Barry Foley

Rapid confirmatory method for the determination of 11 nitroimidazoles in egg using liquid chromatography tandem mass spectrometry

A rapid confirmatory method has been developed and validated for the simultaneous identification, confirmation and quantitation of 11 nitroimidazoles in eggs by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method is validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ), tinidazole (TNZ) and ternidazole (TRZ). MNZ, DMZ and RNZ have been assigned a recommended level (RL) of 3 microg kg(-1) by the Community Reference Laboratory (CRL) in Berlin. The developed method described in this study is easily able to detect all the nitroimidazole compounds investigated at this level and below. Egg samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid-liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The decision limits (CCalpha) range from 0.33 to 1.26 microg kg(-1) and the detection capabilities (CCbeta), range from 0.56 to 2.15 microg kg(-1). The results of the in ter-assay study, which was performed by fortifying hen egg samples (n=18) on three separate days, show the accuracy calculated for the various analytes to range between 87.2 and 106.2%. The precision of the method, expressed as %CV values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 microg kg(-1)), ranged between 3.7 and 11.3%. A Day 4 analysis was carried out to examine species variances in eggs from different birds such as duck and quail and investigating differences in various battery and free range hen eggs.

Rapid multi-class multi-residue method for the confirmation of chloramphenicol and eleven nitroimidazoles in milk and honey by liquid chromatography-tandem mass spectrometry (LC-MS)

A confirmatory method was developed to allow for the analysis of eleven nitroimidazoles and also chloramphenicol in milk and honey samples. These compounds are classified as A6 compounds in Annex IV of Council Regulation 2377/90 (European Commission 1990) and therefore prohibited for use in animal husbandry. Milk samples were extracted by acetonitrile with the addition of NaCl; honey samples were first dissolved in water before a similar extraction. Honey extracts underwent a hexane wash to remove impurities. Both milk and honey extracts were evaporated to dryness and reconstituted in initial mobile phase. These were then injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and analysed in less than 9 min. The MS/MS was operated in multiple reaction monitoring (MRM) mode with positive and negative electrospray ionization. The method was validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole, dimetridazole, ronidazole, ipronidazole and there hydroxy metabolites hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, and hydroxyipronidazole. The method can also analyse for carnidazole, ornidazole, ternidazole, tinidazole, and chloramphenicol. A recommended level of 3 µg l−1/µg kg−1 for methods for metronidazole, dimetridazole, and ronidazole has been recommended by the Community Reference Laboratory (CRL) responsible for this substance group, and this method can easily detect all nitroimidazoles at this level. A minimum required performance level of 0.3 µg l−1/µg kg−1 is in place for chloramphenicol which the method can also easily detect. For nitroimidazoles, the decision limits (CCα) and detection capabilities (CCβ) ranged from 0.41 to 1.55 µg l−1 and from 0.70 to 2.64 µg l−1, respectively, in milk; and from 0.38 to 1.16 µg kg−1 and from 0.66 to 1.98 µg kg−1, respectively, in honey. For chloramphenicol, the values are 0.07 and 0.11 µg l−1 in milk and 0.08 and 0.13 µg kg−1 in honey. Validation criteria of accuracy, precision, repeatability, and reproducibility along with measurement uncertainty were calculated for all analytes in both matrices.

Development and Validation of a Rapid Multi-class Method for the Confirmation of Fourteen Prohibited Medicinal Additives in Pig and Poultry Compound Feed by Liquid Chromatography Tandem Mass Spectrometry

A confirmatory method has been developed to allow for the analysis of fourteen prohibited medicinal additives in pig and poultry compound feed. These compounds are prohibited for use as feed additives although some are still authorised for use in medicated feed. Feed samples are extracted by acetonitrile with addition of sodium sulfate. The extracts undergo a hexane wash to aid with sample purification. The extracts are then evaporated to dryness and reconstituted in initial mobile phase. The samples undergo an ultracentrifugation step prior to injection onto the LC-MS/MS system and are analysed in a run time of 26 min. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation. The method was validated over three days and is capable of quantitatively analysing for metronidazole, dimetridazole, ronidazole, ipronidazole, chloramphenicol, sulfamethazine, dinitolimide, ethopabate, carbadox and clopidol. The method is also capable of qualitatively analysing for sulfadiazine, tylosin, virginiamycin and avilamycin. A level of 100 microg kg(-1) was used for validation purposes and the method is capable of analysing to this level for all the compounds. Validation criteria of trueness, precision, repeatability and reproducibility along with measurement uncertainty are calculated for all analytes.

