VALIDATIEDOSSIER DOSSIER DE VALIDATION SOP/TRA/ANA/04

Transcription

1 Leuvensesteenweg 17 - B 3080 Tervuren PRO/5.4/02/DOC01/V03 VALIDATIEDOSSIER DOSSIER DE VALIDATION SOP/TRA/ANA/04 11/09/2013 Page 1 of 31

2 PRO/5.4/02/DOC02/V01 HISTORIEK VAN DE VALIDATIEDOSSIER / HISTORIQUE DU DOSSIER DE VALIDATION SOP/TRA/ANA/04 DATUM / DATE FASE WIJZIGING / PHASE - MODIFICATION May 2011 October 2012 Method development November 2012-September 2013 January - July 2014 Method validation, and creation of first version of the document of validation Method validation for extra matrices: -feed based on terrestrial plants (K. Cheyns) - beverages based on terrestrial plants (A. Ruttens); + method validation principles for extensions: see addendum 4 11/09/2013 Page 2 of 31

3 PRO/5.4/02/DOC03/V01 TOEPASSINGSGEBIED VAN DE SOP / DOMAINE D APPLICATION DE LA SOP: Deze methode wordt toegepast op voeding (inclusief dranken) en voeders van plantaardige oorsprong (landplanten). Cette procédure s applique aux alimentations humaines et animales d'origine végétale (plantes terrestres) et aux boisons.. Definitions As Arsenic As species Specific chemical form of As defined according to e.g. its oxidation state or its molecular structure As III Arsenite As v Arsenate As i Inorganic arsenic (=As III +AS V ) Certified reference material (CRM) A certified reference material (CRM) is a material in which a specific analyte content has been specified. In the present study the CRM NMIJ 7503a (rice flour) is used. The material is certified for total As and for inorganic arsenic. Limit of quantification (LOQ) MultiQC Procedure blanc Speciation analysis Supplemented material The limit of quantification (LOQ) is the minimal concentration of an analyte which can be measured in a routine analysis. The limit of quantification for As species is calculated as 10 times the standard deviation of the background signal of the chromatogram control chart of the type Shewart test, or value of a test, corresponding to a complete analytical cycle (preparation+measurement), realized in conditions identical to the sample analysis, but in the absence of the sample analytical activity which identifies and/or quantifies chemical species sample enriched with a known quantity of the analyte of interest 11/09/2013 Page 3 of 31

4 VALIDATIEPARAMETERS / PARAMETRES DE VALIDATION The method concerns a quantitative method of confirmation including the following parameters: -calibration -limit of quantification -specificity -trueness -repeatability, reproducibility and measurement uncertainty -peak resolution 1. Analytical specificity The measurement of 20 blank samples, as described in PRO/5.4/02, to demonstrate analytical specificity is not relevant for our method. Arsenic is a natural constituent of the earth crust and is ubiquitous in all biological matrices. This means that it is impossible to find blank samples. In trace element analysis by means of ICP-MS, analytical specificity can refer to the absence of significant interferences during the measurements. In case of speciation analysis of arsenic, the most important potential interference is caused by chloride, because the 40 Ar 35 Cl + and 38 Ar- 37 Cl polyatomic ions can interfere with the detection of As species on mass 75. On VARIAN ICP-MS this interference can be counteracted by the use of H 2 as a reaction gas. During the As speciation measurements a H 2 flow of 90mL/min is activated (cfr. SOP/TRA/ANA/04). In order to verify a potential disturbance of the As speciation chromatograms by a Cl - interference signal, HCl solutions of various concentrations 0.1%, 0.2%, 0.5%, 1% were injected and analysed by HPLC-ICP-MS. On these chromatograms, at all injected HCl concentrations, a small peak at the retention time of As V is observed. However, even at the highest Cl concentration this peak is more than ten times below the LOQ of the method (for a detail of results see file Asi_interference Cl in 71\ACCREDITATIE\ I ARSENIC INORGANIC \VALIDATIEDOSSIER inorganic arsenic). Most likely the peak originates from the presence of a very low concentration of inorganic As in the acid, and not from a chloride interference on the 75 As signal. Moreover, such high Cl - concentrations are not expected to be present in the extracts. The potential presence of matrix specific effects or matrix specific interferences is investigated and discussed in paragraph 10 for various food samples of plant origin (rice, carrot, champignons). 2. Robustness The parameters that have to be considered are discussed on the page PRO/5.4/02/DOC04/V01 11/09/2013 Page 4 of 31

