métodos de validación

Ochratoxin A in Cereals and PastaDate of Issue: For comment of 32

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

Method Validation & Uncertainty ReportDetermination of Ochratoxin A in Cereals and Pasta using

Immunoaffinity Column Clean Up and HPLC with FluorescenceDetection

Food Laboratories DivisionOntario and Nunavut Region

Health Products and Food BranchFood Chemistry Laboratory

2301 Midland Avenue, Scarborough, Ontario

Prepared by: Date:

Winnie Ng, ChemistFood Chemistry Laboratory

Reviewed/Approved by: Date:

Mohan Mankotia, Unit HeadFood Chemistry Laboratory


Peter Pantazopoulos, Quality ManagerFood Laboratories Division


Robert J. Neil, ChiefFood Laboratories Division

Distribution: For comment


Contents

Summary

Methodology

Method Characteristics1. Limit of detection and quantitation 2. Linearity and range3. Specificity and interferences4. Precision and ruggedness5. Trueness / bias and measurement traceability

Quality Control

Method Development Discussion

Estimate of measurement uncertainty

AppendicesAppendix A: Assessment of LOD and LOQAppendix B: Assessment of linearityAppendix C: Assessment of method precisionAppendix D: Assessment of truenessAppendix E: Estimate of quality control limitsAppendix F: Estimate of measurement uncertainty

AttachmentsReferences


Summary

Methodology

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 standardoperating procedure ONT-SOP-0065.

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

ONT-SOP-0065 Rev.3 - Determination of Ochratoxin A in Bran Cereals and Dry Pasta byImmnunoaffinity 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 isfiltered through Whatman #4 filter paper and 5 mL of the extract is diluted with 55 mL phosphatebuffered saline solution. The mixture is subsequently filtered through Whatman 934-AH microfibreglass filter. 48 mL of the filtrate is then applied to an immunoaffinity column containing antibodiesspecific for ochratoxin A. The ochratoxin A is isolated and eluted from the column with methanol. Themethanol containing the mycotoxin is evaporated and the residue redissolved in one mL of methanol-water-acetic acid (30+70+1,v/v/v). 100 :L of the solution is analyzed and quantified by HPLC withfluorescence 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 forthe limited number of samples analyzed.


Results

The following performance characteristics were established in this method:

1. Limit of detection and quantitation 2. Linearity and range3. Specificity and interferences4. Precision and ruggedness5. 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 anddry pasta.

LOQ was estimated to be about 0.5 ng OTA/g with signal to noise ratio at 10 to 1 for both bran cerealsand 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 todetermine 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


3. Specificity and interferences

Reagent blanks were tested and no significant interference was observed in the ochratoxin A peak regionusing 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 tothe OTA peak was found in a couple of samples containing a very low level of OTA. This smallinterference 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 brancereals, an ochratoxin A peak, with a signal to noise greater than 2:1, was detected in all samples. Attimes, a small shoulder next to the OTA peak was found in a couple of samples containing a very lowlevel 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 ngOTA/g (LOQ).

Five samples of pasta, analyzed using method ONT-SOP-0065, were found to contain ochratoxin A atvarious levels ($0.2 ng OTA/g, estimated signal to noise for LOD is approximately 3:1). The presenceof ochratoxin A in the samples was confirmed by LC-MS/MS and the quantitation of the levels foundwere in good agreement with the levels found by the fluorescence detection.

Based on this limited number of samples analyzed, the method demonstrates good specificity forochratoxin A.

Appendix C1: Intermediate Precision and Specificity


4. Precision and Ruggedness

4.1 Intermediate Precision

Intermediate precision is based on the analysis of five different pasta samples, naturally incurred withochratoxin A, at different concentration levels, by two different analysts, on different dates, on differentHPLC systems (of same make and model) and quantitative confirmation of one set of these extracts byLC-MS/MS by a second laboratory. The relative standard deviations of the three results from eachsample (including the % recovery) are pooled to give an estimate of intermediate precision. The relativestandard deviation is 16.6%. It is noted that potential heterogeneity from the sample preparation stepwas 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 injectionsof 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 brancereals. A bran cereal was artificially spiked (n=5) at three levels at 0.5 ng OTA/g, 3.0ng OTA/g and5.0 ng OTA/g.

