Intra- and inter-laboratory validation of a dipstick immunoassay for the detection of tropane alkaloids hyoscyamine and scopolamine in animal feed

<div><p>Tropane alkaloids (TAs) are toxic secondary metabolites produced by plants of, <i>inter alia</i>, the genera <i>Datura</i> (thorn apple) and <i>Atropa</i> (deadly nightshade). The most relevant TAs are (–)-L-hyoscyamine and (–)-L-scopolamine, which act as antagonists of acetylcholine muscarinic receptors and can induce a variety of distinct toxic syndromes in mammals (anti-cholinergic poisoning). The European Union has regulated the presence of seeds of <i>Datura</i> sp. in animal feeds, specifying that the content should not exceed 1000 mg kg^–1 (Directive 2002/32/EC). For materials that have not been ground, visual screening methods are often used to comply with these regulations, but these cannot be used for ground materials and compound feeds. Immunological assays, preferably in dipstick format, can be a simple and cost-effective approach to monitor feedstuffs in an HACCP setting in control laboratories. So far no reports have been published on immunoassays that are capable of detecting both hyoscyamine and scopolamine with equal sensitivity and that can be used, preferably in dipstick format, for application as a fast screening tool in feed analysis. This study presents the results obtained for the in-house and inter-laboratory validation of a dipstick immunoassay for the detection of hyoscyamine and scopolamine in animal feed. The target level was set at 800 µg kg^–1 for the sum of both alkaloids. By using a representative set of compound feeds during validation and a robust study design, a reliable impression of the relevant characteristics of the assay could be obtained. The dipstick test displayed similar sensitivity towards the two alkaloids and it could be concluded that the test has a very low probability of producing a false-positive result at blank level or a false-negative result at target level. The assay can be used for monitoring of TAs in feedstuffs, but has also potential as a quick screening tool in food- or feed-related poisonings.</p></div

Polar pesticides in food of animal origin: interlaboratory validation to evaluate method fitness-for-purpose of official control

The present work reports on the design, execution and evaluation of results of an interlaboratory validation study aimed at verifying the fitness-for-purpose of a LC-MS/MS method for the detection of polar pesticides in food of animal origin in official control and monitoring programmes. To this scope, five participant laboratories, with relevant expertise, were recruited. After passing a pre-trial test, the participants were asked to analyse test samples of bovine fat, chicken eggs and cow’s milk, contaminated with 11 polar pesticides (group A: Aminomethyl phosphonic acid (AMPA), cyanuric acid, ethephon, glyphosate, fosetyl aluminium, 2-hydroxyethyphosphonic acid (HEPA), maleic hydrazide, N-acetyl-glyphosate, group B: N-acetyl glufosinate (NAG), 3-methylphosphinicopropionic acid (MPP) and glufosinate ammonium) at two different levels (0.05 and 0.25 mg/kg−1 and 0.01 and 0.05 mg/kg−1 for group A and B respectively. The method was based on acidified methanol/water extraction followed by dSPE clean up with C18 sorbent. For LC-MS/MS analysis isotopically labelled standards were used for all targeted analytes. With a couple of exceptions, average recoveries ranged from 85% to 110%, with repeatability (RSDr) ranging from 3% to 25%, and reproducibility (RSDR) from 4% to 26%. The assessment by different laboratories provided also insights on key factors impacting method performance characteristics and its implementation by new users.</p

Determination of urea in pet feed: assessing the suitability of different analytical techniques using proficiency test data

The determination of urea in pet feed at contaminant levels using the spectrophotometric method described in Commission Regulation (EC) No 152/2009 has been reported by several EU laboratories to lack the required selectivity. Whilst urea is not authorised as an additive in pet feed, the control of urea in pet feed is of economic importance, because the addition of urea may unlawfully increase the apparent protein content. To investigate the capabilities of different analytical techniques, a proficiency test was organised where the participants (EU official control laboratories, laboratories from the academia and private laboratories) were free to use their method of choice for analysing three dog feed test materials, two samples of which were spiked with urea. Twenty-one laboratories submitted results using the following techniques: spectrophotometry (Implementing Regulation (EC) No 152/2009), LC-MS/MS, HPLC-UV, enzymatic-colorimetry, gravimetry and an ‘in-house photometric’ method. Only two laboratories that used LC-MS/MS were able to quantify urea accurately in the test material containing a mass fraction of 18.9 mg kg−1 whereas satisfactory results at the level of 258.9 mg kg−1 were obtained by one participant that used an ‘in-house photometric method’ and one that used the enzymatic method, in addition to the five participants using LC-MS/MS. The technique that provided the highest success rate across the three test materials was LC-MS/MS, whereas spectrophotometry, the enzymatic-based and HPLC-UV methods led to overestimated results in addition to a dispersion of results not suitable for compliance analysis. To address the determination of urea in pet feed at low levels, a better performing method than the one described in the legislation is required.</p