5 3. Precision (repeatability reproducibility) 3.1. FOOD of vegetal origin Currently no performance criteria have yet been laid down for inorganic arsenic by the Commission of the European Communities. The performance criteria laid down in Commission Regulation 333/2007 (for amongst others cadmium and lead) were used as a guide in the present document. According to the latter Commission Regulation the performance criterion for precision is HORRAT R < 2. The data used to calculate HORRAT R are the data from CRM NMIJ7503A (rice flour; certified for total As at 98±7 µg/kg, As III at 71.1 ± 2.9 µg/kg and As V at 13.0 ±0.9 µg/kg; total inorganic As can be calculated as 84.1±3.0 µg/kg) introduced in the MultiQC between 31/10/2012 and 21/02/2013 as these data represent reproducibility conditions. The HORRAT R values for inorganic arsenic (cfr. Table 1) are below 2 HORRAT R = Observed RSD R /RSD R Horwitz Table 1. HORRAT R values based on the CRM NMIJ 7503 a (rice flour) Asi µg/kg Mean 90.1 SD 3.7 RSD R 4.1 RSD Horwitz 22 HORRAT R Solid food of vegetal origin Repeatability and reproducibility (intermediate precision) were calculated based on different samples of plant origin being: rice, carrots and champignons, supplemented with stock solutions containing inorganic arsenic (spiked as As III ), at two different levels and measured on three different days. The spiking levels were: For carrot and champignon: Natural level of inorganic arsenic (2.4 µg/kg for carrot, 1.6 µg/kg for champignon) 96 µg/kg 192µg/kg For rice: Natural level of inorganic arsenic (136 µg/kg) 272 µg/kg 476 µg/kg The calculations can be found in ValMethTerv 1.4 inorganic As (71\ACCREDITATIE\ARSENIC INORGANIC\V01\repe-repro). The theoretical natural level was determined as the average of the data for the unspiked samples as determined in a preliminary experiment. 11/09/2013 Page 5 of 31

6 Table 2. Overview of repeatability and reproducibility of Asi in food matrices of vegetal origin (solid). Matrix Level RSD r RSD rw Rice Low Medium High Carrot Low Medium High Champignon Low Medium High The long term reproducibility (intermediate precision) will be calculated when sufficient data will be available. Addenda: -template ValMethTerv 1.4 inorganic arsenic in rice -template ValMethTerv 1.4 inorganic arsenic in carrot -template ValMethTerv 1.4 inorganic arsenic in champignon Beverages of vegetal origin Repeatability and reproducibility (intermediate precision) were calculated based on the repeated analyses of routine samples of beverages of vegetal origin. Eight real samples of vegetal milk and 7 real samples of wine were analysed twice (each time in duplicate) on day one and once (in duplicate) on day 2. The repeatability (RSD r ) and intermediate precision (RSD rw ) for beverages of vegetal origin are calculated in the measurement uncertainty template of the FASFC (MU_cal_v03_2 Asi plant_food solid+liquid_v2010.xlsm). Table 3. Repeatability and reproducibility of Asi in beverages of vegetal origin. Matrix RSD r RSD rw Wine and vegetal milk (real samples) The repeatability and reproducibility (intermediate precision) for Asi in beverages (wine, vegetal milk) are comparable to the repeatability and reproducibility of Asi in solid food samples of vegetal origin. Addenda: -template MU_cal_v03 2 Asi plant_food solid+liquid_ v2010.xlsm 3.2. FEED of vegetal origin Currently no performance criteria have yet been laid down for inorganic arsenic in feed by the Commission of the European Communities. The performance criteria laid down in Commission Regulation 333/2007 (for amongst others cadmium and lead in food) were again used as a guide in the present document. According to the latter Commission Regulation the performance criterion for 11/09/2013 Page 6 of 31

7 precision is HORRAT R < 2. The data used to calculate HORRAT R are the data from CRM NMIJ7503A (rice flour; certified for total As at 98±7 µg/kg, As III at 71.1 ± 2.9 µg/kg and As V at 13.0 ±0.9 µg/kg; total inorganic As can be calculated as 84.1±3.0 µg/kg) introduced in the MultiQC between 31/10/2012 and 04/07/2014 as these data represent reproducibility conditions. The HORRAT R values for inorganic arsenic are below 2 (cfr. Table 4). HORRAT R = Observed RSD R /RSD R Horwitz Table 4. HORRAT R values based on the CRM NMIJ 7503 a (rice flour): period 31/10/ /07/2014. Asi µg/kg Mean 83.0 SD 7.4 RSD R 8.9 RSD Horwitz 22 HORRAT R 0.41 Repeatability and reproducibility (intermediate precision) were calculated based on different feed samples of vegetal origin being: lucerne, palm kernel, fodder beet and a complete feed for chicken (Gramix), supplemented with stock solutions containing inorganic arsenic (spiked as As III ), at three different levels and measured on three different days. The spiking levels were: 250 µg/kg (330 µg/kg for lucerne, the natural concentration) 2000 µg/kg 4000 µg/kg The calculations can be found in ValMethTerv 1.4 As inorganic FEED (71\ACCREDITATIE\ARSENIC INORGANIC\V02\Precision-accuracy). Table 5. Overview of repeatability and reproducibility of Asi in feed matrices of vegetal origin (solid). Matrix Level RSD r RSD rw Lucerne Low Medium High Palm kernel Low Medium High Fodder beet Low Medium High Gramix Low Medium High /09/2013 Page 7 of 31