An integrated approach to the toxicity assessment of Irish marine sediments: validation of established marine bioassays for the monitoring of Irish marine sediments.

This paper describes the ecotoxicological evaluation of marine sediments from three sites around Ireland representative of a range of contaminant burdens. A comprehensive assessment of potential sediment toxicity requires the consideration of multiple exposure phases. In addition to the evaluation of multi-exposure phases the use of a battery of multi-trophic test species has been advocated by a number of researchers as testing of single or few organisms may not detect toxicants with a specific mode of action. The Microtox solid phase test (SPT) and the 10-d acute amphipod test with Corophium volutator were used to assess whole sediment toxicity. Porewater and elutriates were assessed with the Microtox acute test, the marine prasinophyte Tetraselmis suecica, and the marine copepod Tisbe battagliai. Solvent extracts were assayed with the Microtox and T. battagliai acute tests. Alexandra Basin was identified as the most toxic site according to all tests, except the Microtox SPT which identified the Dunmore East site as being more toxic. However, it was not possible to correlate the observed ecotoxicological effects with a specific and/or class of contaminants based on sediment chemistry alone. Therefore porewaters found to elicit significant toxicity (Dunmore East and Alexandra Basin) with the test battery were selected for further TIE assessment with T. battalgiai and the Microtox system. The results of this study have important implications for risk assessment in estuarine and coastal waters in Ireland, where, at present the monitoring of sediment and water quality is predominantly reliant on chemical analysis alone.

Determination of eleven coccidiostats in animal feed by liquid chromatography–tandem mass spectrometry at cross contamination levels

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 19 min. 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.

Development and validation of a rapid method for the determination and confirmation of 10 nitroimidazoles in animal plasma using liquid chromatography tandem mass spectrometry.

A rapid LC-MS/MS method has been developed and validated for the simultaneous identification, confirmation and quantitation of 10 nitroimidazoles in plasma. The method validated in accordance with Commission Decision (CD) 2002/657/EC is capable of analysing for metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ) and ternidazole (TRZ) which are rarely analysed by modern methods. MNZ, DMZ and RNZ have a recommended level (RL) of 3 ng mL(-1) which this method is easily able to detect for all the nitroimidazole compounds. Plasma samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid-liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The decision limits (CCalpha) range from 0.5 to 1.6 ng mL(-1) and the detection capabilities (CCbeta), range from 0.8 to 2.6 ng mL(-1). The results of the inter-assay study, which was performed by fortifying bovine plasma samples (n=18) on three separate days, show the accuracy calculated for the various analytes range between 101% and 108%. The precision of the method, expressed as CV% values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 ng mL(-1)), ranged between 4.9% and 15.2%. A day 4 analysis was carried out to examine species variances in animals such as avian, ovine, porcine and equine.

An integrated approach to the toxicity assessment of Irish marine sediments: application of porewater Toxicity Identification Evaluation (TIE) to Irish marine sediments.

An integrated approach to the ecotoxicological assessment of Irish marine sediments was carried out between 2004 and 2007. Phase I Toxicity Identification Evaluation (TIE) of sediment porewaters from two sites on the east coast of Ireland were conducted. Initial Tier I screening of three Irish sites identified the need for TIE after significant toxicity was observed with Tisbe battagliai and the Microtox assay at two of the assayed sites (Alexandra Basin and Dunmore East). Porewaters classified as toxic were characterised using four manipulations, ethylenediaminetetraacetic acid (EDTA) chelation, sodium thiosulphate addition, C(18) Solid Phase Extraction (SPE) and Cation Exchange (CE) SPE. Prior to initial testing, and TIE manipulations, all porewater samples were frozen at -20 degrees C for several months until required. After initial Tier I testing Alexandra Basin porewater was classified as highly toxic by both assays while Dunmore East porewater only warranted a TIE with T. battagliai. Results of TIE manipulations for Alexandra Basin porewater and the Microtox Basic test were inconclusive. The toxicity of the porewater in this assay was significantly reduced after freezing. Three experimental episodes were conducted with one month between each for the Alexandra Basin porewater. After each month of freezing the baseline toxicity was further reduced in the Microtox assay, therefore it was not possible to draw accurate conclusions on the nature of the active contaminants in the sample. However, toxicity to T. battalgiai did not change after storage of the porewater. The C(18) and CE SPE decreased the toxicity of Alexandra Basin porewater to the copepod indicating that both organic and cationic compounds (e.g. metals) were active in the sample. Dunmore East porewater was assayed with T. battalgiai and again a combination of organic and inorganic compounds were found to be partly responsible for the observed toxicity (C(18), CE SPE and EDTA reduced toxicity). Results from these TIEs provide insight into the complexity of interpreting marine TIE data from porewater studies where mixtures of unknown substances are present.