For the five replicates of each of the three levels spiked, the average recovery for the 0.5 ng OTA/g was88% 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% witha 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 thecourse of this validation study.

1. Time (more than two months)2. Analysts (2)3. Various matrices (pasta & bran) at various concentration levels4. Various HPLCs (2), balances and volumetric apparatus5. It is also noted that heterogeneity of the sample from the sample preparation step is also taken intoaccount since bulk naturally incurred samples were prepared and analyzed.


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 valuescarried by reference materials, especially by certified reference materials and/or participation ininterlaboratory 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 potassiumdichromate 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 Z- score a

1 CRM Wheat -0.72 RM Barley -0.8

a Details are provided in Appendix D3

Two different reference materials, purchased from two different sources were analysed by two differentanalysts.

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 Commodity Z- score

1 Wheat +0.8 2 Wheat +0.8

Two analysts participated in an international proficiency test involving 94 participants from 32countries.FAPAS (Food Analysis Performance Assessment Scheme), Round 1729, February 2004. Ochratoxin Ain cereal.

Appendix D4: Trueness / bias evaluation from an interlaboratory proficiency test


Quality Control

Method Development Discussion

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).

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-0044-Guidelines for Control Charts.

See details on the in-house reference material evaluation and the control limits for ochratoxin A inAppendix E: Estimate of quality control limits

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). Asmall shoulder is noted that interfered with the ochratoxin A peak on several sample analyses. Theresolution of the chromatography may be improved with slight strength change in mobile phasecomposition to eliminate the interference.

2. Extra filtration step with Whatman’s 934-AH microfibre glass filter was needed. ONT-SOP-0065Rev. 1 - Sample Preparation and Extraction: encountered with eluting difficulty through IAC (pluggedup 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 fibreafter dilution with PBS and before IAC clean-up.

3. Grinding and Sample Homogeneity: The grinding and homogenizing of bran cereals is accomplishedwith a food processor. Dry pasta, however, must be ground using a Retsch Mill ZM100 with a 0.5 mmsieve. Some samples, depending on their shape and size, may require pre-crushing by using the foodprocessor.


Estimate of measurement uncertainty

Tables, Figures, Appendices

Attachements

Reported Value ± Expanded uncertainty 1

Ochratoxin A ± 45%

1 The expanded uncertainty is calculated using a coverage factor of 2 which gives a level of confidenceof approximately 95%. (i.e., The “true” value is within ±45% of the reported value, 95% of the time.)

Appendix F: Estimate of measurement uncertainty

Appendix A: Assessment of LOD and LOQAppendix B: Assessment of linearityAppendix C: Assessment of method precisionAppendix D: Assessment of truenessAppendix E: Estimate of quality control limitsAppendix F: Estimate of measurement uncertainty


References

1 ONT-SOP-0065 – Rev. 2 Determination of Ochratoxin A in Bran Cereals and Dry Pasta

2 AOAC Official Method 2000.03. "Determination of Ochratoxin A in Barley - Immunoaffinity byColumn HPLC", (First Action 2000). JAOAC Vol. 83, No. 6, 2000

3. “Liquid Chromatographic Method with Immunoaffinity Column Cleanup for Determination ofOchratoxin A in Barley; Collaborative Study, JAOAC Vol. 83, No. 5, 2000

4. ONT-FLD-0020 – Statistics: Uncertainty of Analytical Results

5. ONT-SOP-0044 – Guidelines for Control Charts

6. EURACHEM / CITAC Guide Quantifying Uncertainty in Analytical Measurement, SecondEditionhttp://www.measurementuncertainty.org/

7. Development and Harmonisation of Measurement Uncertainty Principles Part (d): Protocol foruncertainty evaluation from validation data; V J Barwick and S L R Ellison, January 2000, HTTP://www.vam.org.uk/