8 Addenda: -template ValMethTerv 1.4 As inorganic FEED 4. Accuracy/ recovery 4.1 FOOD of vegetal origin Solid food of vegetal origin Accuracy indicates the deviation between the mean value found and the true value. Currently only a rice certified reference material is available in which the concentrations of As III and As V are certified. For rice the determination of accuracy is based on the difference between the sum of the certified values (As i =As III + As V ) and the measured value of As i. For the other matrices accuracy of the method is illustrated by calculating the recovery of spiked samples. This can be determined by applying the inorganic arsenic analysis method to samples to which known amounts of inorganic arsenic species have been added. The accuracy is then calculated from the test results as a percentage of the analyte recovered. The data that have been used to determine the accuracy in the vegetable samples, are the measurement results of spiked samples used for precision calculation. The calculations can be found in Accuracy calculations inorganic As SEPT.xlsx (71\ACCREDITATIE\ARSENIC INORGANIC\V01\precision-accuracy). For the certified rice sample the bias of the measured value compared to the certified value is statistically significant. Due to the fact that the uncertainty on the certified value and the uncertainty on the measurement of that material are both very small (1.8% and 0.7% respectively), even a small bias easily becomes significant. Given the fact that the bias is included in the calculation of the measurement uncertainty (see 7. Measurement uncertainty ), we consider a bias of less than 10% acceptable. Table 6: Mean accuracy for inorganic arsenic in different food matrices of vegetal origin. As i Rice 107% Carrot 108% Champignons 109% CODA-CERVA has participated in two proficiency tests (up till July 2014): IMEP-116 in which inorganic arsenic had to be determined in mushrooms. The value reported by CODA-CERVA was 0.42 ± 0.10 mg/kg (k = 2), while the assigned value was ± mg/kg (k = 2). This resulted in a z-score of 1.6 (satisfactory) IMEP-188 in which inorganic As had to be determined in canned food. The value reported by CODA-CERVA was ± mg/kg (k = 2) while the assigned value was ± mg/kg. This resulted in a z-score of (satisfactory). 11/09/2013 Page 8 of 31

9 beverages of vegetal origin Currently no certified reference materials are available with certified values for Asi in beverages of vegetal origin. The accuracy was determined by calculating the recovery of spiked samples. This was performed by applying the inorganic arsenic analysis method (SOP/TRA/ANA 04) to wine and vegetal milk samples to which known amounts of inorganic arsenic (spiked as As III ) were added. The accuracy was calculated from the test results as a percentage of the analyte recovered. The spiking levels were For rice milk: 50 µg/kg 75 µg/kg 200 µg/kg For wine: 79 (10x the natural level) 158 (20x the natural level) 237 (30x the natural level) The calculations can be found in Accuracy calculations inorganic As plant_food solid+liquid+feed.xlsx (71\ACCREDITATIE\ARSENIC INORGANIC\VALIDATIEDOSSIER inorganic arsenic\v02\precision-accuracy). Table 7: Mean accuracy for inorganic arsenic in beverages of vegetal origin. Matrix Recovery As i Wine 101% Vegetal milk 106% 4.2. FEED of vegetal origin Currently no certified reference materials are available with certified values for Asi in feed of vegetal origin. The accuracy was determined by calculating the recovery of the spiked samples used to calculate repeatability and reproducibility. The calculations can be found in Accuracy calculations inorganic As plant_food solid+liquid+feed.xlsx (71\ACCREDITATIE\ARSENIC INORGANIC\VALIDATIEDOSSIER inorganic arsenic\v02\precision-accuracy). Table 8: Mean accuracy for inorganic arsenic in feed samples of vegetal origin. Matrix Recovery As i Lucerne 100% Palm kernel 105% Fodder beet 95% Gramix 98% 5. Beslissingsgrens (CCα) en detectievermogen (CCβ) 5.1. FOOD of vegetal origin As there are no legal limits yet for As species in food of vegetal origin, CCα and CCβ are not relevant. 11/09/2013 Page 9 of 31

