Sampling, sample preparation and analyte isolation

H 3C COOH O CH3 OH OH HOOC O CH3 HOOC O CH 3 OH NH 2 COOH O Sampling, sample preparation and analyte isolation Hans van Egmond, with assistence of Tom Whitaker, Ton van Osenbruggen & John Gilbert Beijing, 12 December 2006 1 SELAMAT China Maize load: Fumonisin = x Fumonisin tolerance? Research Quality control Regulatory Export/Import 2 1

What do we do with this shipload? Official (AOAC, CEN): HPLC/GC, LC/MS, ELISA, FL, TLC sample laboratory analysis 3 Techniques used in mycotoxin regulatory analysis LC GC/MS GC ELISA fluorometry TLC minicolumn frequency of techniques 4 2

What do we do with this shipload? sample laboratory analysis Official (AOAC, CEN): HPLC/GC, LC/MS, ELISA, FL, TLC rapid: ELISA, dipstick, sensor, lateral flow Management decision - valid results 5 Is this shipload okay? The fumonisin concentration in the lot cannot be determined with 100% certainty by taking samples from the lot Uncertainty is due to variablity among sample test results 6 3

Outline of presentation Introduction Variability associated with sampling, sample preparation and analysis Analyte isolation and clean-up Focus on immunoaffinity cleanup Summary 7 Outline of presentation Introduction Variability associated with sampling, sample preparation and analysis Analyte isolation and clean-up Focus on immunoaffinity cleanup Summary 8 4

Lot Avg.= 3.6 mg/kg 0 0 0 1 2 3 3 5 7 15 9 GOOD BAD ACCEPT REJECT x (Seller s Risk) ACCEPT x (Buyer s Risk) REJECT 0 Tolerance mg/kg 10 5

The perfect Operating Characteristic curve Lots Accepted (%) 10 0 Good Lots Bad Lots 0 Tolerance Lot Concentration High 11 100 False Pos. (Seller's Risk) Lots Accepted (%) Good Lots Bad Lots (Buyer's Risk) False Neg. 0 Tolerance Lot Concentration 12 6

Mycotoxin test procedure Variability Distribution Performance evaluation 13 Lot test procedure sample Mill sample preparation analysis test result 14 7

sampling error total error sample preparation error analytical error lot sample preparation analysis 15 s 2 = s 2 + s 2 + s 2 t s sp a s 2 s 2 s 2 spa sp a = + s 2 a = s a 2 16 8

Coefficient of Variation (%) 25 20 15 10 5 0 Coefficient of Variation Total Sampling (1.1 kg) Sample Prep. (25 g, Romer) Analysis (1 aliquot, HPLC) 0 10 20 30 Fumonisin Concentration - C (mg/kg) 17 Variance at 2 mg/kg Test Variance Ratio 1.1 kg 0.111 61.0 Romer, 25 g 0.033 18.2 HPLC 1 0.038 20.8 Total 0.182 100.0 18 9

Variance at 2 mg/kg Test Variance Test Variance 1.1 kg 0.111 5 kg 0.024 Romer, 25 g 0.033 Romer, 25 g 0.033 HPLC 1 0.038 HPLC 1 0.038 Total 0.182 Total 0.095 19 Variance at 2 mg/kg Test Variance Test Variance 1.1 kg 0.111 1.1 kg 0.111 Romer, 25 g 0.033 Romer, 50 g 0.017 HPLC 1 0.038 HPLC 1 0.038 Total 0.182 Total 0.166 20 10

Variance at 2 mg/kg Test Variance Test Variance 1.1 kg 0.111 1.1 kg 0.111 Romer, 25 g 0.033 Romer, 25 g 0.033 HPLC 1 0.038 HPLC 2 0.019 Total 0.182 Total 0.163 21 Variance at 2 mg/kg Test Variance Test Variance 1.1 kg 0.111 5 kg 0.024 Romer, 25 g 0.033 Romer, 50 g 0.017 HPLC 1 0.038 HPLC 2 0.019 Total 0.182 Total 0.060 22 11

Acceptance Probability (%) 100 80 60 40 20 0 OC Curve for 1.1 kg Sample Sample - 1.1 kg Sample Prep. - Romer Mill, 25g Analysis - HPLC Accept/Reject Limit - 2 mg/kg 0 1 2 3 4 5 Fumonisin Concentration (mg/kg) 23 Reduce Both Buyer s and Seller s Risk s Reduce Variability of Test Procedure - Increase Sample Size 24 12

OC curves for 1.1, 2.2, and 4.4 kg samples Acceptance Probability (%) 100 80 60 40 20 0 Sample Prep. - Romer Mill, 25g Analysis - HPLC Accept/Reject Limit - 2 mg/kg 1.1 kg 2.2 kg 4.4 kg 0 1 2 3 4 5 Fumonisin Concentration (mg/kg) 25 Reduce seller s risk Accept/reject limit > tolerance Reduce buyer s risk Accept/reject limit < tolerance 26 13

