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Determination of vitamin A (retinol), vitamin E ((±)- -tocopherol) and ß-carotene in foodstuffs by HPLC-UV

Reasons for amendment of original German version: V2: Summary of methods N03_01, N03_04, N03_08 V3 (11.07.08): Changes to parameters V4 (21.01.10): New calibration, changes in processing Modifications of norm method explained

Name Signature Date First version written by: Melanie Mongili

Translation by: easytrans24 Bei den Mühren 69a 11/2009 D-20457 Hamburg verified (translation): Melanie Mongili verified (professional): Katharina Lorenz verified (norm conformity): Nadine Wollnitz approved: Matthias Lieske

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1 Area of application This method describes a procedure for the determination of vitamin A (retinol), vitamin E (d,l -tocopherol) and ß-carotene in food products. Vitamin A, E and ß-carotene are categorized as fat-soluble vitamins. They are essential, as is the case for all vitamins, i.e., they cannot be synthesized by the body, but must be taken up with food and are therefore added to baby food.

2 Principle of the method All three vitamins are determined with the aid of saponification. The material under investigation is saponified with aqueous ethanolic potassium hydroxide solution and the vitamins thus released are extracted using petroleum ether. After concentration of the extract, the residue is dissolved in methanol and the contents of vitamin A, E and ß-carotene are determined using HPLC-UV/VIS. This method is based on the norm methods DIN EN 12823-1, DIN EN 12823-2 and DIN EN 12822. The following modifications are applied: All 3 norm methods are summarized to one single method. The 3 vitamins are determined from one sample preparation. For 13-cis retinol, -carotine und - tocopherol no calibration is performed No correction factor for HPLC is derived from purity assessment. Saponification is performed by 3 h incubation at 50 °C in a water bath and incubation at room temperature over night. The extraction is performed just once and with 150 ml petrol ether The neutralisation is performed just once and with approx. 400 ml water A defined aliquot of the extract is evaporated on a drying device at 48 °C with nitrogen Only a single determination is performed. The determination is only repeated if there are deviations from the expected value or mistakes during sample preparation are suspected.

3 Definitions and abbreviations

Definitions The vitamin A content is the content of all-trans retinol and 13-cis-retinol determined using the procedure described here. Conversion factors for the calculation of international units (IU) in units of weight: 1 IU vitamin A = 0.300 µg all-trans retinol 0.344 µg all-trans retinol acetate 0.550 µg all-trans retinol palmitate

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1 µg retinol equivalent = 1.00 µg retinol 3.333 IU vitamin A,

The vitamin E content is the content of d,l- -tocopherol determined using the procedure described here. Conversion factors for the calculation of international units (IU) in units of weight: 1 mg d,l- -tocopherol = 0.74 mg -tocopherol equivalent = 1.10 IU vitamin E

The ß-carotene content is the content of ß-carotene determined using the procedure described here which is employed for the calculation of the vitamin A equivalent. 6 µg ß-carotene correspond to 1 µg retinol.

Abbreviations HPLC high performance liquid chromatography BHT butylhydroxytoluene

4 Equipment Laboratory mill Analytical scales, precision ± 0.1mg Brown glass flat-bottomed flask, 250 ml Stir bar Magnetic stirrer Dispenser (50 ml, 100 ml) Graduated cylinder Water bath with reflux condenser Conical separating funnel, brown glass, 500 ml, with PTFE stopcock Mechanical shaker Volumetric flask, narrow neck, 50 ml, 100 ml Glass funnel Phase separation folding filters Pipette Brown glass vials, 6 ml Drying device with nitrogen (evaporation system) Brown glass vials, 1 ml Vial clamp

HPLC equipment composed of a pump, injector with sample loop (20-100 µl), UV-VIS detector, analytical software

HPLC columns: Vitamin A/E: e.g. LiChrosorb RP18, 7 µm (250 x 4.0 mm) ß-carotene: e.g. Spherisorb ODS 2, 5 µm, (100 mm x 4.6 mm) e.g. Vydac 201TP54 (RP18), 5 µm (250 x 4.6 mm)

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UV-VIS spectrophotometer suitable for the measurement of the absorption capacity at predetermined wavelengths with suitable quartz cuvettes, e.g. 1 cm layer thickness

5 Chemicals 5.1 Reagents Purified water L-ascorbic acid Ethanol, denatured, ~ 95 % Ethanol, absolute, = 99 % Potassium hydroxide p.a. Nitrogen, oxygen-free, = 99.1 % Petroleum ether (boiling range 40-60 °C) Methanol for the liquid chromatography Dichloromethane for the liquid chromatography Acetonitrile for the liquid chromatography Ammonium acetate Triethylamine Buthylhydroxytoluene

5.2 Solutions

All solutions and eluents for HPLC are made up using purified water ( 18M DI).

Potassium hydroxide solution (60 %) 600 g potassium hydroxide are dissolved in 1.0 l water.

