Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

Evaluation of an alternative spectroscopic approach for aflatoxin analysis: Comparative analysis of food and feed samples with UPLC-MS/MS

Increasing research has highlighted the effects of changing climates on the occurrence and prevalence of toxigenic Aspergillus species producing aflatoxins. There is concern of the toxicological effects to human health and animal productivity following acute and chronic exposure that may affect the future ability to provide safe and sufficient food globally. Considerable research has focused on the detection of these toxins, based on the physicochemical and biochemical properties of the aflatoxin compounds, in agricultural products for human and animal consumption. As improvements in food security continue more regulations for acceptable levels of aflatoxins have arisen globally; the most stringent in Europe. These regulations are important for developing countries as aflatoxin occurrence is high significantly effecting international trade and the economy. In developed countries analytical approaches have become highly sophisticated, capable of attaining results with high precision and accuracy, suitable for regulatory laboratories. Regrettably, many countries that are affected by aflatoxin contamination do not have resources for high tech HPLC and MS instrumentation and require more affordable, yet robust equally accurate alternatives that may be used by producers, processors and traders in emerging economies. It is especially important that those companies wishing to exploit the opportunities offered by lucrative but highly regulated markets in the developed world, have access to analytical methods that will ensure that their exports meet their customers quality and safety requirements.

This work evaluates the ToxiMet system as an alternative approach to UPLC–MS/MS for the detection and determination of aflatoxins relative to current European regulatory standards. Four commodities: rice grain, maize cracked and flour, peanut paste and dried distillers grains were analysed for natural aflatoxin contamination. For B1 and total aflatoxins determination the qualitative correlation, above or below the regulatory limit, was good for all commodities with the exception of the dried distillers grain samples for B1 for which no calibration existed. For B1 the quantitative R² correlations were 0.92, 0.92, 0.88 (<250 μg/kg) and 0.7 for rice, maize, peanuts and dried distillers grain samples respectively whereas for total aflatoxins the quantitative correlation was 0.92, 0.94, 0.88 and 0.91. The ToxiMet system could be used as an alternative for aflatoxin analysis for current legislation but some consideration should be given to aflatoxin M1 regulatory levels for these commodities considering the high levels detected in this study especially for maize and peanuts

Identification of an unknown b-agonist in feed by liquid chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and -agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new -agonist in a feed extract.

Mis-measure of animal contests.

Contests between rivals placing similar value on the resource at stake are commonly won by the rival having greater 'resource holding potential' (RHP). Mutual assessment of RHP difference between rivals is usually expected as an economical means of resolution; weaker rivals can retreat when they detect their relative inferiority, thereby avoiding costly, futile persistence. Models of contest resolution that entail retreat decisions based on estimates of RHP difference predict that contest duration diminishes as RHP difference between rivals increases because the asymmetry is more readily detected. This prediction appears to have been fulfilled in contests of diverse taxa, generating widespread support for assessment of RHP differences in contests. But few studies have considered alternatives in which each rival simply persists in accord with its own RHP ('own RHP-dependent persistence'). In contests decided by own RHP-dependent persistence, in which costs accrue only through each rival's own actions, weaker rivals retreat first because they are inherently less persistent, and contest duration depends primarily on the weaker (losing) rival's RHP rather than RHP difference between the rivals. We show here that the analyses most commonly used to detect effects of RHP difference cannot discriminate between these alternatives. Because RHP difference between rivals tends to be correlated with RHP of the weaker rival in a pair, a negative relation between RHP difference and contest duration may be generated even when decisions of retreat are not based on estimated RHP difference. Many studies purporting to show a negative relation between RHP difference and contest duration may actually reflect an incidental association between weaker rival RHP and RHP difference. We suggest statistical and experimental approaches that may help to discriminate between effects of weaker rival RHP and true effects of RHP difference. We also discuss whether 'true' negative effects of RHP difference on contest duration always reflect retreat decisions based on estimated RHP differences. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.