8. Harmonized Guidelines for Single Laboratory Validation of Methods of Analysis (IUPACTechnical Report), International Union of Pure and Applied ChemistryPure Appl. Chem., Vol. 74, No. 5, pp. 835–855, 2002. © 2002 IUPAChttp://www.iupac.org/publications/pac/index.html ,http://www.iupac.org/publications/pac/2002/pdf/7405x0835.pdf

9. Accuracy (Trueness and Precision) of Measurement Methods and Results, ISO/DIS 5725,Geneva (1994).

10. Is my calibration linear?Analytical Methods Committee, No. 3., Dec 2000, ©The Royal Society of Chemistry 2000http://www.rsc.org/

11. Handbook for Calculation of Measurement Uncertainty in Environmental Laboratories Edition 2;NORDTEST Report TR 537http://www.nordtest.org/register/techn/tlibrary/tec537.pdf

12. ISO/DTS 21748:2003, Guide to the use of repeatability, reproducibility and trueness estimates inmeasurement uncertainty estimation.

http://www.measurementuncertainty.org/

HTTP://www.vam.org.uk/

http://www.iupac.org/publications/pac/index.html

http://www.iupac.org/publications/pac/2002/pdf/7405x0835.pdf

http://www.rsc.org/

http://www.nordtest.org/register/techn/tlibrary/tec537.pdf


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


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


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


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


Appendix B: Assessment of Linearity

B1: Example Linear Curve

Figure 5 A Typical Calibration Curve from 0.05 - 2.0 ng OTA


(1): %Residual = PredictedTrue

×100

0.5 n

g/ml

1 ng

/ml

2 ng

/ml

5 ng/m

l

10 n

g/ml

20 n

g/ml

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

85%

90%

95%

100%

105%

110%

115%

[Pre

dict

ed v

alue

/ Tru

e va

lue]

x 1

00

Residuals Plot: Assessment of linearitybased on 12 calibration curves

B2: Assessment of linearity

The % residuals (1) were plotted in order to detect and assess any systematic deviations.

The residuals plot is obtained by calculating the percent residual (or percent recovery) of each sampleand plotting it against sample concentration. For a well-behaved data set, the data points should beevenly scattered on both sides of the 100% line. The results (below) are based on 12 calibration curvesover approximately a four-month period.

Calibration level(ng/mL) Average a Relative Standard

Deviation a

0.5 101% 2.21%1 99% 2.33%2 101% 1.78%5 101% 1.01%

10 99% 0.59%20 100% 0.12%

a 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 variancewith increasing calibration level.The use of a linear calibration function is appropriate and does not make a significant contribution to theoverall uncertainty of the measurement.


( ) ( )( ) ( )

RSDRSD n RSD n

n npool =

× −

+ × −

+

− + − +

1 22 1 2 1

1 1

1 2

1 2

.....

.....

RSD = relative standard deviationn = number of values

Appendix C: Assessment of method precision

C1. Intermediate Precision and SpecificityIntermediate precision is based on the analysis of five different pasta samples, naturally incurred withOchratoxin A, at different concentration levels, by two different analysts on different dates, on differentHPLC systems and quantitative confirmation by LC-MS/MS by a second laboratory. The relativestandard deviations of the three results from each sample (including the %Recovery) are pooled to givean estimate of intermediate precision. The relative standard deviation is 16.6%. It is also noted thatpotential heterogeneity from the sample preparation step is also taken into account since bulk naturallyincurred samples were prepared and analyzed.

LC-Fluorescence Analysis Food Chemistry Laboratory

LC-MS/MS ConfirmationOttawa Laboratory

Uncooked Pasta (Sample ID)

Analyst 1HPLC System 8

Feb/4/2004

Analyst 2HPLC System 10

Mar/08/2004

Extract from Analyst 2Mar/16/2004

%RSDng/g ng/g ng/g

Fusilli (#1420) 0.48 0.35 0.46 16.3Macaroni -Alphabets (#1421) 1.24 1.04 1.47 17.2Macaroni (#1422) 0.33 0.29 0.33 7.3Spaghetti (#1424) 0.61 0.57 0.75 14.7Rotini (#1425)a 0.24 0.15 0.22 23.2Spike results 0.94 b 0.56 c 0.79 d -% Recovery 92 b 82 c 114 d 17.1

Intermediate Precision RSDpool e 16.6

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


C2. Pasta Repeatability

Five individual portions of macaroni pasta were spiked and analyzed as one batch (repeatabilityconditions). Duplicate injections of each spike were made in order to also assess instrumentalvariability.