5.3 Standards Vitamin A standard: Retinol, e.g. supplied by Sigma (purity > 95 %) Retinol palmitate, e.g. supplied by Sigma (purity ~ 1,800,000 USP units/g)

Vitamin E standard: d,l -tocoperol, e.g. supplied by Sigma (purity > 96 %)

ß-carotene standard: ß-carotene, e.g. supplied by Sigma (purity > 95%)

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6 HPLC systems The vitamins are separated under suitable HPLC conditions. For example, the following have proven reliable:

HPLC equipment for the determination of vitamin A and E

Injection volume: 50 µl Column: LiChrosorb RP18, 7 µm (250 x 4.0 mm) Eluent: methanol/water (98/2, v/v) Flow rate: 0 min 0.8 ml 5 min 1.0 ml 8.5 - 14 min 1.5 ml 14.5 min 0.8 ml UV detector: 0 8.5 min 326 nm 8.5 15 min 292 nm Run time: 15 min

HPLC equipment for the determination of vitamin ß-carotene

Injection volume: 20 µl Column: Spherisorb ODS 2, 5 µm, (100 mm x 4.6 mm) Vydac 201TP54 (RP18), 5 µm (250 x 4.6 mm) Eluent: acetonitrile/ methanolic ammonium acetate solution/ dichloro- methane (75/20/5, v/v) with the addition of 0.08 g BHT+ 0.085 g triethylamine (per 100 ml eluent) Flow rate: 1.5 ml (isocratic) UV detector: 450 nm (Wolfram lamp) Run time: 19 min

7 Calibration

Two different calibrations are carried out for the determination of the vitamin A content, one using retinol and one using retinol palmitate as the standard. This is required, as lower contents of vitamin A are otherwise determined for retinol palmitate, probably because saponification is incomplete due to the high stability of retinol palmitate. A retinol palmitate standard is saponified for the calibration, in order to compensate for these "losses" during saponification.

7.1 Calibration for the determination of retinol, retinol acetate and d,l- -tocopherol

Comment: Based on the differing quality of the standards supplied by different manufacturers and on our experience, the calibration standards for retinol and vitamin E are obtained exclusively from Sigma. The standards should

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always exhibit a purity of 95 % (for retinol) or 96 % (for vitamin E). Due to stability problems, the purity of the standards must be checked prior to their use for calibration purposes, even though a manufacturer's certificate is available (see 7.1.1 and 7.1.2).

Vitamin A and vitamin E are measured simultaneously on one column. A standard mixture of the two standards is used for the calibration. The stock solutions and the solution for the purity assessment must be made up separately as the extinctions of the two substances overlap in photometric measurements (see 7.1.3).

7.1.1 Assessment of concentration and purity pursuant to the European Standard DIN EN 12823-1 (retinol)

Concentration and purity are checked against ethanol using spectrophotometric measurements on a retinol standard solution of a defined concentration and subsequent calculation of mass concentration.

There is a form that provides the exact details of how to produce the solution for measurement for this assessment (see Appendix 1). This form documents the initial weight, the dilution and the values measured with the photometer. It is filed in the corresponding folder together with the documents on calibration. The retinol standard A2 is used for the assessment of concentration and purity (see 7.1.3 Calibration).

To this end, the absorption capacity of the retinol standard solution A2 is measured against ethanol in a quartz cuvette with a layer thickness of 1 cm at the absorption maximum of 325 326 nm for retinol using a suitable spectrophotometer.

The mass concentration of all-trans retinol in micrograms per millilitre is calculated using the following equation:

A 104 mlg ]/[ 1830

Where: A Absorption maximum at 325 to 326 nm %1 1830 E1cm value for retinol in ethanol

As an additional safeguard, the absorption capacity of the standard solution is measured against ethanol at 300 nm, 325 nm, 350 nm and 370 nm.

The following ratio is determined for each wavelength:

E/E325 (or 326nm)

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The standard can be used for calibration purposes if the ratio for retinol does not exceed 0.602 (at 300 nm), 0.452 (at 350 nm) and 0.093 (at 370 nm). If the contents deviate from the data provided in the certificate, the concentrations of the standard solutions are calculated based on the contents measured photometrically.

7.1.2 Assessment of concentration and purity pursuant to the European Standard DIN EN 12822 (d,l- -tocopherol)

Concentration and purity are checked against methanol using spectrophotometric measurements on a d,l- -tocopherol standard solution of a defined concentration and subsequent calculation of mass concentration.

There is a form that provides the exact details of how to produce the solution for measurement for this assessment (see Appendix 1). This form documents the initial weight, the dilution and the values measured with the photometer. It is filed in the corresponding folder together with the documents on calibration.

In order to assess concentration and purity, 1.0 ml of the stock solution is pipetted into a 100 ml volumetric flask, evaporated under nitrogen and dissolved in 100 ml methanol. The concentration is approx. 8 mg/100 ml. To this end, the absorption capacity of the d,l- -tocopherol standard solution is measured against methanol in a quartz cuvette with a layer thickness of 1 cm at the absorption maximum of 292 nm for d,l- -tocopherol using a suitable spectrophotometer.

The mass concentration, , of d,l- -tocopherol in micrograms per millilitre is calculated using the following equation: A 104 mlg ]/[ 76

Where: A Absorption maximum at 292 nm %1 76 E1cm value for d,l- -tocopherol in methanol

In addition, the absorption capacity should be measured at 255 nm (minimum). At this wavelength, the extinction should be between 6 and 8; otherwise the vitamin has decomposed and the standard cannot be used. If the contents deviate from the data provided in the certificate, the concentrations of the standard solutions are calculated based on the contents measured photometrically.

