The results in the report relate only to the samples tested.

The report shall not be reproduced except in full, without the written approval of the Food Chemistry Laboratory, Ontario and Nunavut Region. The only definitive report is the signed hard copy original. All other transmissions, FAX, electronic or verbal, are subject to confirmation against the original signed hard copy. The total page count may not be accurate after electronic transmission.

Method Validation & Uncertainty Report Determination of Ochratoxin A in Cereals and Pasta using Immunoaffinity Column Clean Up and HPLC with Fluorescence Detection
Food Laboratories Division Ontario and Nunavut Region Health Products and Food Branch Food Chemistry Laboratory 2301 Midland Avenue, Scarborough, Ontario

Prepared by: Winnie Ng, Chemist Food Chemistry Laboratory Reviewed/Approved by: Mohan Mankotia, Unit Head Food Chemistry Laboratory Reviewed/Approved by: Peter Pantazopoulos, Quality Manager Food Laboratories Division Reviewed/Approved by: Robert J. Neil, Chief Food Laboratories Division Distribution: For comment

Date:

Date:

Date:

Date:

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Contents
Summary Methodology Method Characteristics 1. Limit of detection and quantitation 2. Linearity and range 3. Specificity and interferences 4. Precision and ruggedness 5. Trueness / bias and measurement traceability Quality Control Method Development Discussion Estimate of measurement uncertainty Appendices Appendix A: Appendix B: Appendix C: Appendix D: Appendix E: Appendix F: Attachments References Assessment of LOD and LOQ Assessment of linearity Assessment of method precision Assessment of trueness Estimate of quality control limits Estimate of measurement uncertainty

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Summary
This method validated for the analysis of ochratoxin A in bran cereals (Project 4500354) and dry pasta (Project 4500567) is based on AOAC Official Method 2000.03, "Ochratoxin A in Barley Immunoaffinity by Column HPLC"with minor modifications which are outlined in the in-house standard operating procedure ONT-SOP-0065. Note: The values given in this report were calculated using a spreadsheet. The values presented in the tables and equations have been rounded. Reproducing the calculations with the values given may therefore result in slightly differing answers.

Methodology
ONT-SOP-0065 Rev.3 - Determination of Ochratoxin A in Bran Cereals and Dry Pasta by Immnunoaffinity Column Cleanup and HPLC with Fluorescence Detection. 25 g of test sample is extracted with 100 mL of acetonitrile-water (6+4, v/v). The sample extract is filtered through Whatman #4 filter paper and 5 mL of the extract is diluted with 55 mL phosphate buffered saline solution. The mixture is subsequently filtered through Whatman 934-AH microfibre glass filter. 48 mL of the filtrate is then applied to an immunoaffinity column containing antibodies specific for ochratoxin A. The ochratoxin A is isolated and eluted from the column with methanol. The methanol containing the mycotoxin is evaporated and the residue redissolved in one mL of methanolwater-acetic acid (30+70+1,v/v/v). 100 :L of the solution is analyzed and quantified by HPLC with fluorescence detection at 8ex = 333 nm and 8em = 460 nm. The ochratoxin A is eluted with a retention time of about 10.0 min with no significant interference for the limited number of samples analyzed.

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Results
The following performance characteristics were established in this method: 1. Limit of detection and quantitation 2. Linearity and range 3. Specificity and interferences 4. Precision and ruggedness 5. Trueness / bias and measurement traceability 1. Limit of detection and quantitation LOD was estimated to be about 0.2 ng OTA/g with signal to noise ratio at 3 to1 for both bran cereals and dry pasta. LOQ was estimated to be about 0.5 ng OTA/g with signal to noise ratio at 10 to 1 for both bran cereals and dry pasta. For example chromatograms refer to: Appendix A: Assessment of LOD & LOQ 2. Linearity and range A series of OTA standards with concentrations ranging from 0.5 ng/mL to 20 ng/mL were analysed to determine detector linearity. The average correlation coefficient, based on 12 calibration curves by two analysts, is greater than 0.999. For an example of a typical OTA standard curve and assessment of the linearity, refer to: Appendix B: Assessment of linearity

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3. Specificity and interferences Reagent blanks were tested and no significant interference was observed in the ochratoxin A peak region using the method. Bran Cereals A number of bran cereals were analyzed to find an acceptable commodity blank. An ochratoxin A peak, with a signal to noise greater than 2:1, was detected in all samples. At times, a small shoulder next to the OTA peak was found in a couple of samples containing a very low level of OTA. This small interference was insignificant and did not affect the quantitation of peaks greater than 0.5 ng OTA/g (LOQ). Dry Pasta A number of dry pastas were analyzed to find an acceptable commidity blank. As found in the bran cereals, an ochratoxin A peak, with a signal to noise greater than 2:1, was detected in all samples. At times, a small shoulder next to the OTA peak was found in a couple of samples containing a very low level of OTA. It is also noted that the presence of this shoulder is variable even within the same sample. The small interference was insignificant and did not affect the quantitation of peaks greater than 0.5 ng OTA/g (LOQ). Five samples of pasta, analyzed using method ONT-SOP-0065, were found to contain ochratoxin A at various levels ($0.2 ng OTA/g, estimated signal to noise for LOD is approximately 3:1). The presence of ochratoxin A in the samples was confirmed by LC-MS/MS and the quantitation of the levels found were in good agreement with the levels found by the fluorescence detection. Based on this limited number of samples analyzed, the method demonstrates good specificity for ochratoxin A. Appendix C1: Intermediate Precision and Specificity