10 5.2. FEED of vegetal origin There are no legal limits for As species in feed of vegetal origin, although for palm kernel expeller there is a footnote in Directive 2002/32/EC that says that upon request of the competent authorities, the responsible operator must perform an analysis to demonstrate that the content of inorganic arsenic is lower than 2 ppm. For palm kernel, a CCα of 2.3 mg/kg and a CCβ of 2.6 mg/kg are calculated. 6. Calibration (Linearity of the method) The calibration is an external calibration of the linear type. Calibration curves for As V (representing inorganic As in the sample) are presented in the file calibration Asi (71\ACCREDITATIE\ARSENIC INORGANIC\VALIDATIEDOSSIER inorganic arsenic). The concentrations used are the following: 0, 0.5, 1, 2, 5, 10 en 25 pbb. Based on these concentrations 2 different calibration curves were made: one including all concentrations, and a second one including only the 5 lowest concentrations levels. As these low concentrations correspond to the range of expected sample concentrations (taking into account the dilution factor), and as no difference is observed between the 2 calibration lines, calibration range during routine measurements will be limited to the five lowest concentrations. Calibration details are shown in figure 1 below. Observed R 2 value is > 0.99, and residuals are <10%. The random variation of the residuals (Res%) with increasing standard concentrations supports the trueness of the linear model. When higher Asi concentrations are present/expected in the samples, there is no need to extent the calibration range (not up to 25 ppb), because -as illustrated in Figure 1- the calibration curve remains linear also at these higher concentration levels. A. Area 10,500 10,000 9,500 9,000 8,500 8,000 7,500 7,000 6,500 6,000 5,500 5,000 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1, ppb 4 5 Regression equation: y= x R 2 = Name ppb area area(recalc) Res % Point Point /09/2013 Page 10 of 31

11 Point Point Point B. 45,000 40,000 35,000 30,000 Area 25,000 20,000 15,000 10,000 5, ppb Regression equation: y= x R 2 =1.000 Name ppb area area(recalc) Res % Point Point Point Point Point Point Point Figure 1: Examples of As V calibration curves in case of A. calibration range 0-5 ppb, B. calibration range 0-25 ppb. 11/09/2013 Page 11 of 31

12 7. Measurement uncertainty 7.1 FOOD of vegetal origin Solid food of vegetal origin The measurement uncertainty was calculated via the method imposed by the Federal Agency for the Safety of the Food Chain 1. The combined measurement uncertainty is calculated from the intrareproducibility and the bias. %u = (%u Rw ² + %u bias ²) The intra-reproducibility is calculated from the control chart of a certified rice sample (NMIJ 7503 a): %u Rw ² = RSD cc ² The data from section 4 (accuracy) were used to calculate the bias for rice (based on NMIJ 7503a) and for carrot and mushroom (based on recovery experiments). rice: %u bias = %b² + RSD bias ² + %u CRM ² carrot, mushroom : %u bias = (Σ%b i ²)/n with %b i = (x i -C spike, i )/C spike, I and n the number of recovery experiments As only one control material was used, measurement uncertainties were not calculated at different concentration levels with this method. The expanded measurement uncertainty is the product of the combined uncertainty and the coverage factor k = 2, which corresponds to a confidence level of approximately 95%. The calculations of the measurement uncertainties can be found in Measurement uncertainty inorganic arsenic (71\ACCREDITATIE\ ARSENIC INORGANIC\VALIDATIEDOSSIER inorganic arsenic). The combined standard uncertainty u c is 9% for rice, 12% for carrot and 14% for mushroom. The extended uncertainty U is 18% for rice, 24% for carrot and 28% for mushroom. As an alternative, the measurement uncertainty can also be calculated via the ISO/BIP GUM method 2 from the uncertainties related to repeatability (r), intermediate precision (ip) and trueness (t): u c = Uncertainties related to repeatability and intermediate precision are taken from section 3 (see ValMethTerv 1.4 inorganic As ). The uncertainty related to trueness is taken from the accuracy determination (section 4). The uncertainty calculations via the ISO/BIP GUM method are included as an illustration, but will not be used for real samples. The extended uncertainty U based on the ISO/BIP GUM method varies from 10 to 16% at the low level, 13-21% at the median level, and 10-23% at the high level. 1 Procedure Bepaling van de meetonzekerheid voor kwantitatieve chemische analyses. LAB P 508 Meetonzekerheid-v.01-nl. Goedgekeurd 03/11/ ISO/BIPM GUM Evaluation of measurement data Guide to the expression of uncertainty in measurement. JCGM 100:2008 GUM 1995 with minor corrections. 11/09/2013 Page 12 of 31

