Q U A L I T Y I N N O V A T I O N S E R V I C E

Transcription

1 Q U A L I T Y I N N O V A T I O N S E R V I C E Sample preparation challenges for determination of ochratoxin A in wheat Canadian Grain Commission, 2005 Tom Nowicki & Mike Roscoe Grain Research Laboratory Canadian Grain Commission Acknowledgements: GRL Trace Organic Analysis Unit Linda Wicklow Carla Cohen Dainna Drul GRL Management Team Eugene Gawalko Peter Burnett Dave Hatcher CGC Industry Services Randy Dennis Jim Stuart CGC Corporate Services Anh Phan Kevin Morgan Canadian Wheat Board Gord Kurbis Lawrence Klusa

2 Analytical Testing Process lot sample sample preparation chemical analysis Analytical Testing Process sample preparation Primary sample lot sample Laboratory sample Test portion chemical analysis Includes all processes to which the sample is subjected in the Interval between when the primary sample is generated and extraction of a test portion.

3 What is Ochratoxin A? Ochratoxin A is a mycotoxin Commonly referred to as OTA - toxic by-product of a fungi - natural toxin - First isolated and identified in mid-1960 s Ochratoxin A Isocoumarin moiety linked by a peptide bond to phenylalanine MW: C20H18ClNO6 Dechlorinated analogue: - ochratoxin B Soluble in: - polar organic solvents - dilute sodium bicarbonate - water (slightly) Blue/green fluorescence under UV 360 nm

4 Ochratoxin A Global contaminant in: cereals coffee vine fruits cocoa beans Cereals and cereal products are the main contributors to dietary intake. Also detected in: wine beer spices nuts animal products Ochratoxin A OTA production in grains Produced by Penicillium verrucosum (temperate climates) Formed mainly during storage Conditions for OTA production Moisture content >18% Minimum temp 3 ºC Optimum conditions: MC 24% Temp 20 ºC

5 Ochratoxin A Maximum Limits for OTA in Cereal Grains Maximum Limit Country / Agency / Region Year mg/kg (ppb) Canada not established Codex (proposed) EU 2002 / / 5.0 Other countries Ochratoxin A Nature of OTA contamination in bulk wheat shipments Creates many challenges for accurate and precise measurement

6 Ochratoxin A Nature of OTA contamination in bulk wheat shipments In a contaminated lot, the proportion of contaminated kernels is very low. Contaminated kernels are distributed heterogeneous in bulk grain. NUGGET EFFECT MAJOR IMPLICATIONS FOR SAMPLING AND SUBSAMPLING Ochratoxin A Nature of OTA contamination in bulk wheat shipments The NUGGETS have a wide range in OTA concentration: > GRL has observed levels up to 4990 ppb > Mean concentration 1060 ppb Additional 5 ppb OTA complication: = 167 contaminated kernels / kg = 0.5% contamination OTA may be present in dust/dockage as well as contaminated kernels.

7 Determination of OTA in wheat GRL methods Fluorometer Homogenize with MeOH+H2O (80+20,v/v) sample+solvent (1+4, w/v) Dilute with PBS Filter IAC cleanup Eluents: : PBS then H2O [discard] Then 0.1M NaOH [collect] Read on fluorometer LC - FL Shake with acidified CHCl3+celite sample+solvent (1+5.8,w/v) Centrifuge Filter organic layer Concentrate then reconstitute Silica Sep-Pak cleanup Eluents: : DCM then hexane then toluene [discard] then toluene+ Acetic acid (9+1,v,v) [collect] Concentrate then reconstitute Centrifuge then filter LC-FL (C18 with H2O+HCN+MeOH) Analytical variance Trace analysis Analytical testing process sampling (s) sample preparation (sp) chemical analysis (ca) Total Variance = Variance_s + Variance_sp + Variance_ca

8 Analytical variance Trace analysis Analytical testing process For measurements in the low ppb range, sample preparation can be a significant contributor to total variance. sampling (s) sample preparation (sp) chemical analysis (ca) Total Variance = Variance_s + Variance_sp + Variance_ca Analytical variance Trace analysis Lab sample preparation Primary sample mix Analytical testing process sampling (s) divide/subsample Laboratory sample sample preparation (sp) Test portion chemical analysis (ca) Total Variance = Variance_s + Variance_sp + Variance_ca

9 Analytical variance Trace analysis Primary sample Analytical testing process Test portion preparation Laboratory sample grind mix divide/subsample Test portion sampling (s) sample preparation (sp) chemical analysis (ca) Total Variance = Variance_s + Variance_sp + Variance_ca Analytical variance Trace analysis Primary sample Analytical testing process sampling (s) Lab sample (ls) preparation sample preparation (sp) Test portion (tp) preparation chemical analysis (ca) Total Variance Variance_sp = = + (Variance_ls ( ls + + Variance_tp) tp + Variance_ca

10 Determination of OTA in bulk shipments Operational experiences Variance_sp = Variance_ls + Variance_tp Determination of OTA in bulk shipments Operational experiences Repeatability of OTA results for multiple lab samples % RSD y = e -0.32x R 2 = ppb OTA

11 Determination of OTA in bulk shipments Operational experiences Repeatability of OTA results for multiple test portions % RSD ppb OTA Determination of OTA in bulk shipments Operational experiences Conclusions: 1. Variances associated with our current procedures for preparation of lab samples are too high. 2. Variances associated with our current procedures for preparation of test portions are too high. Variance_sp = Variance_ls + Variance_tp