A one-way analysis of variance (ANOVA) was applied to the results in order to separate the errorcomponents. Total method repeatability is estimated as a relative standard deviation of 8.4%.

Duplicate Injections (ng/g)a Cspike - Cnative

c ANOVA

1 2d Instrumental

variabilitye Wet chemistry

variabilityTotal methodrepeatability

Spike1 7.66 7.612 7.72 7.373 7.59 8.08 3.1% 7.8% 8.4%4 6.34 6.35 7.66 7.28

Mean 7.36b Recovery 90%

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


C3. Bran Cereal Repeatability

Spike Recovery Results

Spike Level ng/gLevel OTA (ng/g ) Found n=5 Average

ng/g%

RecoverySD ng/g RSD

1 2 3 4 5

0.5 0.39 0.43 0.46 0.45 0.46 0.44 88 0.03 6.7%

3.0 3.01 2.95 2.90 2.91 2.85 2.92 97 0.06 2.0%

5.0 5.56 5.53 5.53 5.51 5.48 5.52 110 0.03 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 wasused for spiking. The homogeneity of the bran cereal was evaluated prior to use. The sample was alsorun as is (unpsiked) with each batch. The above results are corrected to the level of OTA found in theunspiked cereal in the respective batches. The range of OTA found in ID 1423 was 0.09 - 0.3 ngOTA/g.


Appendix D: Assessment of truenessTwo different reference materials, purchased from two different sources were analysed by two differentanalysts.

D1. Analysis of CRMSpike recovery results and CRM results were analyzed as one batch (repeatability conditions)

CRM specificationsCertified 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) Set InterlaboratoryStandard Deviation a

Ccrm Sti

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

Spike recovery resultsReplicates (ng/g, n=2) a

Mean %Recovery b

1 2

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

CRM ResultsReplicates (ng/g, n=5) b Mean

Cobs

SDSobs

%RSDa a b c c

Uncorrected for recovery 7.12 7.0 7.27 5.15 5.13 6.35 1.11 17

Corrected for recovery a 8.09 8.07 8.26 5.85 5.83 7.22 1.26 17a 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.


D2. Analysis of RMSpike recovery results and RM results were analyzed as one batch (repeatability conditions)

RM specificationsReference MaterialProducer: FAPAS (Food Analysis Performance Assessment Scheme), analysed by 78 labsinternationallyCommodity: Barley flour; Reference number: RM Series T1718

Assigned Value (ng/g) Set Interlaboratory Standard Deviation a

Ccrm Sti

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

Spike recovery resultsReplicates (ng/g, n=2) a

Mean %Recovery b

1 2

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

RM ResultsReplicates (ng/g) b Mean

Cobs

SDSobs

%RSDa a b c c

Uncorrected for recovery 6.01 4.87 3.87 3.53 2.99 4.25 1.20 28

Corrected for recovery a 6.35 5.15 4.09 3.73 3.16 4.49 1.26 28a 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.


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 internationalcomparability purposes the values used by this laboratory to evaluate results were also corrected forrecovery.

Analyst (C)RM Commodity % of (C)RM a Z-score b

1 CRM Wheat 88% -0.72 RM Barley 83% -0.8

a (Cobs÷ Ccrm) ×100; where Cobs is corrected for recoveryb Represents a simple evaluation of the methodology based on the well known Z-score used in proficiency testing.