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4. Precision and Ruggedness 4.1 Intermediate Precision

Intermediate precision is based on the analysis of five different pasta samples, naturally incurred with ochratoxin A, at different concentration levels, by two different analysts, on different dates, on different HPLC systems (of same make and model) and quantitative confirmation of one set of these extracts by LC-MS/MS by a second laboratory. The relative standard deviations of the three results from each sample (including the % recovery) are pooled to give an estimate of intermediate precision. The relative standard deviation is 16.6%. It is noted that potential heterogeneity from the sample preparation step was also taken into account as bulk naturally incurred samples were prepared and analyzed. Appendix C1: Intermediate Precision and Specificity 4.2 Pasta Repeatability

Five individual portions of macaroni pasta were spiked and analyzed as one batch. Duplicate injections of each spike were made in order to also assess instrumental variability. The total method repeatability is estimated as a relative standard deviation of 8.4%. Appendix C2: Pasta Repeatability 4.3 Bran Cereal Repeatability

The method was evaluated for accuracy and precision for the determination of ochratoxin A in bran cereals. A bran cereal was artificially spiked (n=5) at three levels at 0.5 ng OTA/g, 3.0ng OTA/g and 5.0 ng OTA/g. For the five replicates of each of the three levels spiked, the average recovery for the 0.5 ng OTA/g was 88% with a relative standard deviation of 6.7%. The average recovery for the 3.0 ng OTA/g was 97% with a relative standard deviation of 2.0%. The average recovery for the 5.0 ng OTA/g was 110% with a relative standard deviation of 0.6%. Appendix C3: Bran Cereals Repeatability 4.4 Ruggedness

The estimates of precision in this report take into account a number of parameters varied during the course of this validation study. 1. Time (more than two months) 2. Analysts (2) 3. Various matrices (pasta & bran) at various concentration levels 4. Various HPLCs (2), balances and volumetric apparatus 5. It is also noted that heterogeneity of the sample from the sample preparation step is also taken into account since bulk naturally incurred samples were prepared and analyzed.

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5. Trueness / bias and measurement traceability 5.1 Measurement traceability Traceability is a key element in the mutual recognition and comparison of test results. The one key to traceability that must be supplied from outside the laboratory is the traceability of values carried by reference materials, especially by certified reference materials and/or participation in interlaboratory comparisons. Two reference materials, purchased from two different sources, were analysed by two different analysts. Two analysts participated in an interlaboratory proficiency test. In addition: The traceability to the optical density of the UV spectrophotometer is provided by the potassium dichromate solutions ranging from 0.0625 - 0.25mM (, = 3160 at 8max = 350nm). Balances are calibrated by a service accredited to ISO/IEC 17025. 5.2 Trueness / bias evaluation from (C)RMs The criterion for acceptance is |z|# 2. The z-scores indicate acceptable results.
Analyst (C)RM Commodity 1 CRM Wheat 2 RM Barley a Details are provided in Appendix D3 Z- score a -0.7 -0.8

Two different reference materials, purchased from two different sources were analysed by two different analysts. Appendix D: Assessment of trueness. 5.3 Trueness / bias evaluation from an interlaboratory proficiency test The criterion for acceptance is |z|# 2. The z-scores indicate acceptable results.
Analyst 1 2 Commodity Wheat Wheat Z- score +0.8 +0.8

Two analysts participated in an international proficiency test involving 94 participants from 32 countries. FAPAS (Food Analysis Performance Assessment Scheme), Round 1729, February 2004. Ochratoxin A in cereal. Appendix D4: Trueness / bias evaluation from an interlaboratory proficiency test

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5.4 Summary evaluation of bias or trend in the analytical system: The criterion for acceptance is |RSZ|# 2. The current RSZ score = 0.07 Y no statistically significant bias or trend in the analytical system. Appendix D5: Summary evaluation of bias (RSZ scores).

Quality Control
The routine monitoring requirements of the test method are outlined in section 5.4 of ONT-SOP-0065, Revision 2, Determination of Ochratoxin A in Bran Cereals and Dry Pasta. An in-house reference material was prepared from a naturally contaminated whole wheat sample. The in-house reference material was tested for homogeneity and quality control limits were established. The established control limits are calculated based on the formulae outlined in ONT-SOP-0044Guidelines for Control Charts. See details on the in-house reference material evaluation and the control limits for ochratoxin A in Appendix E: Estimate of quality control limits

