METHOD Pa e 1 on 7

SeMet determination in selenized yeasts Emission date : by HPLC - ICP MS August 26

1h 2013

METHOD

SeMet determination in selenized yeasts by HPLC - ICP MS

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METHOD Pa e 2 on 7

SeMet determination in selenized yeasts Emission date : by HPLC - ICP MS August 26

1h 2013

U1tra Traces Analyse Aqu ta ii0

Title : SeMet determination in selenized yeasts by HPLC - ICP MS

Writter Name: V. Vacchina Function: lngenieur applications Date: Al.f-~w)Y- 2 ~ 0\S Visa:

Content

Approver Name: F. Seby Function: UT2A responsible f-eL

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Date: ~" ~ !l.6 :.U;:::J\ =>

Visa:

Page

1. APPLICATION RANGE ............................................................................................................................. 3

2. BRIEF DESCRIPTION OF THE METHOD ............................................................................................. 3

3. SAMPLING CONDITIONNING AND STORAGE .................................................................................. 3

4. REACT ANTS AND SOLUTIONS .............................................................................................................. 3

5. ST AND ARDS ................................................................................................................................................ 3

6. MATERIAL AND EQUIPMENT ................................................................................................................ 3

7. PREPARATION OF THE SOLUTIONS ................................................................................................... 4

7.1. PREPARATION OF THE BUFFER T .... ....... .. ......... ........ .... ............ .... .. ........... .. .... ... .......... .. ...... .. ... ...... ... .. 4 7.2. PREPARATION OF THE EXTRACTION SOLUTION E ... .... ..... ... ... ............ .......... .... ........ .. ........ ... ......... ..... .4

8. EXTRACTION .............................................................................................................................................. 5

9. ANALYSIS BY HPLC - ICP-MS ................................................................................................................ 5

9.1. PREPARATION OF THE MOBILE PHASE .. .... ........ .. .. ....... ... ..... ..... ... .. .. .. .... ...... ... ... .... ....... .. .. ....... .. .... ... .. .. 5 9.2. SEPARATION CONDITIONS .... .. ................. .. .. .. .. .............. .. .... .. .. .. .. .... ....... .... .. .......... ......... ... ... .. ......... ... .. 5 9.3. ACQUISITION PARAMETERS IN ICP MS ............... ...... .. ... .... .. .... ... ..... .. .. .. ............ ...... ..... .... ............... ... . 5 9.4. CALIBRATION ......... ...... .... ......... .. ...... .... .... ........ ... .. ... ....... ... ........... ..... .... .. ...... .. .. .. ... ...... .. ... ........... ...... . 6 9.5. ANALYSIS SEQUENCE ......... ...... .... .. ......... .. .. .. ...... ... .... ......... ..... .. .. ...... ... ........ .... ... ..... .. .. .. ... ......... .. ..... ... . 6

10. QUANTIFICATION ................................................................................................................................ 6

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METHOD Pa e 3 on 7

SeMet determination in selenized yeasts Emission date : by HPLC - ICP MS August 261

h 2013 Ultra Traces Analyses Aqu11a .ne

1. Application range

This method describes the determination of selenomethionine (SeMet) in selenized yeasts.

2. Brief description of the method

A representative sample uptake of the sample is extracted by a mixture of proteolytic enzymes. SeMet

is determined by the coupling of Liquid Chromatography with Inductively Coupled Plasma Mass

Spectrometry (HPLC - ICP MS) and quantified by external calibration.

3. Sampling, conditioning and storage

The samples are stored at 4°C in plastic bags.

4. Reagents and solutions

The reactants used are of analytical grade.