13 Recently, a new template of the FASFC is available that has to be implemented in the near future to calculate the measurement uncertainty of analytes within the framework of official controls. To be consistent, the new template is also applied on the existing data for food of vegetal origin (solids). The data that have been used in the template to calculate the measurement uncertainty are: Sheet validation : the measurement results of the spiked rice, carrot and champignon samples (section and ) Sheet control chart (1) : no data Sheet reference material (2) : the analysis results of the CRM NMIJ 7503a from the period 31/10/ /02/2013 Sheet PT (3) : the result of PT IMEP-116 The template checks for outlying values, and whether a single measurement uncertainty can be calculated over the whole concentration range (taking into account the precision and bias at different concentration levels). If this is the case, the template calculates several measurement uncertainties in different ways (using different formulas and/or different sets of data). The final measurement uncertainty is the mean of the calculated measurement uncertainties, excluding the lowest and highest value. The inorganic arsenic data that were introduced in the FASFC template allowed for the calculation of a single measurement uncertainty over the spanned concentration range. Calculated extended measurement uncertainties ranged from 22 to 28%. The mean extended measurement uncertainty for solid samples of vegetal origin is 24.7%. Addenda: -template MU_cal_v03 2 Asi plant_food solid_v2010.xlsm Beverages of vegetal origin The new FASFC template has been applied on the data for food of vegetal origin (beverages +solids). The basis was the filled in template for the solid food of vegetal origin, to which data for beverages of vegetal origin were added. The data that have been used in the template to calculate the measurement uncertainty are: Sheet validation : the measurement results of the spiked rice, carrot, champignon, wine and vegetal milk samples (section , and ) Sheet control chart (1) : the analysis results of the CRM NMIJ 7503a from the period 18/12/ /07/2014 Sheet reference material (2) : the analysis results of the CRM NMIJ 7503a from the period 31/10/ /02/ additional results from the period 15/04/ /12/2013 Sheet PT (3) : the result of PT IMEP-116 Sheet real samples : the repeated analyses of real wine and vegetal milk samples (see section 3.1.2) The inorganic arsenic data that were introduced in the FASFC template allowed for the calculation of a single measurement uncertainty over the spanned concentration range. Calculated extended measurement uncertainties ranged from 18 to 23. The mean extended measurement uncertainty for solid and liquid food samples is 20.6%. 11/09/2013 Page 13 of 31

14 Given the larger amount of data under reproducibility conditions on which the measurement uncertainty for solid food samples + beverages is based, the value of 21% will be applied to all food samples of vegetal origin (including beverages). Addenda: -template MU_cal_v03 2 Asi plant_food solid+liquid_v2010.xlsm 7.2. FEED of vegetal origin The new FASFC template has been applied on the data for feed of vegetal origin. The basis was the filled in template for the solid foods of vegetal origin, to which data for feed of vegetal origin have been added. The data that have been used in the template to calculate the measurement uncertainty are: Sheet validation : the measurement results of the spiked rice and of the four feed samples (section and 3.2.) Sheet control chart (1) : the analysis results of the CRM NMIJ 7503a from the period 18/12/ /07/2014 Sheet reference material (2) : the analysis results of the CRM NMIJ 7503a from the period 31/10/ /02/ additional results from the period 15/04/ /12/2013 Sheet PT (3) : the result of PT IMEP-116 The inorganic arsenic data that were introduced in the FASFC template allowed for the calculation of a single measurement uncertainty over the spanned concentration range. Calculated extended measurement uncertainties ranged from 18 to 20%. The mean extended measurement uncertainty is 19.0%. This value will be applied to feed samples of vegetal origin. Addenda: -template MU_cal_v03 2 Asi plant_food solid+feed_v2010.xlsm 8. Quantification limits The limit of quantification (LOQ) is the minimal concentration of an analyte which can be measured in a routine analysis. The limit of quantification for inorganic arsenic corresponds to 10 times the standard deviation of the background signal of the chromatogram. An LOQ of 0.02 µg/l was calculated in the previous validation report of SOP 04. For the current validation report the LOQ is calculated starting from chromatograms of the relevant matrices (and not from the CRM as has been done before). Standard deviations of the background signal of the chromatograms were calculated for a zone of 0.5 minute at the end of the chromatogram. In this way, extra variation caused by tailing effects of high As V peaks are avoided. Average calculated LOQ values (µg/kg) obtained on different measurement days are shown in table 4. Table 4. Calculated LOQ values in solution (µg/l) As V Average signal (c/s) Average standard deviation (c/s) /09/2013 Page 14 of 31