Method Development Discussion

Including issues that may impact on method performance. 1. Potential sample matrix effect on bran cereal samples with low level ochratoxin A (.0.2 ng/g). A small shoulder is noted that interfered with the ochratoxin A peak on several sample analyses. The resolution of the chromatography may be improved with slight strength change in mobile phase composition to eliminate the interference. 2. Extra filtration step with Whatmans 934-AH microfibre glass filter was needed. ONT-SOP-0065 Rev. 1 - Sample Preparation and Extraction: encountered with eluting difficulty through IAC (plugged up columns) with large particulate matter (formed after initial filtrate was diluted with PBS solution). The procedure was modified by adding an extra filtration step with Whatman 934 microfibre glass fibre after dilution with PBS and before IAC clean-up. 3. Grinding and Sample Homogeneity: The grinding and homogenizing of bran cereals is accomplished with a food processor. Dry pasta, however, must be ground using a Retsch Mill ZM100 with a 0.5 mm sieve. Some samples, depending on their shape and size, may require pre-crushing by using the food processor.

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Estimate of measurement uncertainty

Reported Value Ochratoxin A

1

Expanded uncertainty 1 45%

The expanded uncertainty is calculated using a coverage factor of 2 which gives a level of confidence of approximately 95%. (i.e., The true value is within 45% of the reported value, 95% of the time.) Appendix F: Estimate of measurement uncertainty

Tables, Figures, Appendices

Appendix A: Appendix B: Appendix C: Appendix D: Appendix E: Appendix F: Assessment of LOD and LOQ Assessment of linearity Assessment of method precision Assessment of trueness Estimate of quality control limits Estimate of measurement uncertainty

Attachements

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References
1 2
3.

ONT-SOP-0065 Rev. 2 Determination of Ochratoxin A in Bran Cereals and Dry Pasta AOAC Official Method 2000.03. "Determination of Ochratoxin A in Barley - Immunoaffinity by Column HPLC", (First Action 2000). JAOAC Vol. 83, No. 6, 2000 Liquid Chromatographic Method with Immunoaffinity Column Cleanup for Determination of Ochratoxin A in Barley; Collaborative Study, JAOAC Vol. 83, No. 5, 2000 ONT-FLD-0020 Statistics: Uncertainty of Analytical Results ONT-SOP-0044 Guidelines for Control Charts EURACHEM / CITAC Guide Quantifying Uncertainty in Analytical Measurement, Second Edition http://www.measurementuncertainty.org/ Development and Harmonisation of Measurement Uncertainty Principles Part (d): Protocol for uncertainty evaluation from validation data; V J Barwick and S L R Ellison, January 2000, HTTP://www.vam.org.uk/ Harmonized Guidelines for Single Laboratory Validation of Methods of Analysis (IUPAC Technical Report), International Union of Pure and Applied Chemistry Pure Appl. Chem., Vol. 74, No. 5, pp. 835855, 2002. 2002 IUPAC http://www.iupac.org/publications/pac/index.html , http://www.iupac.org/publications/pac/2002/pdf/7405x0835.pdf Accuracy (Trueness and Precision) of Measurement Methods and Results, ISO/DIS 5725, Geneva (1994). Is my calibration linear? Analytical Methods Committee, No. 3., Dec 2000, The Royal Society of Chemistry 2000 http://www.rsc.org/ Handbook for Calculation of Measurement Uncertainty in Environmental Laboratories Edition 2; NORDTEST Report TR 537 http://www.nordtest.org/register/techn/tlibrary/tec537.pdf ISO/DTS 21748:2003, Guide to the use of repeatability, reproducibility and trueness estimates in measurement uncertainty estimation.

4. 5.
6.

7.

8.

9. 10.

11.

12.

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Appendix A: Assessment of LOD and LOQ

Figure 1 Chromatogram of Bran Cereal Spiked with 0.2 ng OTA/g, an OTA standard and reagent blank

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Figure 2 Chromatogram of Bran Cereal spiked with 0.5 ng OTA/g and unspiked bran cereal

* bran cereal (unspiked) contains an estimated level of about 0.13 ng OTA/g

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Figure 3 Chromatogram of Dry Pasta with 0.3 ng/g naturally occurring OTA

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Figure 4 Chromatogram of Dry Pasta Spiked with 0.5 ng OTA/g

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Appendix B: Assessment of Linearity

B1: Example Linear Curve Figure 5 A Typical Calibration Curve from 0.05 - 2.0 ng OTA

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B2: Assessment of linearity The % residuals (1) were plotted in order to detect and assess any systematic deviations.
(1): %Residual = Predicted 100 True

The residuals plot is obtained by calculating the percent residual (or percent recovery) of each sample and plotting it against sample concentration. For a well-behaved data set, the data points should be evenly scattered on both sides of the 100% line. The results (below) are based on 12 calibration curves over approximately a four-month period.
Residuals Plot: Assessment of linearity
based on 12 calibration curves 115%

[Predicted value/ True value] x 100

110%

105%
l l /m l

ng /m

ng /m

ng /m

0. 5

ng

100%

10

95%

90%

85%

Six point linear calibration curve ploted on a log scale (for better viewing)

Calibration level (ng/mL) 0.5 1 2 5 10 20

a

Average a 101% 99% 101% 101% 99% 100%

Relative Standard Deviation a 2.21% 2.33% 1.78% 1.01% 0.59% 0.12%

based on n=12 points per calibration level

Conclusions: The data points are evenly scattered on both sides of the 100% line and exhibit a decreasing variance with increasing calibration level. The use of a linear calibration function is appropriate and does not make a significant contribution to the overall uncertainty of the measurement.

