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Quality control in microscopic meat and bone meal analysisJ. Vancutsem and M. Lekens (FLVVT, Tervuren)

Contrarily to classical chemical analysis by means of HPLC or GC, the introduction of a satisfactory quality con-trol of a series of analyses is not obvious in the case of microscopic analysis. As a matter of fact, a microscopic determination is a once-only interpretation of a microscopic section by a microscopy expert. So, in addition to a well-trained expert, the quality and representativeness of the slide are extremely important factors that must be taken into account when performing the analyses. Furthermore, efforts should be made to achieve maximum control over these factors. Therefore, when introducing quality control of microscopic analyses, the FLVVT focused on avoiding cross contamination during the preparation of the microscopic slide. During the analysis as such, contamination of the sample to be analysed should also be strictly avoided. And finally, the continuous training and inspection of analysts deserves great attention as well.

Cross contamination

The risk of cross contamination of the analysis sample appears as soon as the sample enters the preparation room of the lab, where a multitude of matrices are being ground. Some of these matrices are composed of 100% meat and bone meal, e.g. the samples that must undergo GTH analysis. On the other hand, feedstuffs for bovine ani-mals must not contain any meat and bone meal at all, meaning that specific measures should be taken in order to avoid cross contamination. In addition, the EURL-AP found that levels of 0,0025% of meat and bone meal can be satisfactorily detected by laboratories. This means that for a sample amount of 500 g (according to Annex I of Regulation 152/2009), a transfer of 10 mg may already yield a false positive result. This point of attention is not uni-que : similar findings are made with respect to the analysis of medicated feed followed by the analysis of residues of these substances in feed, for which similar measures must be taken. The FLVVT developed a series of measures to avoid cross contamination during sample preparation. The samples intended for meat and bone meal analysis are ground separately from other samples and the order of working operations is mentioned on the work chart. Moreover, a blank load of maize should be used after every sample in order to remove any possible sample resi-dues (see photograph 1).

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Photograph 1: Grinding device for samples for the analysis of meat and bone meal

Extra attention to avoid cross contamination is also required during the analysis itself. During sieve fraction analy-sis, the samples should be passed through a 0.25 mm sieve. It is possible that a sample containing e.g. authorised fish meal, is sieved prior to a feed for bovine animals in which the presence of fish meal is not allowed. Therefore, the order in which the samples are sieved must be documented in a chart so that any possible contamination can be traced back. Cleaning the sieves is also a critical step, which is why legal provisions have been made with respect thereto. Regulation (EC) No 51/2013 says : “Sieves shall be cleaned by using a brush with stiff synthetic hairs.

A final cleaning of sieves with acetone and compressed air is recommended after sieving of fatty material like fishmeal.” It is important that a brush with synthetic hairs is used, since e.g. a brush with pig’s bristles may cause contamina-tion when some bristles come off which may lead to a false positive result.This regulation also gives a detailed description of how to clean separation funnels used for sedimentation: “Separation funnel pieces shall be disassembled before cleaning. Separation funnel pieces and glassware shall be pre-

washed manually and then washed in a washing machine.”

Another measure consists in monitoring environment (air) contamination. Materials of animal origin may be scattered into the air as dust particles when used either as analysis sample or reference material. The occurrence of contamination during the analysis may be checked by placing Petri dishes on the working surface and testing them for the presence of products of animal origin at regular intervals.

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Control samples

By taking measures to avoid cross contamination during sample preparation, the FLVVT chose not to include the control sample in the preparation step, but only in the analysis as such. Given the amendment in Regulation (EC) No 51/2013, according to which the method for determining meat and bone meal is now a purely qualitative method, the control sample is used for checking if the sedimentation was performed in a correct manner. The weight of the sediment is mentioned on the control chart and evaluated on the basis of statistical criteria (such as Nordtest TR 569, NEN 6603).In addition, a sample used as reference sample for the microscopic analysis is included as well. For this purpose, the sediment of a spiked sample of fishmeal containing e.g. 1% of meat and bone meal accompanies each series of analyses. The analyst must be able to make the distinction between meat and bone meal and fishmeal in this sample (see photograph 2).

Photograph 2 : Microscopic analysis of meat and bone meal

Bibliography:

P. Veys, G. Berben and V. Baeten. (2010). CRL-AP Proficiency Test 2009 (32 p)

COMMISSION REGULATION (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed

COMMISSION REGULATION (EU) No 51/2013 of 16 January 2013 amending Regulation (EC) No 152/2009 as re-gards the methods of analysis for the determination of constituents of animal origin for the official control of feed

H. Hovind, B. Magnusson, M. Krysell, U. Lund and I. Mäkinen (2011). Nordtest rapport TR 569 Internal quality con-trol. Handbook for chemical laboratories

NEN 6603: Environment and food – Internal quality control by the use of control charts with chemical and micro-biological analyses)

jeroen.vancutsem@favv.be