7.1.3 Production of the calibration solutions

There is a form that provides the exact details on the concentrations of the individual solutions for the production of the calibration solutions (see Appendix 1). This form documents the initial weights and the dilution steps. It is filed in the corresponding folder together with the chromatograms and the calibration curve.

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Retinol stock solution: About 80 mg retinol are weighed into a 100 ml volumetric flask (not brown glass) at a precision of ± 0.1 mg, dissolved in ethanol and the solution is made up to the mark. Add a few ml ethyl acetate if the standard does not dissolve. The stock solution contains about 80 mg/100 ml retinol.

Vitamin E stock solution: About 800 mg d,l- -tocopherol are weighed into a 100 ml volumetric flask (not brown glass) at a precision of ± 0.1 mg, dissolved in ethanol and the solution is made up to the mark. Attention: Brown glass volumetric flasks should not be used in the production of the stock solutions as it is not possible to see whether the standard is fully dissolved.

Comment: The stock solution for vitamin A is diluted again to 1/200 in ethanol, the stock solution for vitamin E to 1/100 in methanol (after evaporating off the ethanol) and spectrophotometrically assessed for concentration and purity, as described under points 7.1.1 and 7.1.2. Whether or not the standard can be used as a calibration standard can only be determined based on the values obtained from the photometric determination of purity. This means that the dilutions in the following are only produced, or calculations of their contents are made, if the results of the determination of purity confirm a purity of > 95 %.

Dilutions made from the stock solutions:

Standard mixture A/E 1: 5.0 ml of the retinol stock solution and 5.0 ml of the tocopherol stock solution are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with ethanol. The standard solution contains about 4 mg/100 ml retinol and 40 mg/100 ml tocopherol.

Standard mixture A/E 2: 10.0 ml of the standard mixture A/E 1 are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with ethanol. The standard mixture A/E 2 contains about 0.4 mg/100 ml retinol and 4 mg/100 ml tocopherol.

Calibration solutions and working standard (dilution from standard mixture A/E 2):

Standard mixture A/E 3: 5.0 ml of the standard mixture A/E 2 are pipetted into a 10 ml volumetric flask and the solution is made up to the mark with ethanol. The standard mixture A/E 3 contains about 0.2 mg/100 ml retinol and 2 mg/100 ml tocopherol.

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Standard mixture A/E 4: 25.0 ml of the standard mixture A/E 2 are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with ethanol. The standard mixture A/E 4 contains about 0.1 mg/100 ml retinol and 1 mg/100 ml tocopherol.

Standard mixture A/E 5: 20.0 ml of the standard mixture A/E 2 are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with ethanol. The standard mixture A/E 5 contains about 0.08 mg/100 ml retinol and 0.8 mg/100 ml tocopherol.

Standard mixture A/E 6 (working standard): 10.0 ml of the standard mixture A/E 2 are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with ethanol. The standard solution A6 contains about 0.04 mg/100 ml retinol and 0.4 mg/100 ml tocopherol. This solution is used as a working standard for regular checks on the HPLC equipment.

Standard mixture A/E 7: 2.0 ml of the standard mixture A/E 2 are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with ethanol. The standard mixture A/E 7 contains about 0.008 mg/100 ml retinol and 0.08 mg/100 ml tocopherol.

The standard solutions A/E 3 to A/E 7 are used for the calibration. The solutions are analysed in triplicate. A calibration curve is produced with the aid of the software and a corresponding response factor is calculated. The chromatograms thus obtained and the calibration curves are filed together with the form in the "calibration" folder.

The working standard (standard mixture A/E 6) is decanted into vials that are sealed and stored in the refrigerator at 8 °C. The standard mixture A/E 2 can also be stored in the refrigerator and has a shelf life of up to one year if protected from light. This solution can be diluted and used to make working standard if the working standard is used up prematurely.

A new calibration is required once the working standard has been used up or its concentration deviates from the nominal concentration by more than 5%; however, it should be conducted once a year.

7.2 Calibration for the determination of retinol palmitate

Comment: Based on the differing quality of the standards supplied by different manufacturers and on our experience, the calibration standard for retinol palmitate is obtained exclusively from Sigma. The standard should always exhibit a purity of ~ 1,800,000 USP units/ g. Due to stability problems, the purity of the standard must be checked prior to its use for calibration purposes, even though a manufacturer's certificate is available (see 7.2.1).

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7.2.1 Assessment of concentration and purity pursuant to the European Standard DIN EN 12823-1 (retinol palmitate)

Concentration and purity are checked against isopropanol using spectrophotometric measurements on a retinol palmitate standard solution of a defined concentration and subsequent calculation of mass concentration.

There is a form that provides the exact details of how to produce the solution for measurement for this assessment (see Appendix 2). This form documents the initial weight, the dilution and the values measured with the photometer. It is filed in the corresponding folder together with the documents on calibration.

The retinol palmitate standard P3 is used for the assessment of concentration and purity (see 7.2.2 Calibration).