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20

ng /m

ng

/m

Appendix C: Assessment of method precision

C1. Intermediate Precision and Specificity Intermediate precision is based on the analysis of five different pasta samples, naturally incurred with Ochratoxin A, at different concentration levels, by two different analysts on different dates, on different HPLC systems and quantitative confirmation by LC-MS/MS by a second laboratory. The relative standard deviations of the three results from each sample (including the %Recovery) are pooled to give an estimate of intermediate precision. The relative standard deviation is 16.6%. It is also noted that potential heterogeneity from the sample preparation step is also taken into account since bulk naturally incurred samples were prepared and analyzed.
LC-Fluorescence Analysis Food Chemistry Laboratory Analyst 1 Analyst 2 HPLC System 8 HPLC System 10 Feb/4/2004 Mar/08/2004 ng/g ng/g 0.48 0.35 1.24 1.04 0.33 0.29 0.61 0.57 0.24 0.15 0.94 b 0.56 c b 92 82 c LC-MS/MS Confirmation Ottawa Laboratory Extract from Analyst 2 Mar/16/2004 %RSD ng/g 0.46 1.47 0.33 0.75 0.22 0.79 d 114 d Intermediate Precision RSDpool e 16.3 17.2 7.3 14.7 23.2 17.1 16.6

Uncooked Pasta (Sample ID) Fusilli (#1420) Macaroni -Alphabets (#1421) Macaroni (#1422) Spaghetti (#1424) Rotini (#1425)a Spike results % Recovery

Poor chromatography. Noisy and small shoulder peak observed on Feb 4, 2004 and Mar 8, 2004, respectively. Spiked to #1420 @ 0.5 ng OTA/g; %Recovery = (0.94-0.48)/0.5 100= 92 c Spiked to #1425 @ 0.5 ng OTA/g; %Recovery = (0.56-0.15)/0.5 100 = 82 d Spiked to #1425 @ 0.5 ng OTA/g; %Recovery =(0.79-0.22) /0.5 100 = 114 e The relative standard deviation of the three results from each sample (including the %Recovery) are pooled using the following formulae, to give an estimate of the intermediate precision.
b

RSDpool =

2 RSD12 ( n1 1) + RSD 2 ( n 2 1) +..... ( n1 1) + ( n2 1) +.....

RSD = relative standard deviation n = number of values

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C2. Pasta Repeatability Five individual portions of macaroni pasta were spiked and analyzed as one batch (repeatability conditions). Duplicate injections of each spike were made in order to also assess instrumental variability. A one-way analysis of variance (ANOVA) was applied to the results in order to separate the error components. Total method repeatability is estimated as a relative standard deviation of 8.4%.
Duplicate Injections (ng/g) a Cspike - Cnative 1 Spike 1 2 3 4 5
b a c d

ANOVA Total method repeatability

2 7.61 7.37 8.08 6.3 7.28 7.36 90%

Instrumental variability

Wet chemistry variability

7.66 7.72 7.59 6.34 7.66 Mean Recovery

3.1%

7.8%

8.4%

A suitable blank matrix was not found. Individual portions of a naturally contaminated dry pasta, Macaroni #1422 was spiked with ochratoxin A. The results were calculated as Cspike - Cnative where Cspike is the concentration of the spiked sample and Cnative is the concentration of the unspiked sample. Cnative = 0.24ng/g. b [Mean Spike level] 100; Spike level = 8.16 ng/g c Analysis of Variance is expressed as a relative standard deviation d Variability between injections from the same vial e Variability between spikes

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C3. Bran Cereal Repeatability

Spike Recovery Results

Spike Level ng/g 0.5 3.0 5.0 Level OTA (ng/g ) Found n=5 1 0.39 3.01 5.56 2 0.43 2.95 5.53 3 0.46 2.90 5.53 4 0.45 2.91 5.51 5 0.46 2.85 5.48 Average % ng/g Recovery 0.44 2.92 5.52 88 97 110 SD ng/g 0.03 0.06 0.03 RSD 6.7% 2.0% 0.6%

Each of the spike levels was analyzed in batches and each consisted of five replicates (n=5). A suitable blank matrix was not found and a bran cereal (ID 1423) containing low level of ochratoxin A was used for spiking. The homogeneity of the bran cereal was evaluated prior to use. The sample was also run as is (unpsiked) with each batch. The above results are corrected to the level of OTA found in the unspiked cereal in the respective batches. The range of OTA found in ID 1423 was 0.09 - 0.3 ng OTA/g.