Z C Cobs crm

ti=

−

÷

SC = Value of C S laboratory standard deviation (

crm

obs

ti

(C)RM= Mean of replicate analysis of (C)RM = Set inter 95% tolerance interval 2)

(corrected for recovery)

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


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 32countries.FAPAS (Food Analysis Performance Assessment Scheme), Round 1729, February 2004.Ochratoxin Ain cereal. The commodity was milled wheat grain. The assigned value was 9.1 ng/g.

Analyst %Recovery Lab Value Final Reported Results a Z-score

1 84 8.9 10.6 0.82 110 11.7 10.6 0.8

a 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 inanalytical systems. The RSZ score is normally calculated from the Z scores of the last n PT rounds (n istypically 4 rounds). In this particular case it is based on two (C)RMs and two Proficiency Test results bytwo analysts.

Analyst Analysis Commodity Z-scores a

1 CRM Wheat -0.72 RM Barley -0.81 PT Wheat +0.82 PT Wheat +0.8

RSZ score b + 0.07

a See Appendix D3 and D4 for z-scoresb

RSZ score = =∑ Ζ ii

n

n1

Reference: Analytical Methods Committee; AMC Technical Brief No.16; Apr 2004; © Royal Society of Chemistry2004http://www.rsc.org/pdf/amc/brief16.pdf

http://www.rsc.org/pdf/amc/brief16.pdf


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 findinga naturally contaminated sample with ochratoxin A that might be used as a control sample for qualitycontrol purposes in routine analyses. The pasta samples and organic whole wheat, more than 3 kg wereput 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 wereground in a Brau Multipractic 100 food processor before putting through the Retch Mill ZM100 as thelatter 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 containabout 1.0 ng OTA/g, an appreciable level which can be used as a control sample. In order to establishthe homogeneity of the sample, the 3 kg ground sample was mixed again manually in the large plasticbag for at least an additional twenty minutes. Five replicates were analyzed and the level of ochratoxinfound 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 astandard deviation of 0.37 ng OTA/g and a relative standard deviation of 27%. The data clearly showedthe 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 inthe Retsch Mill ZM100 again using a 0.5 mm sieve. The entire sample was hand mixed in a largeplastic bag and about two equal portions were transferred into two large glass jars. Each jar, containingapproximately 1.5 kg of the finely ground sample, was placed into a vertical stainless steel tumbler andmixed for about 24 hours. Each jar had about 30% empty space which allowed thorough mixing. Thejars 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 referencematerial) 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 ngOTA /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 thatthe 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 1Lplastic 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 thefreezer. The label is marked that it is not to be used until its contents analyzed for n=5 as done for jar1577-A.

The remainder of 1577 whole wheat kernels of about 7 kg is kept sealed in its original sac and stored inthe freezer in sample control. The sample can be prepared and used as an OTA control sample in thesame manner.


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 generatedover time and through the routine use of the analytical method.

Control sample ID1577-A ResultsReplicates

Meann=5

SD %RSD

1 2 3 4 5

Level of OTA found ng/g 1.49 1.47 1.72 1.57 1.44 1.54 0.11 7.2

X Chart Control Limits

Control Limit: 99% confidence 1.54 ± 0.51 ng/g

Warning Limit: 95% confidence 1.54 ± 0.31 ng/g

Calculations of control limits for the X charts were based on formula 1 outlined in ONT-SOP-0044Rev. 2 -Guidelines for Control Charts:

Control Limit =mean ± (k(n-1, 99%) × F) = 1.54 ± (4.6 × 0.11) = 1.54 ± 0.51Warning Limit =mean ± (k (n-1, 95%) × F) = 1.54 ± (2.78 × 0.11)= 1.54 ± 0.31

where:k = the value determined from the Student’s t-distribution tables for a given confidence level with

(n-1) degrees of freedom.n = number of replicates, degrees of freedom = n-1F = standard deviationmean= average result

R Chart Control Limits : applicable to duplicate measurements (n=2)

Upper Control Limit (UCL) 3.6 (F ) = 3.6(0.11) = 0 .40 ng/g, or RPD at 26%

Upper Warning Limit (UWL) 2.8(F ) = 2.8 (0.11) = 0. 31 ng/g, or RPD at 20%

F= standard deviationRPD= 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.


