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C O D A-TERVUREN - C E R V A-TERVUREN

Leuvensesteenweg 17 - B 3080 Tervuren

PRO/5.4/02/DOC01/V03

VALIDATIEDOSSIER

DOSSIER DE VALIDATION

SOP/TRA/ANA/0y


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PRO/5.4/02/DOC02/V01

HISTORIEK VAN DE VALIDATIEDOSSIER / HISTORIQUE DU DOSSIER DE VALIDATION

SOP/TRA/ANA/0y

DATUM / DATE

FASE – WIJZIGING / PHASE - MODIFICATION

January 2013 – October 2013

November 2013-January 2014

Method development

Method validation, and creation of first version of the

document of validation


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PRO/5.4/02/DOC03/V01

TOEPASSINGSGEBIED VAN DE SOP / DOMAINE D’APPLICATION DE LA SOP:

This method is applicable for Se-enriched premixes for feed and for Se-enriched human food supplements.

These matrices, although they are different in end-use, are very similar as both matrices include mineral

carriers. These substrates are commonly enriched in Se with selenite, seleno-methionine or seleno-methionine

from enriched yeasts.

Precision and trueness of the method is calculated based on three different matrix-relevant samples, being: a

feed premix enriched with selenite (PREMIX), a food supplement enriched with a Se-enriched yeast (SUP1) and

another food supplement not enriched with both species (SUP2). The samples were amended with stock

solutions containing SeIV or SeMet, at three different levels, and were measured on three different days. As the

PREMIX sample is enriched with selenite, further spikings with selenite are done for this specie. The SUP1 sample

is enriched with a seleno-methionine enriched yeast so further spikings are done with seleno-methionine. Extra

spikings for both species are done in SUP2.

Definitions

Se Selenium

Se species Specific chemical form of Se defined according to e.g. its oxidation

state or its molecular structure

SeIV

Selenite

SeMet Selenomethionine

SeVI

Selenate

MeSeCys Methyl-seleno-cysteïne

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 SELM-

1 (seleno-yeast) is used. The material is certified for total Se and

Selenomethionine but not for other individual Se species.

Limite of quantification (LOQ) 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 Se species is calculated as 10 times the standard

deviation of the background signal of the chromatogram

MultiQC control chart of the type ‘Shewart’

Procedure blanc 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

Speciation analysis analytical activity which identifies and/or quantifies chemical species

Supplemented material Sample enriched with a known quantity of the analyte of interest


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VALIDATIEPARAMETERS / PARAMETRES DE VALIDATION

The method concerns a quantitative method of confirmation including the following parameters:

1. Analytical specificity

2. Robustness

3. Precision: repeatability and reproducibility

4. Trueness

5. Calibration

6. Measurement uncertainty

7. Quantification limits

8. Decisions related to the repeatability of results

9. 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. Selenium 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 selenium, the most important potential

interference is caused by the argon gas, because the 40

Ar40

Ar polyatomic ions can interfere with the detection of

Se species on mass 80. Therefore, Se is quantified on mass 78. Also at this mass there is a polyatomic

interference from 38

Ar38

Ar, although less pronounced. On VARIAN ICP-MS this interference can be counteracted

by the use of H2 as a reaction gas. During the Se speciation measurements a H

2 flow of 80mL/min is activated.

This gas can react with the polyatoms by means of a charge transfer or an atom transfer. The possible

interference that is left on the measurement will be visible as a higher background signal and will not influence

the peak height.

2. Robustness

The parameters that have to be considered are discussed in addendum 1

3. Precision (repeatability –reproducibility)

Currently no performance criteria have yet been laid down for selenium species 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 HORRATR < 2. The data used to calculate HORRAT

R are the

data from CRM SELM-1 (Selenium enriched-yeast; certified for total Se (2059 ± 64 mg/kg) and SeMet (1372 ±

58 mg Se/kg) but not for other Se species) between 22/11/2013 and 22/01/2014 as these data represent

reproducibility conditions. The HORRATR values are below 2 for SeMet (cfr. Table 1).