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Appendix D: Assessment of trueness

Two different reference materials, purchased from two different sources were analysed by two different analysts. D1. Analysis of CRM Spike recovery results and CRM results were analyzed as one batch (repeatability conditions) CRM specifications Certified Reference Material; Certifying Body: Institute for Reference Materials and Measurements (IRMM); Producer: Community Bureau of Reference (BCR); Commodity: Ground and sieved wheat; Reference number: CRM 472
Certified Value (ng/g) Ccrm
a

Set Interlaboratory Standard Deviation a Sti

8.2 1.4 From Table 7.5 of certification report. Derived by dividing the 95% tolerance interval by 2 (2.80527 ng/g 2 )

Spike recovery results

Replicates (ng/g, n=2) a 1
a

Mean

%Recovery b

7.2 7.13 7.17 88 A suitable blank matrix was not found. Individual portions of a naturally contaminated dry pasta (Rotini #1425) were spiked with ochratoxin A. The results were calculated as Cspike - Cnative where Cspike is the concentration of the spiked sample and Cnative is the concentration of the unspiked sample. Cnative = 0.18 ng/g b [Mean Spike level] 100; Spike level = 8.16 ng/g

CRM Results
Replicates (ng/g, n=5) b a Uncorrected for recovery 7.12 a 7.0 b 7.27 c 5.15 c 5.13 Mean Cobs 6.35 SD Sobs 1.11 %RSD 17

Corrected for recovery a 8.09 8.07 8.26 5.85 5.83 7.22 1.26 17 a Corrected for recovery = Uncorrected for recovery 0.88 (recovery) b The CRM comes packaged in separate pouches: a, b and c represent analytical results from different pouches.

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D2. Analysis of RM Spike recovery results and RM results were analyzed as one batch (repeatability conditions) RM specifications Reference Material Producer: FAPAS (Food Analysis Performance Assessment Scheme), analysed by 78 labs internationally Commodity: Barley flour; Reference number: RM Series T1718
Assigned Value (ng/g) Ccrm
a

Set Interlaboratory Standard Deviation Sti

5.4 1.2 From FAPAS report. Derived by dividing the 95% tolerance interval by 2 (2.4 ng/g 2 )

Spike recovery results

Replicates (ng/g, n=2) a 1
a

Mean

%Recovery b

4.62 5.22 4.92 94.6 A suitable blank matrix was not found. Individual portions of a naturally contaminated dry pasta (Macaroni #1422) were spiked with ochratoxin A. The results were calculated as Cspike - Cnative where Cspike is the concentration of the spiked sample and Cnative is the concentration of the unspiked sample. Cnative = 0.30 ng/g b [Mean Spike level] 100; Spike level = 5.2 ng/g

RM Results
Replicates (ng/g) b a Uncorrected for recovery 6.01 a 4.87 b 3.87 c 3.53 c 2.99 Mean Cobs 4.25 SD Sobs 1.20 %RSD 28

Corrected for recovery a 6.35 5.15 4.09 3.73 3.16 4.49 1.26 28 a Corrected for recovery = Uncorrected for recovery 0.946 (recovery) b The RM comes packaged in separate pouches: a, b and c represent analytical results from different pouches.

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D3. Evaluation of (C)RM results The criterion for acceptance is |z|# 2. The z-scores indicate acceptable results. It is noted that the values derived for the (C)RMs were corrected for recovery. For international comparability purposes the values used by this laboratory to evaluate results were also corrected for recovery.
Analyst 1 2
a b

(C)RM CRM RM

Commodity Wheat Barley

% of (C)RM a 88% 83%

Z-score b -0.7 -0.8

(Cobs Ccrm) 100; where Cobs is corrected for recovery

Represents a simple evaluation of the methodology based on the well known Z-score used in proficiency testing. Cobs Ccrm Z= Sti Ccrm = Value of (C)RM

Cobs = Mean of replicate analysis of (C)RM (corrected for recovery) Sti = Set interlaboratory standard deviation (95% tolerance interval 2)

Note: The difficulty of closely matching the matrix and the concentration of a sample with that of a (C)RM is a familiar problem. It is noted that the (C)RMs used here are not perfect matches for the intended use of the method, regarding expected commodities and concentration levels. They are instead the best available and the results are to be considered as one part of the whole method validation.

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D4. Trueness / bias evaluation from an interlaboratory proficiency test The criterion for acceptance is |z|# 2. The z-scores indicate acceptable results. Two analysts participated in an international proficiency test involving 94 participants from 32 countries. FAPAS (Food Analysis Performance Assessment Scheme), Round 1729, February 2004.Ochratoxin A in cereal. The commodity was milled wheat grain. The assigned value was 9.1 ng/g.
Analyst 1 2
a

%Recovery 84 110

Lab Value 8.9 11.7

Final Reported Results a 10.6 10.6

Z-score 0.8 0.8

This proficiency test scheme calls for final reported results to be corrected for recovery.