Appendix F: Estimate of measurement uncertainty

F1. Principles

Key principles of uncertainty estimation:

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

representative range of analyte concentrations.)

Planning:

T To be representative of the normal operation of the method, uncertainty estimation is a plannedactivity 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 precision study;T trueness (bias) study;T 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 measurement result of a samplex expected (true) value for y(* +B) the combined bias (systematic error) where * is method bias and B is laboratory biasg random error at within-laboratory reproducibility conditions (intermediate precision)T other factors not adequately addressed

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


Prepare sample Purify sample Quantitatesample

Grind sample

Sub-sample

Extract analyte

IA column

Apply sample

Elute analyte

Concentrate

Filter

Wash

Inject sample

Determineconcentration

Detectorspecificity

Wash volume

Elution volume

Column lotsColumn recoveries

T ime

Prepare stocksolution

Prepare workingcalibrant for

standard curve

Volume

Volume

Sub-sampleweight

Sub-samplehomogeneity

LCChromatography

Mobile phaseComposit ion

Identification of sources of uncertainty

ONT-SOP-0065, Determination of Ochratoxin A in Cerealsand Pasta using Immunoaffinity Column Clean Up andHPLC with Fluorescence Detection

This chart represents basic steps in the analytical method.The list to the right of each step represents potential sourcesof uncertainty or variables that may impact this step.

Underlined = may require special consideration since itimpacts at many stages of the method or is judged asignificant potential source of error.

General aspects applicable to all stages:Variable solvents, reagents, calibration of pipettes andbalances, equipment, analyst, cross-contamination, etc...

Standardcurvelinearity

Expiry datesStorage

Flow rate

Flow rate ofNitrogen

T emperature

Prepareglassware

Preparestandards

Wash

Analyteconcentration


Calibration

Storage VolumeDilution

Dilute

Silanize vials

Variabletreatment ofglassware

Mobile phaseFlow rate


Sample type

Sampletype

Sampletype

Sampletype

Sample type

Flow rate ofNitrogen

Filter Analyteconcentration

Polytroncarryover

Extractiontime

Sampletype

T ime Amount Sample type

Extractionvolume

Filter sample

Volume

ColumnT emperature

Volume

Volume


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

F2. Summary of Uncertainty Estimate

Reported Value ± Expanded uncertainty 1

Ochratoxin A ± 45%

1 The expanded uncertainty is calculated using a coverage factor of 2 which gives a level of confidenceof 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 spikedand analyzed as one batch (repeatability conditions).Duplicate injections of each spike were made in order toalso assess instrumental variability (variable instrumentinjection volumes, chromatography,...). A one-wayanalysis of variance (ANOVA) was applied to the resultsin order to separate the error components. Total methodrepeatability is estimated as a relative standard deviationof 8.4%. See Appendix C2.

Wet chemistry variability 7.8

Operational variability 14.4 Represents the variability that is not accounted for by theestimate of method repeatability (i.e the variability overtime, between analysts, instruments, reagents, volumetrics,sample types and homogeneity...etc...). Derived bysubtracting the repeatability variance from theintermediate precision variance (See Appendix F3)

Bias

CRM + RM 14.2 Estimate from two different reference materials. CRM 472(wheat), RM (barley). See Appendix F4.

Calibration

UV spectrophotometer 4.2 Based on the maximum allowable precision and bias of theUV spectrophotometer calibration. See Appendix F5.

22.3 Combined relative standard uncertainty (1)

45 Expanded relative standard uncertainty (2)

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


( ) ( )Operational variability 16.6 8.4= − =2 2 14 4.

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, naturallyincurred with Ochratoxin A, at different concentration levels, by two different analysts ondifferent dates, on different HPLC systems and quantitative confirmation by LC-MS/MS by asecond 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 toAppendix C2). An estimate of the operational variability can be derived by subtracting themethod 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 theestimate of method repeatability.


tPre(Rm)

Recovery =

=−1 Recovery

CC

obs

crm

( )RmRecovery

=−

+1 2

2

kRmPre( )

Rm Rm=

Pre( )Recovery

F4. Bias Estimate - Estimated by analysis of a CRM and RM.