HORRATR = RSD

R (=CV% in multiQC)/RSD

R Horwitz


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Table 1. HORRATR value based on the SELM-1 (Selenium enriched-yeast)

SeMet

mg Se/kg

Mean 1296.796

SD 93.071

RSDR 7.18

RSDHorwitz

5.39

HORRATR 1.33

Repeatability and reproducibility (intermediate precision) were calculated based on the three different matrix-

relevant samples: PREMIX, SUP1 and SUP2. The first two samples were spiked with one specie and the last

sample was spiked with both species. Spikings and measurements are repeated at three different days for each

sample.

The theoretical natural level was determined as the average of the data for the unspiked samples as determined

in a preliminary experiment. This theoretical natural concentrations for Se IV and SeMet are summarized in

Table 2. The final spiking levels of SeIV and SeMet are summarized in Table3.

Table 2. Natural concentrations (mg/L) of Se IV and SeMet in different samples

mg/L PREMIX SUP1 SUP2

SeIV 12 <LOQ <LOQ

SeMet <LOQ 137 <LOQ

Table 3. Three spike levels (mg/L) of SeIV and SeMet in different samples

PREMIX SUP1 SUP2

SeIV

level 1 12 - 5

level 2 24 - 10

level 3 120 - 50

SeMet

level 1 - 137 5

level 2 - 210 13

level 3 - 280 50

The calculations can be found in the files “ValMethTerv_SeXX_XXX.xlsx” (71\ACCREDITATIE\SELENIUM

SPECIATION\VALIDATIEDOSSIER) and are added in addendum.

The long-term reproducibility (intermediate precision) will be calculated when sufficient data will be available.

Addendum 2-5:

ValMethTerv_SeIV_premix.xlsx

ValMethTerv_SeMet_sup1.xlsx

ValMethTerv_SeIV_sup2.xlsx

ValMethTerv_SeMet_sup2.xlsx

4. Accuracy/ recovery

Accuracy indicates the deviation between the mean value found and the true value. Only one certified reference

material (SELM-1, seleno-yeast) is available which is certified for SeMet. For this matrix the accuracy of SeMet is

based on the difference between the certified value and the measured value of SeMet. Over 9 timepoints, this


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accuracy was 95% on average (see file “Accuracy calculations SeSpec.xlsx” (71\ACCREDITATIE\SELENIUM\

SPECIATION\VALIDATIEDOSSIER). No CRM is available for SeIV.

The accuracy of the method for more relevant matrices is illustrated by calculating the recovery of spiked

samples. This can be determined by applying the method to samples to which known amounts of SeIV and/or

SeMet have been added. The accuracy is then calculated from the test results as a percentage of analyte

recovered.

The data that have been used to determine the accuracy in the premix/food supplement samples, are the

measurement results of spiked samples used for precision calculation (Table 4).

All calculations can be found in “Accurancy calculations SeSpec.xlsx” (71\ACCREDITATIE\SELENIUM

SPECIATION\VALIDATIEDOSSIER).

Table 4: Mean accuracy for different Se species in different matrices.

Premix SUP1 SUP2

SeIV

89% - 105%

SeMet - 97% 91%

5. Beslissingsgrens (CCα) en detectievermogen (CCβ)

As there are no legal limits for Se species in premixes and food supplements, CCα and CCβ are not relevant.

6. Calibration (Linearity of the method)

The calibration is an internal calibration of the linear type. Calibration curves for each selenium species are

presented in each analysis file. As example, the calibration curves are given for the measurement of SUP2-d3 in

figure 1-2 below (see file ‘Calibration SpecSe’ (71\ACCREDITATIE\SELENIUM\SPECIATION

\VALIDATIEDOSSIER)). Observed R2

values are > 0.99, and residuals are <15%. The random variation of the

residuals (Res%) with increasing standard concentrations supports the trueness of the linear model.

Figure 1: internal calibration curve for SeMet in a premix sample


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Figure 2: internal calibration curve for SeIV in a premix sample

7. Measurement uncertainty

The measurement uncertainty was calculated from the intra-reproducibility and from the bias (ubias

), which is

calculated from a certified reference material or recovery experiments1

:

uc = √

Uncertainties related to intra-reproducibility can be calculated from the controlchart of a matrix-matching CRM,

or they can be calculated from the repeatability (r) and intermediate precision (ip) as determined in section 3.

The bias can as well be calculated from a matrix-matching CRM, or from recovery experiments if no CRM is

available. 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%.