D5. Summary evaluation of bias (RSZ scores): The criterion for acceptance is |RSZ|# 2. The current RSZ score = 0.07 Y no statistically significant bias or trend in the analytical system. RSZ scores: The Rescaled Sum of Z scores has the useful property of demonstrating a persistent bias or trend in analytical systems. The RSZ score is normally calculated from the Z scores of the last n PT rounds (n is typically 4 rounds). In this particular case it is based on two (C)RMs and two Proficiency Test results by two analysts.
Analyst 1 2 1 2 Analysis CRM RM PT PT Commodity Wheat Barley Wheat Wheat RSZ score b
a b

Z-scores a -0.7 -0.8 +0.8 +0.8 + 0.07

See Appendix D3 and D4 for z-scores

RSZ score =

i =1

Reference: Analytical Methods Committee; AMC Technical Brief No.16; Apr 2004; Royal Society of Chemistry 2004 http://www.rsc.org/pdf/amc/brief16.pdf

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Appendix E: Estimate of quality control limits

E1. Preparation of Control Sample Six pasta and a sample of organic whole wheat were purchased and analyzed for the purpose of finding a naturally contaminated sample with ochratoxin A that might be used as a control sample for quality control purposes in routine analyses. The pasta samples and organic whole wheat, more than 3 kg were put through the Retsch Mill ZM100 using 1.0 mm sieve. The ground samples were placed individually into large plastic bags and manually mixed. Note that the odd and large shape pasta samples were ground in a Brau Multipractic 100 food processor before putting through the Retch Mill ZM100 as the latter was incapable of handling the original shapes and size of the pasta. The samples were analyzed in a singlet and found that the organic wheat (ID 1577) was found to contain about 1.0 ng OTA/g, an appreciable level which can be used as a control sample. In order to establish the homogeneity of the sample, the 3 kg ground sample was mixed again manually in the large plastic bag for at least an additional twenty minutes. Five replicates were analyzed and the level of ochratoxin found ranged from 0.86 ng OTA/g to 1.74 ng OTA/g. The average was 1.35 ng OTA/g for n=5, with a standard deviation of 0.37 ng OTA/g and a relative standard deviation of 27%. The data clearly showed the repeatability was unacceptable and homogeneity appeared to be the problem. In an effort to render the wheat sample (1577) more homogenous, the 3 kg ground sample was ground in the Retsch Mill ZM100 again using a 0.5 mm sieve. The entire sample was hand mixed in a large plastic bag and about two equal portions were transferred into two large glass jars. Each jar, containing approximately 1.5 kg of the finely ground sample, was placed into a vertical stainless steel tumbler and mixed for about 24 hours. Each jar had about 30% empty space which allowed thorough mixing. The jars were labelled as 1577-A and 1577-B. Five replicates were analyzed from the jar labelled 1577-A. A bran cereal (ID1423), two spiked samples (at 1.0 ng OTA/g) and # 1381 CRM 472 (a reference material) were analyzed with the five replicates of the wheat. The level of OTA for the five replicates ranged from 1.44 to 1.72 ng OTA/g, with an average of 1.54 ng OTA /g, a s.d. of 0.11 and r.s.d of 7.2%. The % recoveries for the duplicate spikes were 95% and 101%, with and average of 98%. The level of OTA in the wheat cereal #1381 (reference material CRM 472) was analyzed to be 7.09 ng OTA/g. It is certified to contain 8.2 ng OTA/g. The results demonstrate that the contents in jar 1577-A can be used as a control sample. For ease of storage and handling, the contents of jar 1577-A were transferred into three separate 1L plastic containers that were labelled and stored in the laboratory freezer. The contents of jar 1577-B were transferred into a large plastic container, labelled and stored in the freezer. The label is marked that it is not to be used until its contents analyzed for n=5 as done for jar 1577-A. The remainder of 1577 whole wheat kernels of about 7 kg is kept sealed in its original sac and stored in the freezer in sample control. The sample can be prepared and used as an OTA control sample in the same manner.

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E2. Data & Calculations of Control Limits for X and R Charts

Note: It is understood that these are estimates. Control limits will be recalculated from data generated over time and through the routine use of the analytical method. Control sample ID1577-A Results
Replicates 1 Level of OTA found ng/g 1.49 2 1.47 3 1.72 4 1.57 5 1.44 Mean n=5 1.54 SD %RSD

0.11

7.2

X Chart Control Limits Control Limit: 99% confidence Warning Limit: 95% confidence 1.54 0.51 ng/g 1.54 0.31 ng/g

Calculations of control limits for the X charts were based on formula 1 outlined in ONT-SOP-0044 Rev. 2 -Guidelines for Control Charts: Control Limit =mean (k(n-1, 99%) F) = 1.54 (4.6 0.11) = 1.54 0.51 Warning Limit =mean (k (n-1, 95%) F) = 1.54 (2.78 0.11)= 1.54 0.31 where: k= the value determined from the Students t-distribution tables for a given confidence level with (n-1) degrees of freedom. n= number of replicates, degrees of freedom = n-1 F= standard deviation mean= average result R Chart Control Limits : applicable to duplicate measurements (n=2) Upper Control Limit (UCL) Upper Warning Limit (UWL) 3.6 (F ) = 3.6(0.11) = 0 .40 ng/g, or RPD at 26% 2.8(F ) = 2.8 (0.11) = 0. 31 ng/g, or RPD at 20%

F= standard deviation RPD= relative percent difference between measurements

Calculations of predicting repeatability of control limits for the R chart were based Formula 2 outlined in ONT-SOP-0044 Rev. 2 -Guidelines for Control Charts.