Explanation Calculations(Refer to F4.1 for data used in the calculations)

ResultsCRM RM

Step 1 Do preliminary Rm estimate = Pre(Rm)Rm is an estimate of the mean methodrecovery uncertainty obtained from theanalysis of a CRM or RM. (Basic propagation of error)

Pre(

S

C = Mean of replicate analysis of CRM

C = Certified value of CRM

= Standard Deviation of replicate analysis of CRM

n = number of replicate analysis of CRM

U(C = Standard deviation of the certified value of CRM

obs

crm

obs

crm)

Rm CC

Sn C

U CC

obs

crm

obs

obs

crm

crm) ( )= ×

×+

22

2

0.077 0.103

Step 2 Do significance test (t-test).Evaluate whether the recovery is significantlydifferent from 1 (100%). When the degrees offreedom are unknown, for example if there isa contribution from the uncertainty in thevalue of a reference material, compare t withk, the coverage factor that will be used in thecalculation of the expanded uncertainty. Inthis uncertainty estimate k=2.

2.93 2.06

Step 3 Do final Rm estimate based on the results ofsignificance test (t-test).

The t-test from Step 2 indicates this is a Case 3, since t>2 and inthe normal application of this method, recovery correction is notapplied.

Case 1: t # 2The significance test indicates that therecovery is not significantly different from 1so there is no reason to correct analyticalresults for recovery.

Rm = Pre(Rm) N/A N/A

Case 2: t > 2 & Corrected for recovery.As a correction factor is being applied, R m isexplicitly included in the calculation of theresult.

N/A N/A

Case 3: t > 2 & Not corrected for recovery.The significance test indicates that therecovery is statistically significantly differentfrom 1, but in the normal application of themethod no recovery correction is applied.The uncertainty is increased and then treatedas Case 1.

0.136 0.148

Step 4 Final estimate of biasAverage or pool the estimated uncertainties Average of the two estimated uncertainties

(0.142)14.2%


F4.1 Data used for the bias estimate

Reference Values (ng/g) Laboratory Results (ng/g)c Ccrm U(Ccrm) Cobs f Sobs g n h

CRM a 8.2 0.5 d 6.354 1.11 5 RM b 5.4 0.2 e 4.251 1.20 5

a Wheat, CRM 472b Barley flour, FAPAS reference material analyzed by 78 labs internationallyc Assigned value of (C)RM from reportd Standard uncertainty from certification report. Derived by dividing the 95% confidence interval by 2 (1.0 ÷ 2 ) e Standard uncertainty from FAPAS reportf Mean of replicate analysis of (C)RM (uncorrected for recovery)g Standard deviation of replicate analysis of (C)RMh 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 recoverycorrection is not applied.


5 3% ÷

( ) ( )3 2.92 2+

F5. Calibration of Standards

The uncertainty is based on the maximum allowable precision and bias of the UV spectrophotometercalibration provided by the potassium dichromate solutions.

Allowable range % Standard uncertainty

%RSD (max repeatability) < 3% 3% Correction factor (max bias) 1 ± 0.05 2.9% a

Total % standard uncertainty 4.2%a 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 curveNonlinearity would have contributed to the observed precision and is therefore inherently included inthe precision estimate. Refer also to Appendix B: Assessment of Linearity

Sample heterogeneity and matrix affectsPotential heterogeneity from the sample preparation step is taken into account since bulk naturallyincurred samples were prepared, sub-sampled and analyzed.Matrix affects are taken into account since in both the precision and bias estimates various matriceswere used.

Refer also to the following documents for typical in-house uncertainty estimates since during this studyvarious 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”.

Volumetrics 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 volumetricuncertainty.

2. ONT-FLD-0020 – Statistics: Uncertainty of Analytical Results.Section 5A; Attached spreadsheet and PDF file for typical in-house estimates of “volumetric uncertainties”.

métodos de validación

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