As there is no certified reference material available for SeIV in feed premixes and food supplements,

uncertainties related to intra-reproducibility are calculated from the repeatability and intermediate precision

data (see “ValMethTerv_SeIV_XX” files & section 3), and uncertainties related to bias are calculated from the

data from section 4 (recovery experiments for accuracy calculation). All calculations can be found in

“Measurement uncertainty SeIV.xlsx”. The expanded measurement uncertainties, calculated at different

concentration levels, are presented in Table 5.

Table 5: Expanded measurement uncertainties for SeIV in different matrices at different concentration levels.

Concentration level Premix SUP2

10 mg/kg 50% 38%

20 mg/kg 29% 34%

100 mg/kg 13% 10%

1

Calculated as described in “Procedure Bepaling van de meetonzekerheid voor kwantitatieve chemische

analyses. LAB P 508 Meetonzekerheid-v.01-nl. Goedgekeurd 03/11/2008”.


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SeMet is certified in the CRM SELM-1, which is selenium-enriched yeast. Hence this CRM can be used to

calculate the uncertainties related to intra-reproducibility and bias for SeMet in the selenium-enriched food

supplements. As the SeMet concentrations in SELM-1 are very high (1372 ± 58 mg Se/kg) and the matrix is not

quite relevant, we also chose to calculate these uncertainties by using the repeatability and intermediate

precision data (see “ValMethTerv_SeMet_XX” files & section 3) and the recovery experiment data from section 4.

All calculations can be found in “Measurement uncertainty SeMet.xlsx”. The expanded measurement uncertainty

for the determination of SeMet in food supplements or feed premixes equals 19% when calculated solely based

on the CRM. When based on the CRM (intra-reproducibility) and recovery experiments (bias), the expanded

measurement uncertainty varies from 18% in SUP1 to 19% in SUP2. The expanded measurement uncertainties,

when calculated at different concentration levels using repeatability and intermediate precision data and the

data from recovery experiments, are presented in Table 6.

Table 6: Expanded measurement uncertainty for SeMet in different matrices at different concentration levels.

Concentration level SUP1 SUP2

10 mg/kg - 19%

20 mg/kg - 14%

100 mg/kg - 10%

175 mg/kg 18% -

250 mg/kg 10% -

320 mg/kg 9% -

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 each Se species corresponds to 10 times the standard deviation of the

background signal of the chromatogram. Standard deviations of the background signal of SELM-1

chromatograms were calculated in different zones of the chromatogram, because we wanted to include the

potential effect of the mobile phase gradient. For SeIV

, LOQ was based on the background signal 0.5 minutes

after SeIV

peak elution. In this region there are still small unidentified peaks present in yeast-based matrices.

Nevertheless this region is chosen to incorporate the presence of these peaks in the LOQ.

For SeMet was based on the background signal 0.5 minutes after SeMet peak elution.

Average calculated LOQ values (µg/kg) for the various species obtained on different measurement days are

shown in table 4 (values rounded to 0.5 unity).

Table 5. Calculated LOQ values in solution (µg/L) for the different Se species

SeIV

SeMet

Average signal (c/s) 355 234

Average standard deviation (c/s) 92 19

Relative standard deviation (RSD) 25% 8.1%

LOQ in solution (µg/L) 2 0.5

The calculations are detailed in the file ‘LODLOQ calculations’ (71\ACCREDITATIE\ SELENIUM

SPECIATION\VALIDATIEDOSSIER). These LOQ values translate into 4 mg/kg for SeIV

and 1 mg/kg for SeMet in

a matrix (final dilution factor of 200).

These calculated LOQ values need to be confirmed by measuring solutions with similar concentration levels.


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9. 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 duplicate, 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 needs to be <25%.

-When the coefficient of variation is higher than 25%, the analysis will be repeated in 3 replicates.

10. Peak resolution

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 3). 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 3: 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 the 4 Se species is presented in Figure

4. Values for retention times and peak width at 13.4% are presented in the same Figure. Using these values,

peak resolutions, Rs, can be calculated. The resolution between SeIV

and SeVI

is 5.2 and between the SeVIbis

and

SeMet is 12.4. Rs values >1.3 are characteristic for well separated peaks.


