Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry

Ractopamine (RCT) is a member of the beta-2-agonist (beta-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The People's Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A.

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 (BC) 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, BC, 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 BC 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. (C) 2011 Elsevier B.V. All rights reserved.

UTILISATION OF MOLLUSC RESOURCES IN N. IRELAND

Mussel beds in N. Ireland contain about 3–400 000 tonnes of molluscs of which 6–7% is edible flesh. Most of these resources are not suitable for direct human consumption and attempts are currently being made at Queen's University, to perfect a process to separate mussel shell and flesh aimed at the production of high grade products from a low grade source. This report deals with the likely consequences of large scale harvesting of mussels and offers possible alternatives.

Development of a rapid, multi-class method for the confirmatory analysis of anti-inflammatory drugs in bovine milk using liquid chromatography tandem mass spectrometry

A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous identification, confirmation and quantitation of seven licensed anti-inflammatory drugs (AIDS) in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. Two classes of AIDS were investigated, corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs). The developed method is capable of detecting and confirming dexamethasone (DXM), betamethasone (BTM), prednisolone (FRED), tolfenamic acid (TV), 5-hydroxy flunixin (5-OH-FLU). meloxicam (MLX) and 4-methyl amino antipyrine (4-MAA) at their associated maximum residue limits (MRLs). These compounds represent all the corticosteroids and NSAIDs licensed for use in bovine animals producing milk for human consumption. These compounds have never been analysed before in the same method and also 4-methyl amino antipyrine has never been analysed with the other licensed NSAIDs. The method can be considered rapid as permits the analysis of up to 30 samples in one day. Milk samples are extracted with acetonitrile; sodium chloride is added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is finally evaporated to dryness and reconstituted in a water/acetonitrile mixture and determination is carried out by LC-MS/MS. Decision limit (CC alpha) values and detection capability (CC beta) values have been established for each compound. (C) 2009 Elsevier B.V. All rights reserved.

Detection of Paralytic Shellfish Toxins by a Solid-Phase Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 µg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Occurrence and Partitioning of Cadmium, Arsenic and Lead in Mine Impacted Paddy Rice: Hunan, China

Paddy rice has been likened to nictiana sp in its ability to scavenge cadmium (Cd) from soil, whereas arsenic (As) accumulation is commonly an order of magnitude higher than in other cereal crops. In areas such as those found in parts of Hunan province in south central China, base-metal mining activities and rice farming coexist. Therefore there is a considerable likelihood that lead (Pb), in addition to Cd and As, will accumulate in rice grown in parts of this region above levels suitable for human consumption. To test this hypothesis, a widespread provincial survey of rice from mine spoilt paddies (n = 100), in addition to a follow-up market grain survey (n = 122) conducted in mine impacted areas was undertaken to determine the safety of local rice supply networks. Furthermore, a specific Cd, As, and Pb biogeochemical survey of paddy soil and rice was conducted within southern China, targeting sites impacted by mining of varying intensities to calibrate rice metal(loid) transfer models and transfer factors that can be used to predict tissue loading. Results revealed a number of highly significant correlations between shoot, husk, bran, and endosperm rice tissue fractions and that rice from mining areas was enriched in Cd, As, and Pb. Sixty-five, 50, and 34% of all the mine-impacted field rice was predicted to fail national food standards for Cd, As, and Pb, respectively. Although, not as elevated as the grains from the mine-impacted field survey, it was demonstrated that metal(loid) tainted rice was entering food supply chains intended for direct human consumption.

Association between tortilla consumption and human urinary fumonisin B1 levels in a Mexican population

Fumonisins are mycotoxins produced by Fusarium spp. and commonly contaminate maize and maize products worldwide. Fumonisins are rodent carcinogens and have been associated with human esophageal cancer. However, the lack of a valid exposure biomarker has hindered both the assessment of human exposure and the evaluation of disease risk. A sensitive liquid chromatography-mass spectrometry method to measure urinary fumonisin B1 (FB1) following extraction on Oasis MAX cartridges was established and applied to urine samples from women in a cohort recruited in Morelos County, Mexico. Urinary FB1 was compared with dietary information on tortilla consumption. FB1 recovery in spiked samples averaged 94% as judged by deuterium-labeled FB1 internal standard. Urinary FB1 was determined in 75 samples from women selected based on low, medium, or high consumption of maize-based tortillas. The geometric mean (95% confidence interval) of urinary FB1 was 35.0 (18.8-65.2), 63.1 (36.8-108.2), and 147.4 (87.6-248.0) pg/mL and the frequency of samples above the detection limit (set at 20 pg FB1/mL urine) was 45%, 80%, and 96% for the low, medium, and high groups, respectively. Women with high intake had a 3-fold higher average FB1 levels compared with the "low intake" group (F = 7.3; P = 0.0015). Urinary FB1 was correlated with maize intake (P-trend = 0.001); the correlation remained significant after adjusting for age, education, and place of residence. This study suggests that measurement of urinary FB1 is sufficiently sensitive for fumonisin exposure assessment in human populations and could be a valuable tool in investigating the associated health effects of exposure.