15 Relative standard deviation (RSD) 5.5% LOQ in solution (µg/l) 0.1 The calculations are detailed in the file LOD_LOQ calculations (71\ACCREDITATIE\ ARSENIC INORGANIC \Validatiedossier inorganic As\V02\LOD_LOQ)) These calculated LOQ values were verified by measuring As V solutions with similar concentration levels (0.1 µg/l, 0.2 µg/l, 0.3 µg/l and 0.4 µg/l) in triplicate. The results are shown in de file Val Asi_LOQ injecties (cfr. 71\ACCREDITATIE\ ARSENIC INORGANIC\VALIDATIEDOSSIER inorganic arsenic\lod_loq). Concentrations of 0.1 µg/l were measured accurately taking into account the calculated measurement uncertainties, which confirms the suitability of the calculated LOQ-value. A summary of LOQ values, at different dilution factors of the matrix is presented in table 5. Table 5. Final LOQ values after recalculation to concentration in matrix (µg/kg) taking into account the dilution factor (DF) As V LOQ in solution LOQ in matrix for DF=5 LOQ in matrix for DF=10 LOQ in matrix for DF =20 LOQ in matrix for DF= Decisions related to the repeatability of results Because of the potential heterogeneity of biological samples, a homogenisation step is included before subsamples are taken for analysis. Indeed, heterogeneity of the sample influences the repeatability of results (variation among subsamples). Each analysis will therefore be performed in 2 replicates, and the following criteria will be taken into account:-values of the reference sample need to fall within the limits specified in the file Accept CRM. These values take into account the extended measurement uncertainty of the analysis. -To be accepted, measurement results have to be higher than the LOQ. Results below this value will be reported as <LOQ. - Analyses will be performed in 2 replicates. The coefficient of variation (VC) needs to be VC 20% for CRM values mg/kg and VC 15% for CRM values > mg/kg. In real samples, the coefficient of variation needs to be 30% for results mg/kg and VC 20% for results > mg/kg. These values are based on the repeatability limit. -When the coefficient of variation is higher than those values, the analysis will be repeated in 2 replicates. 10. Peak resolution 11/09/2013 Page 15 of 31

16 Peak resolution (Rs) can be calculated according to the formula Rs= 2(Tr2-Tr1)/(W1+W2), with Tr2 en Tr1 retention times of two consecutive peaks, and W2 en W1 baseline width of the corresponding peaks. Baseline peak width (W) corresponds to the distance between the baseline intercepts of tangent lines to the front slope and the back slope of a peak (Figure 6). This baseline peak width, W, is considered equivalent to the peak width at 13.4 % peak height, or for Gaussian peaks can be approached by taking 1.7* peak width at 50% peak height. Figure 6: Illustration of the baseline peak witdt, W, which corresponds to the distance between the baseline intercepts of tangent lines to the front slope and the back slope of the peak. A typical chromatogram of a 5 ppb standard solution containing a mix of 4 As species (AB, DMA, MMA, As V ) is presented in Figure 7. 60,000 55,000 50,000 45,000 40,000 c/s AsB DMA solb As b_5ppb_130416_29.data 35,000 30,000 25,000 MMA As5 20,000 15,000 10,000 5,000 SPW 0.70 STH STH # Name Time [Min] Quantity [ppb] Height [c/s] Area [c/s.min] Area % [%] Width 13.4% [Min] 1 AsB DMA MMA As Figure 7: Chromatogram of a 5 ppb standard solution containing a mix of 4 As species (AB, DMA, MMA, As V ) RT [min] 11/09/2013 Page 16 of 31

17 Values for retention times and peak width at 13.4% are presented in Figure 7. For the current method, describing the determination of Asi in food of plant origin, only the resolution of the peak-pair MMA-As V is important. Using the values presented in Figure 7 the resolution, Rs, of this peak-pair corresponds to a value Rs= 2.3, indicating an adequate separation. Rs values >1.5 are characteristic for well separated peaks. 11/09/2013 Page 17 of 31