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# Name

Time

[Min]

Quantity

[ppb]

Height

[c/s]

Area

[c/s.Min]

Area %

[%]

Width

13.4% [Min]

1 Se4 3.35 10.73 25962.8 4887.8 20.247 0.28

2 Se6 5.07 10.42 13384.5 3648.6 15.114 0.38

3 MeSeCys 5.85 10.32 77319.5 6171.9 25.567 0.12

4 Se6bis 6.93 10.7 14254.5 4081.9 16.909 0.35

5 SeMet 13.36 11.68 12740.5 5350.2 22.163 0.69

Figure 4: Chromatogram of a 5 ppb standard solution containing a mix of the 4 Se species.

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Addendum 1: PRO/5.4/02/DOC04/V01

FACTOREN DIE DE JUISTHEID EN BETROUWBAARHEID VAN DE BEPROEVINGEN BEÏNVLOEDEN

SOP/TRA/ANA/0y

Parameter Composants character Control

1.Equipement

and laboratorium

material

mixer

non relevant if clean and

working

mixer is cleaned with bi-distilled water after use

balances relevant

maintenance by external firm 1x/year and daily

verification

pipettes relevant

control by external firm 2x/year and

maintenance by external firm 1x/year

glassware for

standards

non relevant if clean flasks stored in bi-distilled water

ICP-MS relevant

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).

HPLC pumps relevant

intervention by firm when a pump related

problem is observed (see logbook ICP-MS)

HPLC

autosampler

non relevant if working

and injection volume

constant

check injection volume 1x/year (RSD<0.5%)

2. Products and

samples

milliQ water

non relevant if produced

daily fresh

via maintenance mentioned in logbook and

daily verification

Standards for

Se species

relevant daily fresh preparations of spike dilutions

HFBA/MeOH

solution for

mobile phase

relevant

constant use of same mark

daily fresh preparation

1° routine

samples

2° ringtest

samples

relevant

1° verification of extraction efficiency in sample

(total As in extract versus total As after

mineralisation)

2° contrôle of Z-score of PT-Test

C8 column

relevant

(physic-chemical

characteristiques of the

column)

constant use of same column mark

cleaning or replacement of column if problems

are observed (high pressure, bad peak

separation, tailing of peaks,…)

3.Method

principles

Temperature

of HPLC-

chain

non relevant because of

thermostatisation by oven

control of oven temperature 1x/year

4.Room

temperature

Room

temperature

of LC-ICP-MS

laboratory

non relevant

daily tuning of ICP-MS signal with a tuning

solution optimises signal in function of daily

conditions (cfr SOP/TRA/ANA/02)

5.Staff

Analists,

technical

responsible

relevant

first, second, third line controls

formation of people

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Addendum 2

CALCULATION of REPEATABILITY, WITHIN-LAB REPRODUCIBILITY, CCa, CCb and RECOVERY (reminder : formulas, with "E" = "sum of" :

ACCORDING TO 2002/657/EC, ISO 11843-1 and Van Loco & Beernaert (2004) SSrepl = Ed Er (Y-Ymoy(level,day))2

Srep2 = Srepl2 = MSrepl = SSrepl/(n-ndays)

DATA : RESULTS AND GRAPHS : SSdays = nrepl E(Ymoy(level,day)-Ymoy(level))2

MSdays = SSdays / (ndays-1)

Analyte : SeMet linear regression : recovery intercept R2 Sdays2 = (MSdays-MSrepl) / nrepl

Method : 103.2% 25.10 0.9863 Srw2 = Sdays2 + Srep2 )

Matrix : food supplement Se-enriched yeast warning : this is only the "repe-repro"

MRL : (Maximum Residue Limit) CCa, CCb : t(26,0.05) Yc CCa CCb part of the MU and does not include lab bias

unit : mg/kg mg/kg % of MRL mg/kg % of MRL (possibly different for different samples)

Nrepl : 3 (number of replicates) 1.706 40.90 15.31 #DIV/0! #DIV/0! #DIV/0!