To this end, the absorption capacity of the retinol palmitate standard solution P3 is measured against isopropanol in a quartz cuvette with a layer thickness of 1 cm at the absorption maximum of 325 326 nm for retinol using a suitable spectrophotometer.

The mass concentration, , of retinol palmitate in IU per gram is calculated using the following equation:

A 1900200 gIU ]/[ E

Where: A Absorption maximum at 325 to 326 nm %1 1900 E1cm value for retinol palmitate in isopropanol 200 Dilution factor E Initial weight [g]

As an additional safeguard, the absorption capacity of the standard solution is measured against isopropanol at 300 nm, 325 nm, 350 nm and 370 nm.

The following ratio is determined for each wavelength:

E/E325 (or 326nm)

The standard can be used for calibration purposes if the ratio for retinol palmitate does not exceed 0.602 (at 300 nm), 0.452 (at 350 nm) and 0.093 (at 370 nm). If the contents deviate from the data provided in the certificate, the concentrations of the standard solutions are calculated based on the contents measured photometrically.

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7.2.2 Production of the calibration solutions

There is a form that provides the exact details on the concentrations of the individual solutions for the production of the calibration solutions (see Appendix 2). This form documents the initial weights and the dilution steps. It is filed in the corresponding folder together with the chromatograms and the calibration curve.

Retinol palmitate stock solution: About 150 mg retinol palmitate are weighed into a 100 ml volumetric flask (not brown glass) at a precision of ± 0.1 mg, dissolved in isopropanol and the solution is made up to the mark.

Attention: Brown glass volumetric flasks should not be used in the production of the stock solution as it is not possible to see whether the standard is fully dissolved. Comment: The stock solution is diluted again to 1/200 in isopropanol and spectrophotometrically assessed for concentration and purity, as described under point 7.2.1. Whether or not the standard can be used as a calibration standard and can only be determined based on the values obtained from the photometric determination of purity. This means that the dilutions in the following are only produced, or calculations of their contents are made, if the results of the determination of purity confirm a purity of > 80 %.

Dilutions made from the stock solutions:

Standard solution P 1: 5.0 ml of the retinol palmitate stock solution are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with isopropanol. The standard solution contains about 4 mg/100 ml retinol.

Standard solution P 2: 20.0 ml of the standard solution P 1 are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with isopropanol. The standard solution P 2 contains about 0.8 mg/100 ml retinol.

Standard solution PV 1 The standard P 2 is processed like a sample for the production of the calibration solutions. To this end, 5 ml of the standard solution P 2 are used for saponification and then extracted with 150 ml petroleum ether (see sample processing 8.3 8.4). This is how the standard solution PV 1 (standard solution after saponification) is obtained, with contents of ~ 30 µg/100 ml.

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Calibration solutions (dilutions made from standard solution PV 1):

Standard PV 2: 8.0 ml of the standard solution PV 1 are pipetted into a brown glass vial and the solvent is evaporated off under nitrogen. The residue is then taken up in 1.0 ml methanol and transferred to an HPLC vial. The standard PV 2 contains approx. 0.2 mg/100 ml retinol.

Standard PV 3: 4.0 ml of the standard solution PV 1 are pipetted into a brown glass vial and the solvent is evaporated off under nitrogen. The residue is then taken up in 1.0 ml methanol and transferred to an HPLC vial. The standard PV 3 contains approx. 0.1 mg/100 ml retinol.

Standard PV 4: 2.0 ml of the standard solution PV 1 are pipetted into a brown glass vial and the solvent is evaporated off under nitrogen. The residue is then taken up in 1.0 ml methanol and transferred to an HPLC vial. The standard PV 4 contains approx. 0.05 mg/100 ml retinol.

Standard PV 5: 1.0 ml of the standard solution PV 1 are pipetted into a brown glass vial and the solvent is evaporated off under nitrogen. The residue is then taken up in 1.0 ml methanol and transferred to an HPLC vial. The standard PV 5 contains approx. 0.02 mg/100 ml retinol.

Standard PV 6: 1.0 ml of the standard solution PV 1 are pipetted into a brown glass vial and the solvent is evaporated off under nitrogen. The residue is then taken up in 4.0 ml methanol and transferred to an HPLC vial. The standard PV 6 contains approx. 0.006 mg/100 ml Retinol.

The standard solutions PV 2 to PV 6 are used for the calibration. The solutions are analysed in triplicate. A calibration curve is produced with the aid of the software and a corresponding response factor is calculated. The chromatograms thus obtained and the calibration curve are filed together with the form in the calibration folder.

None of the retinol palmitate standard solutions can be stored as the standard solution PV 1, in particular, is not stable. The standard mixture A/E 6 from the calibration with retinol is used as the working standard in daily checks on HPLC measurements (see 7.1.3).

A new calibration should be carried out once a year.

7.3 Calibration for the determination of ß-carotene

Comment: Based on the differing quality of the standards supplied by different manufacturers and on our experience, the calibration standard for ß-carotene is obtained exclusively from Sigma. The standard should always exhibit a purity of 95 %. In addition to the availability of the manufacturer's

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certificate, purity must be assessed prior to use of the standard for the calibration (7.3.1).