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Appendix F: Estimate of measurement uncertainty

F1. Principles Key principles of uncertainty estimation:

T T

The studies must be representative of normal operation of the method. The studies must cover the complete method, (a representative range of sample matrices and a representative range of analyte concentrations.)

Planning:

To be representative of the normal operation of the method, uncertainty estimation is a planned activity that incorporates data from various sources such as method development, validation, training and routine internal quality control data.

The stages in the quantification of measurement uncertainty are as follows:

T T T

precision study; trueness (bias) study; identification and evaluation of other uncertainty contributions not adequately covered by the precision and trueness studies.

General error model: This model is a simplification of the model presented in the ISO guide [Ref 12] y = x + (* +B) + g + T y x (* +B) g T measurement result of a sample expected (true) value for y the combined bias (systematic error) where * is method bias and B is laboratory bias random error at within-laboratory reproducibility conditions (intermediate precision) other factors not adequately addressed

Note: The values given in this report were calculated using a spreadsheet. The values presented in the tables and equations have been rounded. Reproducing the calculations with the values given may therefore result in slightly differing answers.

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Identification of sources of uncertainty

ONT-SOP-0065, Determination of Ochratoxin A in Cereals and Pasta using Immunoaffinity Column Clean Up and HPLC with Fluorescence Detection This chart represents basic steps in the analytical method. The list to the right of each step represents potential sources of uncertainty or variables that may impact this step. Underlined = may require special consideration since it impacts at many stages of the method or is judged a significant potential source of error. General aspects applicable to all stages: Variable solvents, reagents, calibration of pipettes and balances, equipment, analyst, cross-contamination, etc... Grind sample
T ime Amount Sample type

LC
Chromatography

Sub-sample
Sub-sample weight Sub-sample homogeneity

Mobile phase Composition

Mobile phase Column T emperature Flow rate

Extract analyte
Extraction Extraction Polytron Filter Sample Analyte type concentration volume carryover time

Inject sample

Volume

Dilute

Volume

Determine concentration
Standard Detector curve specificity linearity Sample type Analyte concentration

Prepare glassware

Prepare standards

Prepare sample

Purify sample

Quantitate sample

Wash
Variable treatment of glassware

Prepare stock solution

Storage Volume Calibration

IA column
Expiry dates Column lots Column recoveries

Silanize vials

Prepare working calibrant for standard curve

Filter sample
Volume Storage Volume Dilution Flow rate of Nitrogen Sample type

Apply sample

Flow rate

Wash
Wash volume Sample type Analyte concentration

Elute analyte
Elution volume Sample type Analyte concentration

Concentrate
T ime T emperature Flow rate of Nitrogen Volume

Filter

Sample type

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F2. Summary of Uncertainty Estimate Reported Value Ochratoxin A

1

Expanded uncertainty 1 45%

The expanded uncertainty is calculated using a coverage factor of 2 which gives a level of confidence of approximately 95%. (i.e the true value is within 45% of the reported value, 95% of the time.) The following represents a quantified breakdown of the major sources of uncertainty.
Breackdown of uncertainty sources % Standard uncertainty Comments

Precision Instrumental variability 3.1 Five individual portions of macaroni pasta were spiked and analyzed as one batch (repeatability conditions). Duplicate injections of each spike were made in order to also assess instrumental variability (variable instrument injection volumes, chromatography,...). A one-way analysis of variance (ANOVA) was applied to the results in order to separate the error components. Total method repeatability is estimated as a relative standard deviation of 8.4%. See Appendix C2. Represents the variability that is not accounted for by the estimate of method repeatability (i.e the variability over time, between analysts, instruments, reagents, volumetrics, sample types and homogeneity...etc...). Derived by subtracting the repeatability variance from the intermediate precision variance (See Appendix F3)

Wet chemistry variability

7.8

Operational variability

14.4

Bias CRM + RM Calibration UV spectrophotometer 4.2 22.3 45 Based on the maximum allowable precision and bias of the UV spectrophotometer calibration. See Appendix F5. Combined relative standard uncertainty (1) Expanded relative standard uncertainty (2) 14.2 Estimate from two different reference materials. CRM 472 (wheat), RM (barley). See Appendix F4.

2 2 2 (1): Combined uncertainty = ( Precision) + ( Bias) + ( Calibration) ...

(2): The combined uncertainty is multiplied by a coverage factor of 2. This gives a 95% confidence interval, (i.e the true value is within 45% of the reported value, 95% of the time.)