Protease type XIV from Streptomyces griseus

Lipase type VII from Candida rugosa

Others

Ultrapure water resistivity : 18.2 Mn.cm

Tris(hydroxymethyl)aminomethane 99.9+%

Hydrochloric acid (HCI) 37%

Dithiothreitol (OTT) ~ 99%

Acetic acid 99.7%

Ammonium citrate ~97%

5. Standards and Certified Reference Materials

The daughter standard solutions are prepared from the certified powder described below :

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METHOD Pa e 4 on 7

SeMet determination in selenized yeasts Emission date : by HPLC - ICP MS August 261

h 2013

Species Purity Se Met ~99%

The SElM-1 CRM described below is used which each series of samples :

Reference SELM-1

6. Material and equipment

- variable volume micropipettes

- stainless steel spatula

Provider Certified value

NRC 1383 ± 63 mg/kg

- balance: PRECISA model XT220A balance equipped with a printer or equivalent

- glass flasks of 22 ml

- glass bottles of 1 l

- thermostatically controlled and shaking water bath

- centrifuge

- glass vials of 2 ml for HPlC

- chromatographic column: Hamilton PRPX-100 (250 mm x 4 mm) equipped with a guard column

- separation system : HPlC

- detection system : Inductively Coupled Plasma Mass Spectrometer (ICP-MS) Agilent technologies

7500 CX Series or equivalent

7. Preparation of the solutions

7 .1. Preparation of the buffer T

- Weigh 3.63 g de tris(hydroxymethyl)aminomethane in a glass bottle of 1 land fill with ultrapure water.

-Adjust the pH at 7.5 by adding 2 ml of HCI.

7.2. Preparation of the extraction solution E

- Weigh (10 x n) mg of lipase and (20 x n) mg of protease (with n = number of tubes)

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METHOD Pa e 5 on 7

SeMet determination in selenized yeasts Emission date : by HPLC - ICP MS August 26

1h 2013

- Add (5 x n) ml of the buffer T

This solution is prepared just before use.

8. Extraction

- Weigh 200 mg of sample in a glass tube of 22 ml and print the weight tickets. Each sample is

prepared in duplicate.

- Add 5 ml of E and print the weight tickets.

- Shake 16h in the water bath at 37°C.

- Centrifuge (3000 rpm, 5 min) and collect the supernatant.

- Repeat twice the extraction with E on the residue and mix all the supernatants.

- Add 0.2 M of DTT on the mixture of supernatants.

Each series of extraction should contain at least 1 blank and 1 CRM (SELM-1 ).

9. Analysis by HPLC - ICP MS

9.1. Preparation of the phase mobile

Weigh 1.22 g of ammonium citrate in a 1 L glass bottle, fill with ultrapure water and adjust the pH at 4.8

by addition of 600 µl of acetic acid.

9.2. Separation conditions

- column : Hamilton PRPX-100 equipped with a guard column

- flow : 1 mUmin

- volume injected : 100 µL

- mobile phase : isocratic during 20 min

9.3. Acquisition parameters in ICP MS

- isotopes monitored : 1sse, nse, 1sse, sose and s2se

- integration time : 0.15 sec

- drainage with the peristaltic pump : 0.4 rps

- reaction gas : H2 (flow to be optimized)

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METHOD Pa e 6 on 7

SeMet determination in selenized yeasts Emission date : by HPLC - ICP MS August 26

1h 2013

U1tra Traces Analyoes Aquitaine

- acquisition time : 1200 sec

9.4. Calibration

Quantification is made by external calibration. The calibration range is chosen according to the expected

concentration.

9.5. Analysis sequence

- inject the blank

- inject the calibration curve in the rising concentrations order

- inject the samples

- inject the CRM

- inject one point of the calibration curve (medium range) at the end of the sequence or each 10

samples if necessary

10. Quantification

Quantification is made from peaks area from the isotope 1ase.

The regression line is marked. The correlation coefficient should be higher than 0.995 to validate the

calibration.

For the CRM, the tolerance is of 15% to validate the full analytical sequence.

The concentration calculation is made according to the following equation :

C =Ax m exr

p m ech

with : C concentration in the sample

A peak area

p slope of the calibration curve

mext mass of the extractant added

mech sample uptake

The concentration given corresponds to the average of the concentrations obtained for each replicate.

The uncertainty given corresponds to twice the standard deviation of the concentrations obtained for

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Analytical method related to authorised feed additive - 3b817

each replicate.

METHOD Pa e 7 on 7

SeMet determination in selenized yeasts Emission date : by HPLC - ICP MS August 26

1h 2013

• END OF DOCUMENT •

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Analytical method related to authorised feed additive - 3b817