7.3.1 Assessment of concentration and purity pursuant to the European Standard DIN EN 12823-1

Concentration and purity are checked against n-hexane using spectrophotometric measurements on a ß-carotene standard solution of a defined concentration and subsequent calculation of mass concentration.

There is a form that provides the exact details of how to produce the solution for measurement for this assessment (see Appendix 3). This form documents the initial weight, the dilution and the values measured with the photometer. It is filed in the corresponding folder together with the documents on calibration.

In order to assess concentration and purity, 10.0 ml of stock solution I is pipetted into a 100 ml volumetric flask and the solution is made up to the mark with 100 ml n-hexane. The concentration is approx. 0.7 mg/100 ml. (see also 7.3.2 Calibration).

To this end, the absorption capacity of the ß-carotene solution is measured against n-hexane in a quartz cuvette with a layer thickness of 1 cm at the absorption maximum of 453 455 nm for ß-carotene using a suitable spectrophotometer (e.g. RPM002). The mass concentration, , of ß-carotene in micrograms per millilitre is calculated using the following equation:

A 104 mlg ]/[ 2592

Where: A Absorption maximum at 453 to 455 nm %1 2592 E1cm value for ß-carotene in n-hexane

As an additional safeguard, the absorption capacity of the standard solution is measured against n-hexane at 453 nm, 455 nm, 340 nm and 483 nm.

The following ratio is determined for each wavelength:

E/E453 (or 455nm)

The standard can be used for calibration purposes if the ratio for ß-carotene is > 15 (at 340 nm) and between 1.14 1.18 (at 483 nm). If the contents deviate from the data provided in the certificate, the concentrations of the standard solutions are calculated based on the contents measured photometrically.

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7.3.2 Production of the calibration solutions

There is a form that provides the exact details on the concentrations of the individual solutions for the production of the calibration solutions (see Appendix 3). This form provides an exact documentation of the initial weights and the dilution steps. It is filed in the corresponding folder together with the chromatograms and the calibration curve.

ß-carotene stock solution I: About 70 mg ß-carotene are weighed into a 100 ml volumetric flask (notbrown glass) at a precision of ± 0.1 mg, dissolved in n-hexane/ dichloromethane (80/20, v/v) and the solution is made up to the mark. The solution is then diluted 1/10 with n-hexane. The stock solution contains about 7 mg/100 ml. Attention: Brown glass volumetric flasks should not be used here as it is not possible to see whether the standard is fully dissolved. All further dilutions are carried out in a brown glass volumetric flask. Comment: The stock solution I is diluted again to 1/10 in n-hexane and spectrophoto- metrically assessed for concentration and purity, as described under point 7.3.1. Whether or not the standard can be used as a calibration standard and can only be determined based on the values obtained from the photometric determination of purity. This means that the dilutions in the following are only produced, or calculations of their contents are made, if the results of the determination of purity confirm a purity of > 95%.

ß-carotene stock solution II: 10.0 ml of stock solution I are pipetted into a 100 ml volumetric flask and the solvent is evaporated off under nitrogen. The residue is dissolved in 100 ml eluent (6.2). The stock solution II contains about 0.7 mg/100 ml.

Calibration solutions and working standard (dilutions made from stock solution II):

ß-carotene standard solution C1: 10.0 ml of stock solution II are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with eluent. The standard solution C1 contains about 0.07 mg/100 ml. ß-carotene standard solution C2: 5.0 ml of stock solution II are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with eluent. The standard solution C2 contains about 0.035 mg/100 ml.

ß-carotene standard solution C3 (working standard): 3.0 ml of stock solution II are pipetted into a 100 ml volumetric flask and the solution is made up to the mark with eluent. The standard solution C3 contains about 0.02 mg/100 ml. This solution is used as a working standard for regular checks on the HPLC equipment.

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ß-carotene standard solution C4: 1.0 ml of stock solution II is pipetted into a 100 ml volumetric flask and the solution is made up to the mark with eluent. The standard solution C4 contains about 0.007 mg/100 ml.

The standard solutions C 1 to C 4 are used for the calibration. The solutions are analysed in triplicate. A calibration curve is produced with the aid of the software and a corresponding response factor is calculated. The chromatograms thus obtained and the calibration curve are filed together with the form in the calibration folder.

The working standard (standard solution C3) is decanted into vials that are sealed and stored in the freezer at -4 °C. Stock solution I is highly unstable and cannot be stored in the refrigerator. A new calibration must be conducted if the working standard is used up prematurely.

A new calibration is otherwise only required if the working standard has been used up or its concentration deviates from the nominal concentration by more than 5 %. However, it should be conducted once a year.

8 Conduct General note: Vitamin A, E and ß-carotene are sensitive to UV radiation and oxidizing agents (such as, e.g. oxygen). It is therefore important to protect the samples from exposure to UV light (e.g. through the use of brown glass or aluminium foil) and oxygen (by working under nitrogen, especially during the saponification) as much as is possible.

8.1 Preparation / processing The sample material will need to be chopped up and homogenized by milling if it is a cereal. All other samples will generally not require milling unless they are inhomogeneous or if they contain coarse-grained material.