12 Determination of OTA in bulk shipments Operational experiences Questions raised centered around: 1. Lab sample weight? 2. What grinder should be used? 3. Mixing procedure for the ground grain? 4. Subsampling technique? 5. Test portion weight? 6. Number of test portions? Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) Grind on LM3100 Mill 20 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg Test portions Grind on Romer Mill 20 kg wheat Mix Subsample Ten 2 kg portions (lab samples) Grind 5 on Romer mill 5 on LM3100 mill Mix ground wheat Subsample test portions (20 per lab sample) Analyze test portion for OTA (IA-FL method)

13 Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) Grind on LM3100 Mill 20 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg Test portions Grind on Romer Mill Note: The the total weight of OTA in any given lab sample is the same whether it is ground on a Romer Mill or a LM3rinder. OTA recovery from ground wheat is not likely much different for samples ground on the Romer and LM3rinders. Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) Grind on LM3100 Mill 20 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg Test portions Grind on Romer Mill Therefore: For each primary sample, we can combine the results for the two grinders to calculate the average mean OTA level in the 20 kg sample and the variability of OTA concentrations in the 10 lab samples.

14 Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) Grind on LM3100 Mill 20 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg Test portions Grind on Romer Mill ppb OTA Mean of 20 Lab Test Portions Sple# PS#1 PS# Avg. Mean %RSD Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) Lab sample preparation Lab samples: 2 kg % R S D Relationship between ppb OTA and %RSD PS# 2 PS# OTA, ppb %RSD = 66% at 5.0 ppb ppb OTA Mean of 20 Lab Test Portions Sple# PS#1 PS# Avg. Mean %RSD

15 Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) Lab sample preparation Lab samples: % of weight of primary sple. Relationship between relative lab sample weight and %RSD %RSD R2 = 0.99 R2 = 0.99 Mean OTA 2.3 ppb Mean OTA 3.7 ppb Lab Sample Weight (% of primary sample) ppb OTA Mean of 20 Lab Test Portions Sple# PS#1 PS# Avg. Mean %RSD Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) Lab sample preparation Lab samples: % of weight of primary sple. %RSD Relationship between %RSD and ppb OTA for lab sample weights of 2 to 10 kg kg kg kg 75 8 kg kg Average mean ppb OTA in 20 kg Lot ppb OTA Mean of 20 Lab Test Portions Sple# PS#1 PS# Avg. Mean %RSD

16 Determination of OTA in 2 kg ground wheat Subsampling study Two primary samples (PS) Test portion preparation Determination of OTA in 2 kg ground wheat Subsampling study Two primary samples (PS) Grind on LM3100 Mill 20 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg Test portions Grind on Romer Mill PS#1 ppb % Lab sple# OTA RSD Mean

17 Determination of OTA in 2 kg ground wheat Subsampling study Two primary samples (PS) Grind on LM3100 Mill 20 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg 2 kg Test portions Grind on Romer Mill PS#2 ppb % Lab sple# OTA RSD Mean Determination of OTA in 2 kg ground wheat Subsampling study Two primary samples (PS) Test portion preparation Relationship between ppb OTA and %RSD 120 Romer Mill LM3rinder 100 Romer Mill %RSD OTA, ppb LM3rinder

18 Determination of OTA in 20 kg samples of wheat Subsampling study Two primary samples (PS) %RSD Test portion preparation Relationship between %RSD and ppb OTA for analysis of 1 to 5 test portions ( each) single test portion 2 test portions 3 test portions 4 test portions 5 test portions Average mean ppb OTA LM3rinder Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Sample preparation Primary sample Laboratory sample Test portion Variance_sp = Variance_ls + Variance_tp

19 Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Laboratory sample preparation Primary sample Relative weight of lab sample Variance_sp = Variance_ls + Variance_tp Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Test portion preparation Laboratory sample Variance_sp = Variance_ls + Variance_tp

20 Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Grinder / particle size Test portion preparation Laboratory sample Variance_sp = Variance_ls + Variance_tp Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Grinder / particle size Test portion preparation Mixing Laboratory sample Variance_sp = Variance_ls + Variance_tp

21 Determination of OTA in bulk shipments Additional study Key factors affecting accuracy and repeatability of OTA results: Grinder / particle size Test portion preparation Mixing study %RSD vs mixing time Mixing Laboratory sample Variance_sp = Variance_ls + Variance_tp Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Grinder / particle size Test portion preparation Mixing Laboratory sample Subsampling technique Variance_sp = Variance_ls + Variance_tp

22 Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Grinder / particle size Test portion preparation Mixing Subsampling technique Laboratory sample Relative weight of test portion(s) Variance_sp = Variance_ls + Variance_tp Determination of OTA in bulk shipments What have we learned? Key factors affecting accuracy and repeatability of OTA results: Mixing Subsampling technique Grinder / particle size Laboratory sample Relative weight of test portion(s) Test portion preparation Number of test portions Variance_sp = Variance_ls + Variance_tp

23 Certifying cargo shipments for OTA Proposed approach to sample preparation 10 kg laboratory sample (1/2-1/3 split of official composite sample). extended mixing and more meticulous subsampling. test portion. Cargo composite Sample Subsample to produce 10 kg laboratory sample Grind entire 10 kg Mix Subsample 1 kg Extract entire 1 kg Analyze test portions Total Variance = Variance_s + (Variance_ls + Variance_tp) + Variance_ca Certifying cargo shipments for OTA Another possibility 10 kg laboratory sample (1/2-1/3 split of official composite sample) 1 kg test portion Cargo composite Sample Subsample to produce 10 kg laboratory sample Grind entire 10 kg Mix Subsample 1 kg Extract entire 1 kg Analyze test portions Total Variance = Variance_s + (Variance_ls( + Variance_tp) + Variance_ca

24 Conclusion Analytical testing process sampling (s) sample preparation (sp) chemical analysis (ca) Total Variance = Variance_s + Variance_sp + Variance_ca

25 grainscanada.gc.ca Canadian Grain Commission, 2005