Use of Caged Nucella Lapillus and Crassostrea Gigas to Monitor Tributyltin‐Induced Bioeffects in Irish Coastal Waters

Caging studies have been previously reported to be useful for providing valuable information on biological effects of mollusks over short periods of time where resident species are absent. The degree of imposex in caged dog whelk (Nucella lapillus), was measured using the vas deferens sequence index (VSDI) and the Relative Penis Size Index (RPSI) and the extent of shell thickening in caged Pacific oyster (Crassostrea gigas) was investigated at t = 0 and t = 18 weeks. Nucella lapillus, when provided with mussels as a food source at the control site at Omey Island on the west Irish coast, did not demonstrate imposex features, whereas those transplanted to port areas did. Dunmore East exhibited the highest level of imposex (3.25 VDSI and 2.37 RPSI). Shell thickening was evident in C. gigas transplanted to Dunmore East, with low effects evident at the control location, Omey Island, and Dublin Bay at t = 18 weeks. Dry weight whole-body concentrations of organotins were most elevated in all species held at Dunmore East compared with other locations. Greatest delta15N and delta13C enrichment was observed within the tissues of the predatory N. lapillus in all three test sites. Increased assimilation in the Dublin Bay oysters might have been influenced by the presence of more nutrients at this location. Surficial sediment organotin levels were most elevated in the Dunmore East <2-mm fraction (22,707 microg tributyltin/kg dry weight), whereas low organotin levels were determined from Dublin and Omey Island sediments. The valuable application of cost-effective caging techniques to deliver integrated biological effects and chemical measurements in the absence of resident gastropod populations in potential organotin/tributyltin hotspot locations is discussed.

Primary aromatic amine migration from polyamide kitchen utensils: method development and product testing

A rapid and sensitive LC-MS/MS method for the identification, quantification and confirmation of six primary aromatic amines (PAAs) was developed and validated to ISO 17025:2005. From a literature survey, 57 frequently used PAA compounds were identified and subsequently reduced to six – aniline, 4,4′-MDA, 3,3′-DMB, 2,4-TDA, 2,6-TDA and o-T – based on results from migration studies on a range of utensils. Low LOQs of between 0.075 and 0.496 µg l−1 were determined for the six analytes, thereby quantifying well below the legal limit of 10 µg kg−1 total PAAs. Furthermore, low measurement uncertainties were calculated for the analytical method, in the range of 3.15–3.20%. Mean recoveries were between 98% and 102% and spanned over ±12% at 95% CI. Following the analysis of 84 black polyamide kitchen utensils, the migration of PAAs detected was significant and is therefore of concern. The six analytes identified, quantified and confirmed in this survey could be utilised as possible markers for the identification of PAA migration, thereby improving the time and cost-efficiency of food control laboratories.

In vitro screening of organotin compounds and sediment extracts for cytotoxicity to fish cells.

The present study reports an in vitro screening method for contaminants in sediment samples utilizing an RTG-2 cell line. This technique integrates cytotoxicity testing with analytical chemistry with the aim of achieving a toxicity evaluation of the sediment sample. The toxic effect of individual organotin (OT) compounds and their presence in the sediment sample is the focus of the present study; however, other contaminants are also discussed. The following OT compounds: tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT), and a sediment solvent extract are exposed to the RTG-2 fish cell line. Both the alamar blue (AB) and neutral red (NR) assays are used to assess cytotoxicity after 24-h and 96-h exposure. Methodology for preparation of a sediment solvent extract suitable for biological testing and analytical determination is also described. With the RTG-2 cells, the AB and NR assays had comparable sensitivity for each individual OT compound exposure after 24 h, with TPT being the most toxic compound tested. The individual OT compound concentrations required to induce a 50% toxic effect on the cells (369 ng ml⁻¹ TBT, 1,905 ng ml⁻¹ DBT) did not equate to the concentrations of these contaminants present in the sediment extract that induced a 50% effect on the cells (294 ng ml⁻¹ TBT, 109 ng ml⁻¹ DBT). The solvent extract therefore exhibited a greater toxicity, and this suggests that the toxic effects observed were not due to OT compounds alone. The presence of other contaminants in the solvent extract is confirmed with chemical analysis, warranting further toxicity testing of contaminant mixtures and exposure to the cell line to further elucidate a complete toxicity evaluation.