New insights into the causes of human illness due to consumption of azaspiracid contaminated shellfish

Azaspiracid (AZA) poisoning was unknown until 1995 when shellfish harvested in Ireland caused illness manifesting by vomiting and diarrhoea. Further in vivo/vitro studies showed neurotoxicity linked with AZA exposure. However, the biological target of the toxin which will help explain such potent neurological activity is still unknown. A region of Irish coastline was selected and shellfish were sampled and tested for AZA using mass spectrometry. An outbreak was identified in 2010 and samples collected before and after the contamination episode were compared for their metabolite profile using high resolution mass spectrometry. Twenty eight ions were identified at higher concentration in the contaminated samples. Stringent bioinformatic analysis revealed putative identifications for seven compounds including, glutarylcarnitine, a glutaric acid metabolite. Glutaric acid, the parent compound linked with human neurological manifestations was subjected to toxicological investigations but was found to have no specific effect on the sodium channel (as was the case with AZA). However in combination, glutaric acid (1mM) and azaspiracid (50nM) inhibited the activity of the sodium channel by over 50%. Glutaric acid was subsequently detected in all shellfish employed in the study. For the first time a viable mechanism for how AZA manifests itself as a toxin is presented.

Monoclonal antibody development for acrylamide-adducted human haemoglobin; A biomarker of dietary acrylamide exposure

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered, leading to dietary exposure estimates of 30.8 mu g of acrylamide day(-1) for an average 77 kg human male. This is considerably higher than the European legal limit of acrylamide in drinking water, which is approximately 0.2 mu g of acrylamide person(-1) day(-1). A recent study of 62,573 women over 11.3 years has observed an increased risk of postmenopausal endometrial and ovarian cancer (but not breast cancer) with increasing dietary acrylamide intake, demonstrating significant risk to human health. As individual acrylamide exposure is affected by dietary habits, cooking methods, and cigarette consumption; accurate extrapolation from estimated dietary exposure is extremely difficult. Quantifying biomarkers of acrylamide exposure therefore remains the most effective means of rapidly determining individual exposure to acrylamide, and correlating exposure with lifestyle choices. Current methodologies for the analysis of blood biomarkers of acrylamide are focused on expensive, slower chromatographic techniques such as GC and LC coupled to mass spectrometry. This paper describes the first successful development of two monoclonal antibodies specific to acrylamide-adducted haemoglobin (IC50 of 94 ng ml(-1) and 198 ng ml(-1)), that are suitable for use in a high-throughput biomarker immunoassay to determine individual acrylamide exposure. Further development of acrylamide-haemoglobin standards with defined levels of acrylamide adduction will enable a fully quantitative assay, and allow sensitivity comparisons with alternative chromatographic methods of analysis. (C) 2008 Elsevier B.V. All rights reserved.

Biomarkers of fruit and vegetable intake in Human intervention studies a systematic review

Observational evidence consistently shows that consumption of a diet rich in fruit and vegetables may offer protection against diseases such as cardiovascular disease and cancer. Assessment of dietary intake is complex and prone to many sources of error. More objective biomarkers of fruit and vegetable intake are therefore of interest. The aim of this review is to examine the usefulness of the main biomarkers of fruit and vegetable intake to act as objective indicators of compliance in dietary intervention studies. A comprehensive search of the literature was conducted using six databases. Suitable papers were selected and relevant data extracted. The papers were categorized into 3 sub-groups: whole diet interventions; mixed fruit and vegetable interventions; and studies involving individual varieties of fruits or vegetables. Ninety-six studies were included in the review. Overall, the most commonly measured, and most consistently responsive, biomarkers were the carotenoids and vitamin C. Based on the results of this systematic review, it remains prudent to measure a panel of biomarkers in fruit and vegetable intervention studies. The only possible exception to this is “fruit only” intervention studies where assessment of vitamin C alone may suffice.