8.2. Weighing out samples All samples are weighed out into a 250 ml brown glass flat-bottomed flask. The initial weights should never be below 0.5 g or above 10 g for solid samples. Following extraction, the sample must be diluted accordingly if vitamin contents are high (e.g. for vitamin mixtures). The initial weight is generally tailored to the vitamin A content and is selected such that the vitamin A content in the initial weight is at least 0.01 mg, but no more than 0.05 mg. If only vitamin E is of interest in the sample, then the vitamin E content in the initial weight should be at least 0.1 mg, but no more than 1.0 mg.

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If only vitamin ß-carotene is of interest in the sample, then the ß-carotene content in the initial weight should be at least 0.005 mg, but no more than 0.05 mg. The weight table in the following is used for guidance in the selection of the initial weight: Vitamin A vitamin content [mg/100g] initial weight [g] solid samples ~0.100 0.700 5.0 vitamin mixture/effervescent tablet >1.0 600.0 0.5 1.0* vitamin content [mg/100g] initial weight [g] liquid samples ~0.05 0.1 20.0 >0.1 10.0 creams (e.g. Forticream) 0.1 0.2 10.0

Vitamin E vitamin content [mg/100g] initial weight [g] solid samples ~ 2.0 20.0 5.0 vitamin mixture/effervescent tablet >20 0.5 1.0* vitamin content [mg/100g] initial weight [g] liquid samples ~0.5 5.0 20.0 >5.0 10.0 creams (e.g. Forticream) 1.0 2.0 10.0

Vitamin ß-carotene vitamin content [mg/100g] initial weight [g] solid samples ~ 0.05 1,0 5.0 vitamin mixture/effervescent tablet >50 0.5 1.0* vitamin content [mg/100g] initial weight [g] liquid samples ~0.05 0.25 20.0 >0.25 10.0 creams (e.g. Forticream) 1.0 2.0 10.0 * Samples must be further diluted

8.3 Saponification

The samples are initially suspended in 20 to 40 ml of water and left to stand for a short while. Attention: Care must be taken to ensure that no lumps are formed; the sample must be fully wetted with water. While swirling the flask, add 100 ml ethanol, about 1 g sodium ascorbate or L-ascorbic acid and 25 ml potassium hydroxide solution (60%) to the suspended sample. The flat- bottomed flasks are then sealed with a glass stopper and shaken vigorously. The reaction mixture is now heated to boiling point under reflux in a water bath that has been pre-heated to 80 - 90°C and maintained at boiling point for 30 min under a slow flow of nitrogen. Alternatively, the samples can also be heated for 3 hours in a water bath at 50 °C and then left to stand overnight after the saponification is complete. In this case, the samples are sealed with a glass stopper.

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Attention: Ensure that the ground glass joint of the flat-bottomed flask is thoroughly rinsed with water before it is fitted to the reflux condenser, as it will otherwise not be possible to remove the flask from the reflux condenser after saponification. However, if too much water is added, then saponification will no longer be complete. After saponification, rinse the condenser with a little water and cool the contents of the flask to about 15°C. Attention: If oil drops are still visible on the surface of the saponified mixture after saponification, then add more potassium hydroxide solution and extend the duration of saponification.

8.4 Extraction

The saponified solution is transferred quantitatively to a separating funnel through multiple rinsing with water. In order to avoid the formation of emulsions, sufficient water is added to the saponified sample solution to ensure that the alcohol/water ratio in the resulting solution is about 1:1. Following this, 150.0 ml petroleum ether is added and the vitamins are extracted by shaking on a mechanical shaker for approx. 10 minutes.

Attention: The quantity of petroleum ether that is added must be measured accurately.

The aqueous phase is allowed to drain away and discarded after phase separation. Add a little ethanol to dissolve the emulsion if the aqueous and the petroleum ether phases do not separate.

Attention: The two phases must separate before the aqueous phase is allowed to drain away.

The extraction solution is now washed neutrally with purified water through multiple shaking and the washing water is allowed to drain away after the phases have separated. The extraction solution is filtered through a phase separation filter into a 50 ml volumetric flask to remove any potential suspended water drops.

8.5 Concentration

For vitamin A and E and for carotene, 3.0 ml each of the extraction solution are pipetted into a brown glass vial and the solution is evaporated to dryness under nitrogen at 45 °C in the evaporation system.

Attention: Vitamin mixtures and more highly concentrated samples (see 8.2 Weighing out samples) must be diluted accordingly prior to evaporation to dryness. After evaporation to dryness, each residue is taken up in 1.0 ml methanol for the determination of vitamin A and E and ß-carotene. The solutions are transferred to HPLC vials and can be used for the determination.

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8.6 Determination

Vitamin A and E: Vitamin A and vitamin E are measured simultaneously on one column. To this end, 50 µl of the sample and standard solutions (working standard A/E 6) are injected onto the HPLC column and eluted using a methanol/water mixture (98+2, v/v). A flow-rate gradient is used to ensure that the two substances elute at different retention times and to largely eliminate interfering matrix peaks (see 6. HPLC). Furthermore, the wavelength is changed from 326 nm (for retinol) to 292 nm (for tocopherol) after 8.5 min. Comment: The HPLC separation conditions must be selected such that the isomers of all-trans retinol and 13-cis-retinol are combined in one peak.