Ndays : 3 (number of days or analysts)

(Nlev : 3 (number of levels)) SD, U : nom.ccn av.meas. uncertainty (k = 1) (*) U, ext. unc. (k = 2) (**) Umax. (k = 2) (***)

Measured concentrations : mg/kg mg/kg mg/kg % mg/kg % mg/kg %

(NOT corrected for recovery) : 137 165.46 7.76 4.69% 15.52 9.4% 39.4 28.8%

210 245.11 7.38 3.01% 14.77 6.0% 56.7 27.0%

conc. repl : day or analyst : 280 313.03 8.79 2.81% 17.58 5.6% 72.3 25.8%

(µg/kg) 1 2 3 4 5 6 (*) u = sd (**) U = 2 sd (***) from modif. Horwitz for Srw, HorRat < 2x2/3

level 1 : 1 162.3 177.21 167.24 224.567 SSrepl cfr Codex Alim - mth criteria - draft 14.2.08

137 2 149.7 170.59 162.81 37.428 MSrepl

3 166.67 166.48 166.14 6.118 Srepl

4 165.461 average for level

5 211.367 SSdays

6 105.684 MSdays

7 22.752 Sdays2

8 4.770 Sdays

9 7.758 Srw

10

level 2 : 1 240.28 252.94 247.44 80.671 SSrepl

210 2 238.12 250.44 238.89 13.445 MSrepl

3 236.2 249.99 3.667 Srepl


5 273.334 SSdays

6 136.667 MSdays

7 41.074 Sdays2

8 6.409 Sdays

9 7.384 Srw

10

level 3 : 1 305.79 317.58 318.55 270.610 SSrepl

280 2 304.62 321.45 320.71 45.102 MSrepl

3 308.07 299.99 320.46 6.716 Srepl


5 283.384 SSdays

6 141.692 MSdays

7 32.197 Sdays2

8 5.674 Sdays

9 8.792 Srw

10

average level 1 : 159.56 171.43 165.4 average

SumSquares 155.29 58.608 10.665 SumSquares

average level 2 : 238.2 251.69 245.44 average calculations performed by : date

SumSquares 8.3135 4.6888 67.669 SumSquares using template : ValMethTerv ver 1.72

average level 3 : 306.16 313.01 319.91 average template date : 23-sept-13


0

5

10

15

20

2 x Srepl 2 x Sdays 2 x Srw

Uncertainty (ug/kg) (k=2, 95% conf. int.)

137

210

280

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

10%


Uncertainty (%) (k=2, 95% conf. int.)

137

210

280

135

140

145

150

155

160

165

170

175

180

1 2 3 4 5 6 7 8 9 10

0.5 x MRL

225

230

235

240

245

250

255

1 2 3 4 5 6 7 8 9 10

1 x MRL

285

290

295

300

305

310

315

320

325

1 2 3 4 5 6 7 8 9 10

1.5 x MRL

y = 7.2764x + 130.01R² = 0.5586

0

50

100

150

200

250

300

350

0.0 5.0 10.0 15.0 20.0 25.0 30.0

linear regression

of 15

Addendum 3






Analyte : SeMet linear regression : recovery intercept R2 Sdays2 = (MSdays-MSrepl) / nrepl

Method : 100.0% 1.26 0.9978 Srw2 = Sdays2 + Srep2 )

Matrix : food supplement selenate warning : this is only the "repe-repro"








