Comparison of Automatic and Manual sampling for ochratoxin A in Barley.
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1 Comparison of Automatic and Manual sampling for ochratoxin A in Barley. Gunnar Andersson 1, Elisabeth Viktoria Reiter 2, Ebrahim Razzazi Fazeli 2, Per-Anders Lindqvist 3, Per Häggblom 1 1 National Veterinary Institute Sweden 2 Veterinary University of Vienna - Austria 3 Svenska Foder - Sweden
2 Biotracer IP (FP6) Improved biotraceability of unintended micro-organisms and their substances in food and feed chains Feed chain : Mycotoxins and Salmonella Jan 2007 Dec partners - 24 countries - Research Institutes - Universities -SME s
3 Mycotoxin work in Biotracer (feed) Quality control of sampling Evaluation of Eurachem/CITAC guidelines. Comparison of automatic and manual sampling for ochratoxin A in barley Aim: Simplified sampling procedures and better decision making by automatic sampling. Impact of sampling method on total measurement uncertainty. Impact of sample mass and sample preparation methods.
4 Distribution of ochratoxin A in whole grain consignment Current knowledge about distribution Storage mycotoxin -> stratification likely to occur Contamination may be localized into few grains
5 Ochratoxin A in Barley manual v.s. automatic sampling Sampling plan design OTA Monitoring Preparation and analysis Target preparation End-point sampling ANOVA
6 Justification for ANOVA Necessary condition for ANOVA: Toxin concentration in repeated samples from same batch should follow normal distribution. - Not valid for individual incremental samples. - Average concentrations in sufficiently large aggregate samples follow normal distribution (Central limit theorem) - Empirical data (Biselli et al) and samplingtheory used to design sampling plan (4-5 kg aggregate sample sufficient)
7 Preparation of the sampling target (Svenska Foder, Hällekis, Sweden) - Selection of barley with right moisture content - Penicillium verrucosum inocculation - Incubation at ambient temperature & monitoring
8 Monitoring mycotoxin formation Temp. Ochratoxin A
9 Manual sampling Eight alternative sampling patterns of five hits One aggregate sample = 10 kg barley
10 The automatic sampler 8*4,5 kg collected in 1h (max frequency) >100 increments/aggregate sample
11 Automatic sampling Eight aggregate samples from interpenetrating sampling. Incr#18 Incr#17 Incr#16 Incr#15 Incr#14 Incr#13 Incr#12 Incr#11 Incr#10 Incr#9 Incr#8 Incr#7 Incr#6 Incr#5 Incr#4 Incr#3 Incr#2 Incr#1 etc.. #3 #2 #1
12 Sample preparation & analysis Reduction to 4.5 kg, riffle splitter Coarse grinding in RAS-mill Reduction to, riffle splitter Fine milling Forming analytic sample (fractional showelling) Extraction - cleanup - detection
13 Experimental design The duplicate method Manual sampling (8 aggregate samples) Automatic sampling (8 aggregate samples) Reduction RAS-mill Sub sample 1 Mill Sub sample 2, test#1a1 test#1aa sub#1a 4.5kg sub#1a 4.5kg test#1a2 test#1ab Aggregate# 1 (~10kg) test#1b1 test#1ba sub#1b 4.5kg sub#1b 4.5kg test#1b2 test#1bb Aggregate#1 (~4.5kg) test#1a1 test#1aa sub#1a 4.5kg sub#1a 4.5kg test#1a2 test#1ab #1aaa #1aab #1aba #1abb #1baa #1bab #1bba #1bbb #1aaa #1aab #1aba #1abb In total 12*8 = 96 analyses
14 Uncertainty from different sources (at p=0,05) * * *Normal assumption not valid. Concentration range among 8 bulk samples, 2-80 ppb
15 Conclusions - Very large uncertainty from manual sampling used (5 hit pattern) - A 4,5 kg aggregate-sample is sufficient for Ochratoxin A in grain - Error from automatic sampling is in the same range as sample reduction error. - Sample preparation and sub-sampling may introduce large errors. (>40% at p=0.05) - Presumably due to particle segregation - Validated methods essential
16 Thank You for your attention! Gunnar Andersson PhD National Veterinary Institute (SVA) Uppsala, Sweden
17 Duplikatmetoden Standardmetod för QC av provtagning Rekommenderas av CAC, Eurachem, Nordtest Råvaruparti >= 8 partier Samlingsprov1 Samplingsprov 2 Analys 1 Analys 2 Analys 1 Analys 2 Analyseras med variansanalys ANOVA Prov1 Prov2 -> Uppskatta stickprovs osäkerhet Analys 1 Analys 2 -> Uppskatta analytisk osäkerhet mätosäkerhet = stickprovs osäkerhet+ analytisk osäkerhet
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