Martin Danaher

New method for the analysis of flukicide and other anthelmintic residues in bovine milk and liver using liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-residue method for the simultaneous quantification and identification of 38 residues of the most widely used anthelmintic veterinary drugs (including benzimidazoles, macrocyclic lactones, and flukicides) in milk and liver has been developed and validated. For sample preparation, we used a simple modification of the QuEChERS method, which was initially developed for pesticide residue analysis. The method involved extracting sample (10 g) with acetonitrile (10 mL), followed by phase separation from water (salting out) with MgSO(4):NaCl (4:1, w/w). After centrifugation, an aliquot of the extract (1 mL) was purified by dispersive solid-phase extraction with MgSO(4) (150 mg) and C(18) (50mg), prior to LC-MS/MS analysis. Two injections of the same extract were required with the LC-MS/MS instrument to cover the 30 electrospray positive and 8 electrospray negative analytes. The limit of quantitation of the method was 5 microgkg(-1) for 37 analytes (and 10 microgkg(-1) for dichlorvos). The method was successfully validated according to the 2002/657/EC guidelines. Recovery of analytes was typically in the 70-120% range, with repeatabilities and reproducibilities typically <15% in milk and <20% in liver.

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.

Benzimidazole carbamate residues in milk: Detection by Surface Plasmon Resonance-biosensor, using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method for extraction

A surface plasmon resonance (SPR) biosensor screening assay was developed and validated to detect 11 benzimidazole carbamate (BZT) veterinary drug residues in milk. The polyclonal antibody used was raised in sheep against a methyl 5(6)-[(carboxypentyl)-thio]-2-benzimidazole carbamate protein conjugate. A sample preparation procedure was developed using a modified QuEChERS method. BZT residues were extracted from milk using liquid extraction/partition with a dispersive solid phase extraction clean-up step. The assay was validated in accordance with the performance criteria described in 2002/657/EC. The limit of detection of the assay was calculated from the analysis of 20 known negative milk samples to be 2.7mugkg(-1). The detection capability (CCbeta) of the assay was determined to be 5mugkg(-1) for 11 benzimidazole residues and the mean recovery of analytes was in the range 81-116%. A comparison was made between the SPR-biosensor and UPLC-MS/MS analyses of milk samples (n=26) taken from cows treated different benzimidazole products, demonstrating the SPR-biosensor assay to be fit for purpose.

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).

Detection of pyrrolizidine alkaloids in commercial honey using liquid chromatography–ion trap mass spectrometry

Pyrrolizidine alkaloids (PAs) are known secondary plant metabolites which can cause hepatotoxicity in both humans and livestock. PAs can be consumed through the use of plants for food, medicinal purposes and as contaminants of agricultural crops and food. PA contaminated grain has posed the largest health risk, although any PA contamination in our food chain should be recognised as a potential health threat. For this purpose, retail honeys were tested by LC-MS/MS. The method allows for specific identification of toxic retronecine and otonecine-type PAs by comparison to reference compounds via a spectral library. In total, 50 honey samples were matched to the reference spectra within a set of tolerance parameters. Accurate data analysis and quick detection of positive samples was possible. Positive samples contained an average PA concentration of 1260 μg kg(-1) of honey. Good linear calibrations were obtained (R(2)>0.991). LOD and LOQ ranged from 0.0134 to 0.0305 and 0.0446 to 0.1018 μg mL(-1), respectively.

Development and optimisation of an improved derivatisation procedure for the determination of avermectins and milbemycins in bovine liver

A robust procedure has been developed to overcome the instability problems experienced with the fluorescent derivative of eprinomectin. The procedure involves addition of acetic acid, together with the typical reagents methylimidazole and trifluoroacetic anhydride, to produce a fluorescent molecule that can be determined by high performance liquid chromatography (HPLC) with fluorescence detection. Derivatisation is completed in 30 min at 65 degrees C. This derivatisation procedure was shown to be suitable, also, for the related compounds, moxidectin, abamectin, doramectin and ivermectin. A multi-residue method for these compounds in bovine liver has been developed using the derivatisation procedure. Samples are extracted with acetonitrile; followed by clean-up on deactivated alumina and C18 solid phase extraction (SPE) cartridges. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery ranged between 73 and 97%. The intra- and inter-assay variations showed relative standard deviations typically of < 6% and < 14%, respectively. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).

The use of ultra-high pressure liquid chromatography with tandem mass spectrometric detection in the analysis of agrochemical residues and mycotoxins in food - challenges and applications.

In the field of food contaminant analysis, the most significant development of recent years has been the integration of ultra-high pressure liquid chromatography (UHPLC), coupled to tandem quadrupole mass spectrometry (MS/MS), into analytical applications. In this review, we describe the emergence of UHPLC through technological advances. The implications of this new chromatographic technology for MS detection are discussed, as well as some of the remaining challenges in exploiting it for chemical residue applications. Finally, a comprehensive overview of published applications of UHPLC-MS in food contaminant analysis is presented, with a particular focus on veterinary drug residues.

A dual validation approach to detect anthelmintic residues in bovine liver over an extended concentration range

This paper describes a method for the detection and quantification of 38 residues of the most widely used anthelmintics (including 26 veterinary drugs belonging to the benzimidazole, macrocyclic lactone and flukicide classes) in bovine liver using two different protocols for MRL and non-MRL levels. A dual validation approach was adopted to reliably quantify anthelmintic residues over an extended concentration range (1-3000 μg kg(-1)). Sample extraction and purification was carried out using a modified QuEChERS method. A concentration step was included when analysing in the low μg kg(-1) range. Rapid analysis was carried out by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which was capable of detecting residues to <2 μg kg(-1). The method has been single-laboratory validated according to the 2002/657/EC guidelines and met acceptability criteria in all but a few cases. The inclusion of 19 internal standards, including 14 isotopically labelled internal standards, improved accuracy, precision, decision limit (CCα) and detection capability (CCβ).

Stability during cooking of anthelmintic veterinary drug residues in beef

Anthelmintic drugs are widely used for treatment of parasitic worms in livestock, but little is known about the stability of their residues in food under conventional cooking conditions. As part of the European Commission-funded research project ProSafeBeef, cattle were medicated with commercially available anthelmintic preparations, comprising 11 active ingredients (corresponding to 21 marker residues). Incurred meat and liver were cooked by roasting (40 min at 190°C) or shallow frying (muscle 8–12 min, liver 14–19 min) in a domestic kitchen. Raw and cooked tissues and expressed juices were analysed using a novel multi-residue dispersive solid-phase extraction method (QuEChERS) coupled with ultra-performance liquid chromatography-tandem mass spectrometry. After correction for sample weight changes during cooking, no major losses were observed for residues of oxyclozanide, clorsulon, closantel, ivermectin, albendazole, mebendazole or fenbendazole. However, significant losses were observed for nitroxynil (78% in fried muscle, 96% in roast muscle), levamisole (11% in fried muscle, 42% in fried liver), rafoxanide (17% in fried muscle, 18% in roast muscle) and triclabendazole (23% in fried liver, 47% in roast muscle). Migration of residues from muscle into expressed cooking juices varied between drugs, constituting 0% to 17% (levamisole) of total residues remaining after cooking. With the exception of nitroxynil, residues of anthelmintic drugs were generally resistant to degradation during roasting and shallow frying. Conventional cooking cannot, therefore, be considered a safeguard against ingestion of residues of anthelmintic veterinary drugs in beef.

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.