OC curves for 1, 2, and 4 mg/kg accept/reject limits Acceptance Probability (%) 100 80 60 40 20 0 Sample - 1.1 kg Sample Prep. - Romer Mill, 25g Analysis - HPLC 1 mg/kg 2 mg/kg 3 mg/kg 0 1 2 3 4 5 Fumonisin Concentration (mg/kg) 27 Sampling plans developed by Tom Whitaker Mycotoxin maize peanuts cottonseed Commodity almonds wheat barley coffee aflatoxin fumonisin DON ochratoxin 28 14

Evaluation of mycotoxin sampling plans FAO Food & Nutrition Paper 55 Sampling Plans for Aflatoxin Analysis in Peanuts and Corn 29 Outline of presentation Introduction Variability associated with sampling, sample preparation and analysis Analyte isolation and clean-up Focus on immunoaffinity cleanup Summary 30 15

Sampling Extraction Clean-up Concentration Ultimate separation Detection and quantitation Confirmation of identity 31 Extraction from food and feed Choice of suitable solvent (mixtures) - methanol/water - acetonitrile/water - addition of chloroform/hexane for defatting Deconjugation if mycotoxin is bound to matrix (e.g. issue samples) Blending at high speed or shaking (avoid emulsion formation) Filter or centriguge to obtain clear supernatant 32 16

Extraction Need to demonstrate adequate extraction from naturally contaminated materials Spiking of matrices does NOT demonstrate adequate extraction Repeated extraction of same matrix gives some indication of recovery 33 Extraction of mycotoxins from foods and feeds Spiking gives good indication of losses through analytical stages post-extraction Performance of extraction solvents evaluated relative one another, but difficult to measure recovery in absolute terms as true mycotoxin content never known Repeated extraction of same matrix gives some indication of recovery Foods containing radio-labelled mycotoxins can facilitate extraction studies 34 17

Sampling Extraction Clean-up Concentration Ultimate separation Detection and quantitation Confirmation of identity 35 Clean-up for mycotoxin determination Removal of co-extractives Preliminary separation and concentration Various possibilities exist (crude chromatography, adsorption, antibodies) 36 18

Clean-up after extraction Silicagel chromatography SPE columns Ion exchange columns Romer Mycosep columns Immuno affinity columns 37 Solid Phase Extraction On- or off line Based on polar separation Concentration possible 38 19

Ion exchange columns Fumonisins, moniliformin The primary retention mechanism of the compound is based on the electrostatic attraction of the charged functional group Charging can be modified by changing ph 39 Mycosep (1) A & B-trichothecenes, zearalenone Loaded with adsorbents like charcoal, Celite, ion exchange resins Fast and easy to use 40 20

Mycosep (2) 41 Outline of presentation Introduction Variability associated with sampling, sample preparation and analysis Analyte isolation and clean-up Focus on immunoaffinity cleanup Summary 42 21

Immuno affinity columns Advantage IACs: -selective - sample concentration Disadvantage IACs: - no high concentration of organic phase - ph sensitive - relatively expensive, multiple use sometimes possible, but declining recivery Final separation with HPLC or GC 43 Immuno affinity columns 44 22

Comparison of SPE clean-up with affinity column for aflatoxin M 1 in cheese 5 10 15 5 10 15 Fluorescence detector response Aflatoxin M 1 Aflatoxin M 1 Time (minutes) 45 Immuno affinity columns ph can be important ph important: Spike sometimes in acetic acid solution Acid capacity PBS-buffer low 46 23

47 Immuno affinity columns Elute with organic solvent Add sample Clean with water or buffer 48 24

Gilson ASPEC System for Automated Analysis 49 Automated system - ASPEC Sample loaded on to affinity column Column washed Mycotoxin eluted Sample concentrated Injection on to HPLC Full system control processing and analysis 50 25

Gilson ASPEC System 51 Immuno affinity columns available for several Fusarium toxins Zearalenone Fumonisins Deoxynivalenol T-2 & HT-2 toxins 52 26

Immuno affinity columns in combination with HPLC Provide excellent clean-up of complex matrices and coloured food and feed matrices Concentrate the toxin of interest allowing more sensitive detection Successfully validated, accepted and standardized by international organisations 53 CEN working group biotoxins 54 27

Analysis of fumonisins B 1 and B 2 by HPLC Extraction from maize with acetonitrile:water (1:1) Shake 60 min or blend for 1-2 min Filter Purify on SPE, SAX or affinity column Derivatize with o-phthaldialdehyde and 2- mercaptoethanol RP-HPLC with fluorescence detection 55 Cleanup - analysis Conventional cleanup is relatively time consuming LC-MS is rapidly coming up - 1 extraction step - Inject on LC-MS - Expensive but low hands-on costs - Multi method 56 28

Outline of presentation Introduction Variability associated with sampling, sample preparation and analysis Analyte isolation and clean-up Focus on immunoaffinity cleanup Summary 57 Summary Variability in sampling > variability in sampling preparation and analysis Detailed studies on sampling done and sampling plans developed e.g. by Tom Whitaker For clean-up of sample extracts, various possibilities exist: IA-LC technique efficient, validated and frequently used LC-MS is now developing as an alternative technique, but not yet fully validated 58 29