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F3. Precision Estimate Intermediate precision is estimated as a relative standard deviation of 16.6% (Refer to Appendix C1). The estimated precision was based on the analysis of five different pasta samples, naturally incurred with Ochratoxin A, at different concentration levels, by two different analysts on different dates, on different HPLC systems and quantitative confirmation by LC-MS/MS by a second laboratory. The relative standard deviation of the three results from each sample (including the %Recovery) are pooled to give an estimate of intermediate precision. Operational variability. Total method repeatability is estimated as a relative standard deviation of 8.4% (Refer to Appendix C2). An estimate of the operational variability can be derived by subtracting the method repeatability from the intermediate precision. Operational variability represents the variability over time, between analysts, instruments, reagents, volumetrics, sample types and homogeneity...etc... that is not accounted for by the estimate of method repeatability.
Operational variability =

(16.6) 2 ( 8.4) 2

= 14.4

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F4. Bias Estimate - Estimated by analysis of a CRM and RM. Explanation Step 1 Do preliminary Rm estimate = Pre(Rm)
Rm is an estimate of the mean method recovery uncertainty obtained from the analysis of a CRM or RM. (Basic propagation of error)

Calculations
(Refer to F4.1 for data used in the calculations)
Pre( Rm) = Cobs U (Ccrm ) 2 Sobs 2 + 2 Ccrm Ccrm n Cobs

Results CRM RM

Cobs = Mean of replicate analysis of CRM Ccrm = Certified value of CRM

0.077

0.103

Sobs = Standard Deviation of replicate analysis of CRM

n = number of replicate analysis of CRM U(Ccrm) = Standard deviation of the certified value of CRM

Step 2 Do significance test (t-test).

Evaluate whether the recovery is significantly different from 1 (100%). When the degrees of freedom are unknown, for example if there is a contribution from the uncertainty in the value of a reference material, compare t with k, the coverage factor that will be used in the calculation of the expanded uncertainty. In this uncertainty estimate k=2. significance test (t-test).

1 Recovery Pre(Rm) Cobs Recovery = Ccrm t=

2.93

2.06

Step 3 Do final Rm estimate based on the results of The t-test from Step 2 indicates this is a Case 3, since t>2 and in
Case 1: t # 2 The significance test indicates that the recovery is not significantly different from 1 so there is no reason to correct analytical results for recovery. Case 2: t > 2 & Corrected for recovery. As a correction factor is being applied, R m is explicitly included in the calculation of the result. Case 3: t > 2 & Not corrected for recovery. The significance test indicates that the recovery is statistically significantly different from 1, but in the normal application of the method no recovery correction is applied. The uncertainty is increased and then treated as Case 1.

the normal application of this method, recovery correction is not applied.

Rm = Pre(Rm)

N/A

N/A

Rm =

Pre( Rm) Recovery

N/A

N/A

1 Recovery 2 Rm = + ( Pre( Rm)) k

0.136

0.148

Step 4 Final estimate of bias

(0.142) Average of the two estimated uncertainties

Average or pool the estimated uncertainties

14.2%

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F4.1 Data used for the bias estimate

Reference Values (ng/g)

c

Laboratory Results (ng/g) Cobs f 6.354 4.251 Sobs g 1.11 1.20 nh 5 5

Ccrm 8.2 5.4

U(Ccrm) 0.5 d 0.2 e

CRM a RM b
a b

Wheat, CRM 472 Barley flour, FAPAS reference material analyzed by 78 labs internationally c Assigned value of (C)RM from report d Standard uncertainty from certification report. Derived by dividing the 95% confidence interval by 2 (1.0 2 ) e Standard uncertainty from FAPAS report f Mean of replicate analysis of (C)RM (uncorrected for recovery) g Standard deviation of replicate analysis of (C)RM h Number of replicate analysis of (C)RM

Notes: 1. Raw data is in Appendix D: Assessment of trueness 2. Uncorrected Cobs is used for this bias estimate since in the normal application of this method recovery correction is not applied.

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F5. Calibration of Standards The uncertainty is based on the maximum allowable precision and bias of the UV spectrophotometer calibration provided by the potassium dichromate solutions.
Allowable range %RSD (max repeatability) Correction factor (max bias) < 3% 1 0.05 % Standard uncertainty 3% 2.9% a 4.2%

5% 3

Total % standard uncertainty

a

( 3) 2 + ( 2.9) 2

Assumes a uniform (rectangular) distribution

F6. Other Sources of Uncertainty The estimates of precision and bias take into account all analytical steps after sampling. Notes: Linear curve Nonlinearity would have contributed to the observed precision and is therefore inherently included in the precision estimate. Refer also to Appendix B: Assessment of Linearity Sample heterogeneity and matrix affects Potential heterogeneity from the sample preparation step is taken into account since bulk naturally incurred samples were prepared, sub-sampled and analyzed. Matrix affects are taken into account since in both the precision and bias estimates various matrices were used. Refer also to the following documents for typical in-house uncertainty estimates since during this study various balances, volumetric flasks and pipettes have been used. Balances ONT-FLD-0020 Statistics: Uncertainty of Analytical Results. Section 5A; Attached spreadsheet and PDF file for typical in-house estimates of weighing uncertainties. 1. Survey 4500336 Report ; Project FM90, Determination of Aflatoxins in Beer 2000 - 2001; Date of Issue: 2001/09/11; for an in-house estimate of volumetric uncertainty. 2. ONT-FLD-0020 Statistics: Uncertainty of Analytical Results. Section 5A; Attached spreadsheet and PDF file for typical in-house estimates of volumetric uncertainties.

Volumetrics

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