25 27.00 1.15 4.26% 2.30 8.5% 7.3 29.3%




10 2 11.324 10.752 10.274 0.108 MSrepl

3 10.733 10.596 10.545 0.329 Srepl


5 0.577 SSdays

6 0.288 MSdays

7 0.060 Sdays2

8 0.245 Sdays

9 0.410 Srw

10

level 2 : 1 25.743 27.91 27.81 1.002 SSrepl

25 2 26.24 27.912 26.665 0.167 MSrepl

3 25.465 28.135 27.161 0.409 Srepl


5 7.259 SSdays

6 3.629 MSdays

7 1.154 Sdays2

8 1.074 Sdays

9 1.149 Srw

10

level 3 : 1 101.32 100.43 102.82 71.684 SSrepl

100 2 105.64 105.11 99.105 11.947 MSrepl

3 95.684 100.66 99.726 3.456 Srepl


5 3.819 SSdays

6 1.910 MSdays

7 -3.346 Sdays2

8 0.000 Sdays

9 3.456 Srw

10







0

1

2

3

4

5

6

7

8



10

25

100

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%



10

25

100

9

9.5

10

10.5

11

11.5

1 2 3 4 5 6 7 8 9 10

0.5 x MRL

24

25

26

27

28

29

1 2 3 4 5 6 7 8 9 10

1 x MRL

90

92

94

96

98

100

102

104

106

108

1 2 3 4 5 6 7 8 9 10

1.5 x MRL

y = 1.0003x + 1.2561R² = 0.9978

0

20

40

60

80

100

120

0.0 20.0 40.0 60.0 80.0 100.0 120.0

linear regression

of 15

Addendum 4






Analyte : Se IV linear regression : recovery intercept R2 Sdays2 = (MSdays-MSrepl) / nrepl

Method : 97.5% -1.58 0.9966 Srw2 = Sdays2 + Srep2 )

Matrix : Premix feed warning : this is only the "repe-repro"








24 21.83 1.36 6.22% 2.72 12.4% 7.0 29.3%




12 2 9.4087 9.9081 10.771 0.311 MSrepl

3 10.023 11.107 9.4086 0.558 Srepl


5 1.132 SSdays

6 0.566 MSdays

7 0.085 Sdays2

8 0.292 Sdays

9 0.629 Srw

10

level 2 : 1 21.397 23.174 21.373 9.921 SSrepl

24 2 20.319 21.655 21.968 1.653 MSrepl

3 21.147 20.728 24.685 1.286 Srepl


5 4.447 SSdays

6 2.224 MSdays

7 0.190 Sdays2

8 0.436 Sdays

9 1.358 Srw

10

level 3 : 1 111.34 107.85 120.09 187.017 SSrepl

120 2 119.5 120.18 108.86 31.169 MSrepl

3 115.96 117.07 117.99 5.583 Srepl


5 0.700 SSdays

6 0.350 MSdays

7 -10.273 Sdays2

8 0.000 Sdays

9 5.583 Srw

10







0

2

4

6

8

10

12



12

24

120

0%

2%

4%

6%

8%

10%

12%

14%



12

24

120

8.5

9

9.5

10

10.5

11

11.5

1 2 3 4 5 6 7 8 9 10

0.5 x MRL

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10

1 x MRL

100

102

104

106

108

110

112

114

116

118

120

122

1 2 3 4 5 6 7 8 9 10

1.5 x MRL

y = 0.9751x - 1.5784R² = 0.9966

0

20

40

60

80

100

120

140

0.0 50.0 100.0 150.0

linear regression

of 15

Addendum 5






Analyte : Se IV linear regression : recovery intercept R2 Sdays2 = (MSdays-MSrepl) / nrepl

Method : 97.6% -0.41 0.9985 Srw2 = Sdays2 + Srep2 )

Matrix : food supplement selenate warning : this is only the "repe-repro"








20 18.63 1.99 10.66% 3.97 21.3% 5.9 29.3%




10 2 10.054 9.6729 9.6367 0.144 MSrepl

3 9.7268 10.259 10.221 0.379 Srepl


5 0.070 SSdays

6 0.035 MSdays

7 -0.036 Sdays2

8 0.000 Sdays

9 0.379 Srw

10

level 2 : 1 20.579 16.373 20.837 6.961 SSrepl

20 2 20.335 16.025 19.246 1.160 MSrepl

3 18.026 17.334 18.942 1.077 Srepl


5 19.015 SSdays

6 9.508 MSdays

7 2.783 Sdays2

8 1.668 Sdays

9 1.986 Srw

10

level 3 : 1 95.028 94.764 101.35 13.156 SSrepl

100 2 96.61 96.86 98.504 2.193 MSrepl

3 97.866 96.262 97.903 1.481 Srepl


5 18.685 SSdays

6 9.343 MSdays

7 2.383 Sdays2

8 1.544 Sdays

9 2.139 Srw

10







0

1

2

3

4

5



10

20

100

0%

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10

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100

8.6

8.8

9

9.2

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10

10.2

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0.5 x MRL

0

5

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25

1 2 3 4 5 6 7 8 9 10

1 x MRL

90

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94

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100

102

1 2 3 4 5 6 7 8 9 10

1.5 x MRL

y = 0.9759x - 0.406R² = 0.9985

0

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120

0.0 20.0 40.0 60.0 80.0 100.0 120.0

linear regression

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