18 PRO/5.4/02/DOC04/V01 FACTOREN DIE DE JUISTHEID EN BETROUWBAARHEID VAN DE BEPROEVINGEN BEÏNVLOEDEN FACTEURS QUI INFLUENCENT L EXACTITUDE ET LA FIABILITE DES ESSAIS SOP/TRA/ANA/03 Parameter Composants character Control 1.Equipement and laboratorium material 2. Products and samples 3.Method principles mixer balances pipettes glassware for standards microwave teflon tubes for microwave ICP-MS HPLC pumps HPLC autosampler ph-meter milliq water Standards for As species Ammonium carbonate mobile phase 1 routine samples 2 ringtest samples Ion exchange colonne non relevant if clean and working relevant relevant non relevant if clean relevant non relevant if clean relevant relevant non relevant if working and injection volume constant relevant non relevant if produced daily fresh relevant relevant relevant relevant (physic-chemical characteristiques of the column) non relevant because of thermostatisation by oven mixer is cleaned with bi-distilled water after use maintenance by external firm 1x/year and daily verification control by external firm 2x/year and maintenance by external firm 1x/year flasks stored in bi-distilled water control by external firm 3x/year and maintenance by external firm 1x/year. cleaning of tubes with HNO3 25% after use (wash cycle) verification of tube state at each use; if cracks are observed in a tube, it is not used anymore 1 maintenance by external firm 1x/year or interventions mentioned in logbook 2 daily control of sensitivity of the apparatus by use of a tuning solution (cfr SOP/TRA/ANA/02). intervention by firm when a pump related problem is observed (see logbook ICP-MS) check injection volume 1x/year (RSD<0.5%) ph meter is calibrated daily and T dependency is taken into account via maintenance mentioned in logbook and daily verification daily fresh preparations of calibration dilutions constant use of same mark daily fresh preparation 1 verification of extraction efficiency in sample (total As in extract versus total As after mineralisation) 2 contrôle of Z-score of PT-Test constant use of same column mark cleaning or replacement of column if problems are observed (high pressure, bad peak separation, tailing of peaks, ) Temperature of HPLC-chain control of oven temperature 1x/year 4.Room Room daily tuning of ICP-MS signal with a tuning temperature temperature of solution optimises signal in function of daily non relevant LC-ICP-MS conditions (cfr SOP/TRA/ANA/02) laboratory 5.Staff Analists, technical relevant first, second, third line controls 11/09/2013 Page 18 of 31

19 responsible formation of people 11/09/2013 Page 19 of 31

20 Addendum 1: Template ValMethTerv 1.4 inorganic arsenic in rice Notes for user : This page is for up to 5x10x3. Only blue cells are for inputs. N(repl) and N(days) calculated automatically (Check!). For CCa etc see instructions on line 46. CALCULATION of REPEATABILITY, WITHIN-LAB REPRODUCIBILITY, CCa, CCb and RECOVERY ACCORDING TO 2002/657/EC, ISO and Van Loco & Beernaert (2004) DATA : RESULTS AND GRAPHS : Analyte : Asi spiked in rice Slope intercept formulas : SSrepl = Ed Er (Y-Ymoy(level,day))2 Method : 121.8% Srep2 = Srepl2 = MSrepl = SSrepl/(n-ndays) MRL : 272 NB: no MRL available! SSdays = nrepl E(Ymoy(level,day)-Ymoy(level))2 unit : ug/kg t(26,0.05) Yc CCa CCb MSdays = SSdays / (ndays-1) Nrepl : 3 (number of replicates) ug/kg % of MRL ug/kg % of MRL Sdays2 = (Msdays-Msrepl) / nrepl Ndays : 3 (number of days or analysts) % % Srw2 = Sdays2 + Srep2 Nlev : 3 (number of levels)) nom.ccn av.meas. uncertainty (st.dev.) ext. unc. (2 x st.dev.) Measured concentrations : ug/kg ug/kg ug/kg % ug/kg % (NOT corrected for recovery) : % % % % day or analyst : % % level 1 : level 2 : level 3 : replicate : replicate : replicate : SSrepl MSrepl Srepl average for level SSdays MSdays Sdays Sdays Srw SSrepl MSrepl Srepl average for level SSdays MSdays Sdays Sdays Srw SSrepl MSrepl Srepl average for level SSdays MSdays Sdays Sdays Srw Level Level Level % 6% 5% 4% 3% 2% 1% 0% linear regression y = 1.218x R² = Uncertainty Srepl Sdays Srw Uncertainty (%) reps days rw /09/2013 Page 20 of 31