Vitamin ß-carotene: 20 µl of the sample and standard solution (working standard C3) are injected onto the HPLC column and eluted isocratically at a flow rate of 1.5 ml/min using a mixture of dichloromethane/ methanolic ammonium acetate solution/ acetonitrile (20/5/75, v/v) with the addition of 0.08 g BHT and 0.085 g triethylamine per 100 ml. Determination is carried out using VIS detection at 450 nm. Comment: The conditions must be selected such that -carotene is separated from ß-carotene.

9 Analysis

Vitamin A The isomers of all-cis retinol and all-trans retinol are combined in one peak and their sum is the content of vitamin A. Comment: All samples that contain retinol palmitate are analysed using the retinol palmitate calibration (see 7.2). All other samples containing retinol or retinol acetate are analysed using the retinol calibration (see 7.1). Vitamin E: At higher contents, the peak for -tocopherol exhibits a small shoulder that does not need to be separated off. Vitamin ß-carotene Two separate peaks are obtained for -carotene and ß-carotene. The peak for ß-carotene exhibits a small shoulder, the area of which must be included for the determination of content. -carotene elutes approx. 1 minute before ß-carotene and appears as a double peak. If -carotene is present in the sample, then this peak is separated from the ß-carotene peak and the contents are given separately. However, the value for -carotene is only given as a guideline, as no calibration is conducted with -carotene.

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10 Calculation The contents of vitamin A, E and ß-carotene are calculated using the external standard method. A response factor is calculated based on the peak areas of the standard and this is then used to calculate the vitamin content in the sample. The contents of vitamin A, E and ß-carotene are given in mg/100g sample.

Calculation of the response factor from the standard chromatogram:

c(Std.) mg /100ml Re sponsefactor Area

Calculation of the vitamin content based on the response factor:

Area(Pr) Re sponsefactor c Vitamin mg /100 g 100 gW ][

Area Sa c()( Std.)[mg] 150 1mlml c Vita min mg /100 g 100 f Area Std)( 100ml gE 3][ ml

Where: c (vitamin): Vitamin concentration [mg/100g] Area (Std.): Standard peak area Area (Sa) Sample peak area W: Initial weight [g] c (Std): Standard concentration [mg/100ml] 150 ml: Quantity of petroleum ether added 3 ml: Quantity evaporated off 1 ml: Quantity of MeOH or iso-octane added 100: Factor for the calculation per 100 g sample f: Dilution factor dependent on dilution

11 Validation

11.1 Validation data for vitamin A

The current method essentially corresponds to the European Standard prEn 12823-1, Determination of Vitamin A by high performance liquid chromatography Part 1: Measurements of all-trans retinol and 13-cis retinol (Final draft, September 1999) . The following characteristic procedural data were determined to assess and validate the method at CLF:

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11.1.1 Linearity Linearity was assessed with the aid of standard solutions at different concentrations. Two separate calibration curves were produced. To this end, 7 different concentrations ranging between ~ 8 µg/100ml and ~ 200 µg/100ml were measured for retinol. For retinol palmitate, 6 different concentrations ranging between ~ 40 µg/100ml and ~ 200 µg/100ml were measured.

The following correlation coefficients (R²) were calculated:

Retinol Retinol palmitate Correlation coefficient (R²) 0.9999 0.9992

11.1.2 Precision The precision was assessed with repeat analysis (6-fold) of the 3 most frequently occurring matrices: infant formula, milk cereal and PKU. For the infant formula, repeated precision (several determinations, one laboratory, different days, different analysts) was assessed in addition to laboratory precision (repeat determination, one laboratory, one day, one analyst).

The expanded uncertainty (UE) was then calculated from the standard deviation (s), which permits a statement to be made on the confidence interval for the method.

Vitamin A x (n=5) [mg/100g] 0.357 repeated precision s [mg/100g] 0.0106 infant formula CV [%] 3.0 U [mg/100g] 0.030 x (n=6) [mg/100g] 0.352 laboratory precision s [mg/100g] 0.0207 infant formula CV [%] 5.9 U [mg/100g] 0.048 x (n=6) [mg/100g] 1.071 repeated precision s [mg/100g] 0.150 PKU CV [%] 14.0 U [mg/100g] 0.386 x (n=6) [mg/100g] 0.358 repeated precision s [mg/100g] 0.0606 milk cereal CV [%] 16.9 U [mg/100g] 0.156

11.1.3 Accuracy The accuracy was assessed by spiking an infant formula with standard solutions. The sample was initially analysed without spiking and then twice each with spiking in the lower

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and upper working ranges. In addition, a reagent blank was measured. Following this, the recovery rate was determined for the added quantity of standard.

Vitamin A Recovery - mean value (x) [% ] 84.6

11.1.4 Limit of determination The limit of determination was determined based on the signal/noise ratio. Standard solutions of different concentrations were injected to determine the limit of determination. The concentration at which the peak area was still at least 10 times higher than the noise was defined as the limit of determination.

Vitamin A Limit of determination [mg/100 g] 0.02

11.1.5 Reference material Within the framework of routine analysis, the current method is assessed through regular analysis of internal reference material. The internal reference material is always analysed together with the routine samples as a control sample and the results are regularly entered into quality control charts.

