted using a modified QuEChERS method and analysed by positive electrospray ionisation mass spectrometry with all analytes eluted by 2.23 min. The method was validated at the MRL according to Commission Decision (CD) 2002/657/EC criteria. The decision limit (CCα) of the method was in the range of 250.8-287.2, 2554.9-290.8 and 10.9-12.1 μg kg(-1) for liver, muscle and milk, respectively. The performance of the method was successfully verified for triclabendazole in muscle by participating in a proficiency study, the method was also applied to incurred liver, muscle and milk samples.