VALIDATION REPORT (According to the Commission Regulation (EU) No 519/2014) AFLATOXIN B1 ELISA

Transcription

1 VALIDATION REPORT (According to the Commission Regulation (EU) No 519/2014) AFLATOXIN B1 ELISA Beijerinckweg 18 NL-6827 BN Arnhem The Netherlands Phone: info@europroxima.com

2 1. Introduction Aflatoxins (Aflatoxins B1, B2, G1, G2) are a group of structurally related, extremely toxic compounds produced by the Aspergillus moulds. Within the EU, maximum levels (MLs) for a number of mycotoxins have been set by Commission Regulation (EC) 1881/2006 [1]. Concerning the Aflatoxins, this Regulation was amended by the Commission Regulation (EC) No 165/2010 [2]. In this amending Regulation, changes in the MLs were made to align with Codex Alimentarius FAO/WHO standards, and new groups of foodstuffs (oilseeds and apricot kernels) were specified. The MLs for Aflatoxin B1 in most of the foodstuffs for direct human consumption vary from 2 to 12 μg/kg (ppb). For processed cereal based foods and baby foods for infants and young children as well as for dietary foods for special medical purposes intended specifically for infants, the ML for Aflatoxin B1 is set at 0.1 μg/kg (0.1 ppb). EuroProxima has developed and validated the Aflatoxin B1 sensitive ELISA (S) especially for this low measuring ranges in a number of different matrices. This report presents the validation study results for nuts, cereals and dried fruits. The new validation approach was used that complies with the Commission Regulation 519/2014 [3] for the validation of screening methods for mycotoxins. This new Regulation is specifically intended for bioanalytical methods such as ELISA and LFD. The concept of screening target concentration (STC) is used. STC is the concentration of interest for detection of the mycotoxin. For the purpose of this validation the STC was set to 1 ppb what is half of the lowest maximum limit for aflatoxin B1. The aim of the validation was to demonstrate the fitness of purpose of the aflatoxin B1 ELISA as a screening method for the detection of aflatoxin B1 at the level 1 ppb and higher in nuts, cereals and dried fruits. The Regulation requires to determine 2 parameters: cut-off and false suspect rate. Cut-off is the concentration measured in a samples above which the sample is classified as suspect, meaning it may contain mycotoxin at a level higher than STC. Cut-off is calculated from the results obtained for 20 samples spiked at STC by using equation given in the Regulation. Another parameter that needs to be determined in the validation study is false suspect rate. This is calculated based on the results obtained for 20 blank samples and 20 samples spiked at STC. The false suspect rate gives an estimation how often the method will generate false suspect result. The 5 % false suspect rate means that 1sample in 20 can be wrongly classified as suspect. The lower the false suspect rate the better, as it means that lower number of false suspect samples will require analysis by confirmatory method. As also advised by the Regulation, the samples should be spiked and analysed at other levels in order to determine how the method can distinguish between different mycotoxin concentrations. For the purpose of this validation the STC was set at 1 ppb and cut-off and false suspect rate were determined in nuts, cereals and dried fruits. The samples were also spiked at 2 and 4 ppb and analysed to determine the method recovery at different levels. Additionally (not required by the Commission Regulation 519/2014), detection limit (LOD) was calculated based on 20 blank samples for each matrix. 2. Kit characteristics: see manual [18]03.15 The reactivity pattern of the antibody. Cross- reactions: Aflatoxin B1 100% Aflatoxin B2 20% Aflatoxin G1 17% Aflatoxin G2 4% 2

3 3. Scope of validation The validation study was carried out with the Aflatoxin B1 ELISA (). The study included: - Determination of limit of detection (LOD). - Determination of the cut-off and false suspect rate for nuts including peanuts, cereals and dried fruit at screening target concentration (STC) 1 ppb = 0.5 ML. - Determination of the % recovery for nuts including peanuts, cereals and dried fruit spiked with 1, 2 and 4 ppb of Aflatoxin B1. 4. Sample material Non-contaminated nuts: cashew, pistachio, hazelnut, almonds, brazil nuts and peanuts. Non-contaminated cereals: oats, buckwheat, wheat, quinoa, barley and spelt. Non-contaminated dried fruit: apricots, figs, raisins, papaya, dates and currants. 5. Sample treatment Sample preparation was performed according to the manual [18]03.15, chapter Results For determination of cut-off and recovery, samples were spiked with Aflatoxin B1, LGC standardl15331b. Table 1. LOD determined for different matrices. Matrices Nuts* Peanuts Cereals** Spelt Dried LOD n=20 [ppb] LOD: mean of 20 blanks + 3 x SD Table 2. Results of the validation study according to the Commission Regulation (EU) No 519/2014. Matrices Nuts* Peanuts Cereals** Spelt Dried STC [ppb] Cut-off n=20 [ppb] False suspect rate <0.01% <0.01% <0.01% <0.01% <0.01% <0.01% STC: screening target concentration, concentration of interest for detection of aflatoxin B1 in a sample, set to 1 ppb = 0.5 ML Cut-off: the concentration above which samples is classified as suspect, cut-off = mean of spiked samples x SD (at STC) False suspect rate: The t-value corresponding to the event that the result of a negative samples is above the cut-off is calculated from the following equation: t-value = (cut-off- mean blank )/SD blank Then the probability of false suspect rate is calculated from the obtained t-value. 3

4 *n=20: The combination of the 20 samples was as follows cashew (n=4), pistachio (n=4), hazelnut (n=4), almonds (n=4), brazil nuts (n=4). **n=20: The combination of the 20 samples was as follows oats (n=4), buckwheat (n=4), wheat (n=4), quinoa (n=4),barley (n=4). ***n=20: The combination of the 20 samples was as follows apricots (n=4), figs (n=4), raisins (n=4), papaya (n=4), dates (n=4). 4

5 Table 3. Recovery [%] determined for samples spiked at 1, 2 and 4 ppb. Matrices n=20 Nuts* Peanuts Cereals** Spelt Dried Spiked at 1 ppb Spiked at 2 ppb Spiked at 4 ppb *n=20: The combination of the 20 samples was as follows cashew (n=4), pistachio (n=4), hazelnut (n=4), almonds (n=4), brazil nuts (n=4). **n=20: The combination of the 20 samples was as follows oats (n=4), buckwheat (n=4), wheat (n=4), quinoa (n=4),barley (n=4). ***n=20: The combination of the 20 samples was as follows apricots (n=4), figs (n=4), raisins (n=4), papaya (n=4), dates (n=4). 7. Literature 1. Commission Regulation (EC) No 1881/2006 of 19 December 2006 settingmaximum levels for certain contaminants in foodstuffs. Off. J. European Union.L364 (2006) Commission Regulation (EU) No 165/2010 of 26 February 2010 amendingregulation (EC) No 1881/2006 setting maximum levels for certain contaminantsin foodstuffs as regards aflatoxins. Off. J. European Union. L50 (2010) Commission Regulation (EU) No 519/2014 of 16 May 2014 amending Regulation (EC) No 401/2006 as regards methods of sampling of large lots, spices and food supplements, performance criteria for T-2, HT-2 toxin and citrinin and screening methods of analysis. 5