21 Addendum 2: Template ValMethTerv 1.4 inorganic arsenic in carrot Notes for user : This page is for up to 5x10x3. Only blue cells are for inputs. N(repl) and N(days) calculated automatically (Check!). For CCa etc see instructions on line 46. CALCULATION of REPEATABILITY, WITHIN-LAB REPRODUCIBILITY, CCa, CCb and RECOVERY ACCORDING TO 2002/657/EC, ISO and Van Loco & Beernaert (2004) DATA : RESULTS AND GRAPHS : Analyte : Asi spiked in carrots Slope intercept formulas : SSrepl = Ed Er (Y-Ymoy(level,day))2 Method : 104.1% 1.57 Srep2 = Srepl2 = MSrepl = SSrepl/(n-ndays) MRL : 96 NB: no MRL available! SSdays = nrepl E(Ymoy(level,day)-Ymoy(level))2 unit : ug/kg t(26,0.05) Yc CCa CCb MSdays = SSdays / (ndays-1) Nrepl : 3 (number of replicates) ug/kg % of MRL ug/kg % of MRL Sdays2 = (Msdays-Msrepl) / nrepl Ndays : 3 (number of days or analysts) % % Srw2 = Sdays2 + Srep2 Nlev : 3 (number of levels)) nom.ccn av.meas. uncertainty (st.dev.) ext. unc. (2 x st.dev.) Measured concentrations : ug/kg ug/kg ug/kg % ug/kg % (NOT corrected for recovery) : % % % % day or analyst : % % level 1 : level 2 : level 3 : replicate : replicate : replicate : SSrepl MSrepl Srepl average for level SSdays MSdays Sdays Sdays Srw SSrepl MSrepl Srepl average for level SSdays MSdays Sdays Sdays Srw SSrepl MSrepl Srepl average for level SSdays MSdays Sdays Sdays Srw Level Level Level % 8% 6% 4% 2% 0% linear regression y = x R² = Uncertainty Srepl Sdays Srw Uncertainty (%) reps days rw /09/2013 Page 21 of 31

22 C O D A-TERVUREN - C E R V A-TERVUREN Addendum 3: Template ValMethTerv 1.4 inorganic arsenic in mushroom 11/09/2013 Page 22 of 31

23 Addendum 4: Method validation principles applied to extensions of the validation dossier When extending a validation dossier for an existing SOP, the concerned technical responsible decides which validation parameters have to be determined. Depending on the type of extension, different approaches can be followed: a. Extension with new elements: preferentially the same approach as for an initial validation dossier should be followed b. Extension with new matrices: an alternative method can be followed (see below) c. Extension with a new measurement device (i.e. new device of the same type): an alternative method can be followed (see below) Alternative method: For the determination of precision, accuracy or recovery and the measurement uncertainty, following approaches can be followed: Precision In order of availability of the appropriate materials, the precision (repeatability, intermediate precision and/or reproducibility) can be determined as follows 1. The analysis of certified reference materials (under reproducibility conditions) of the matrix for which the extension applies 2. The analysis of routine samples of the matrix for which the extension applies: at least 8 samples should be analysed under repeatability conditions (twice a day) and under reproducibility conditions (two different days). => Extension of SOP04 with beverages of vegetal origin (terrestrial plants) The precision calculated from these data should be compared to the precision calculated in the initial validation dossier. If no suitable CRMs or routine samples are available, the approach of the initial validation dossier should be followed. => Extension of SOP04 with feed Accuracy or recovery In order of availability of the appropriate materials, the accuracy or recovery can be determined as follows: 1. The analysis of proficiency test materials (PT materials): at least 8 PT materials should be analysed 2. The analysis of CRMs: at least 6 analyses of one or multiple CRMs 3. Recovery: known amounts of the analyte are added to an appropriate matrix with a low analyte content: at least six aliquots of the matrix should be prepared: to each time two aliquots, three different concentrations should be added to get a representative range of concentrations => Extension of SOP04 with beverages and feed of vegetal origin Measurement uncertainty The new FASFC template can be used to calculate the extended measurement uncertainty. The basic data are those from the initial validation dossier, to which new data related to the extension are added. If there are no gross differences between the extended measurement uncertainty based on data from the initial validation dossier and the measurement uncertainty of the extended validation dossier, a common measurement uncertainty can be calculated. 11/09/2013 Page 23 of 31

24 Addendum 5: Template MU_cal_v03 2 Asi plant_food solid+liquid_ v2010.xlsm, sheet real samples 11/09/2013 Page 24 of 31

25 C O D A-TERVUREN - C E R V A-TERVUREN Addendum 6: Template ValMethTerv 1.4 Lucerne 11/09/2013 Page 25 of 31

26 C O D A-TERVUREN - C E R V A-TERVUREN Gramix 11/09/2013 Page 26 of 31

27 C O D A-TERVUREN - C E R V A-TERVUREN Palm kernel 11/09/2013 Page 27 of 31

28 C O D A-TERVUREN - C E R V A-TERVUREN Fodder beet 11/09/2013 Page 28 of 31

29 Addendum 7: Template MU_cal_v03 2 Asi plant_food solid_ v2010.xlsm, sheet decision tree 11/09/2013 Page 29 of 31

30 Addendum 8: Template MU_cal_v03 2 Asi plant_food solid+liquid_ v2010.xlsm, sheet decision tree 11/09/2013 Page 30 of 31

31 Addendum 9: Template MU_cal_v03 2 Asi plant_food solid+feed_ v2010.xlsm, sheet decision tree 11/09/2013 Page 31 of 31