11.1.6 Inter-laboratory comparisons A further assessment of the method occurs through regular participation in inter-laboratory comparisons.

11.2 Validation data beta-carotene The current method essentially corresponds to the European Standard DIN EN 12823-2, Determination of Vitamin A by high performance liquid chromatography Part 2: Measurement of ß-carotene (July 2000) . The following characteristic procedural data, listed under points 1-4, were determined to assess and validate the method at CLF:

11.2.1 Linearity Linearity was assessed with the aid of standard solutions at different concentrations. To this end, a calibration curve was produced using 6 different concentrations ranging between ~ 30 µg/100ml and 190 µg/100ml.

Based on this, the following correlation coefficient (r) was calculated:

Beta-carotene Correlation coefficient (R²) 0.9999

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11.2.2 Precision Reproducibility was determined for beta-carotene. To this end, infant formula was analysed by different people over a lengthy time period.

The expanded uncertainty (UE) was then calculated from the standard deviation (s), which permits a statement to be made on the confidence interval for the method.

Beta-carotene x (n=5) [mg/100g] 0.150 laboratory precision s [mg/100g] 0.0104 infant formula CV [%] 6.9 U [mg/100g] 0.025

11.2.3 Accuracy The accuracy was assessed by spiking an infant formula with standard solutions. The sample was initially analysed without spiking and then twice each with spiking in the lower and upper working ranges. In addition, a reagent blank was measured. Following this, the recovery rate was determined for the added quantity of standard.

Beta-carotene Recovery - mean value (x) [% ] 95.7

11.2.4 Limit of determination The limit of determination was determined based on the signal/noise ratio. Standard solutions of different concentrations were injected to determine the limit of determination. The concentration at which the peak area was still at least 10 times higher than the noise was defined as the limit of determination.

Beta-carotene Limit of determination [mg/100 g] 0.06

11.2.5 Reference material Within the framework of routine analysis, the current method is assessed through regular analysis of internal reference material. The internal reference material is always analysed together with the routine samples as a control sample and the results are regularly entered into quality control records.

11.2.6 Inter-laboratory comparisons

A further assessment of the method occurs through regular participation in inter-laboratory comparisons.

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11.3 Validation data Vitamin E The current method essentially corresponds to the European Standard prEn 12822, Determination of Vitamin E by high performance liquid chromatography Measurements of -, -, - and -tocopherol (Final draft, September 1999) . The following characteristic procedural data, listed under points 1-4, were determined to assess and validate the method at CLF. (If not otherwise noted, the data provided under vitamin E refer to -tocopherol).

11.3.1 Linearity Linearity was assessed with the aid of standard solutions at different concentrations. To this end, a calibration curve was produced using 7 different concentrations ranging between ~ 5 µg/100ml and 180 µg/100ml.

Based on this, the following correlation coefficient (r) was calculated:

Vitamin E Correlation coefficient (r) 0.9994

11.3.2 Precision The precision was assessed with repeat analysis (6-fold) of the 3 most frequently occurring matrices: infant formula, milk cereal and PKU. For the infant formula, repeated precision (several determinations, one laboratory, different days, different analysts) was assessed in addition to laboratory precision (repeat determination, one laboratory, one day, one analyst).

The expanded uncertainty (UE) was then calculated from the standard deviation (s), which permits a statement to be made on the confidence interval for the method.

Vitamin E x (n=5) [mg/100g] 6.00 repeated precision s [mg/100g] 0.123 infant formula CV [%] 2.1 U [mg/100g] 0.34 x (n=6) [mg/100g] 6.18 laboratory precision s [mg/100g] 0.389 infant formula CV [%] 6.3 U [mg/100g] 0.90 x (n=6) [mg/100g] 25.30 repeated precision s [mg/100g] 2.28 PKU CV [%] 3.0 U [mg/100g] 5.86

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x (n=6) [mg/100g] 3.03 repeated precision s [mg/100g] 0.103 milk cereal CV [%] 3.4 U [mg/100g] 0.27

11.3.3 Accuracy The accuracy was assessed by spiking an infant formula with standard solutions. The sample was initially analysed without spiking and then twice each with spiking in the lower and upper working ranges. In addition, a reagent blank was measured. Following this, the recovery rate was determined for the added quantity of standard.

Vitamin E Recovery - mean value (x) [% ] 96.9

11.3.4 Limit of determination The limit of determination was determined based on the signal/noise ratio. Standard solutions of different concentrations were injected to determine the limit of determination. The concentration at which the peak area was still at least 10 times higher than the noise was defined as the limit of determination.

Vitamin E Limit of determination [mg/100 g] 0.08

11.3.5 Reference material Within the framework of routine analysis, the current method is assessed through regular analysis of internal reference material. The internal reference material is always analysed together with the routine samples as a control sample and the results are regularly entered into quality control records.

11.3.6 Inter-laboratory comparisons

A further assessment of the method occurs through regular participation in inter-laboratory comparisons.

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9 References 1. European Standard prEn 12823-1 Determination of Vitamin A by high performance liquid chromatography Part 1: Measurements of all-trans retinol and 13-cis retinol, Final draft, September 1999

2. European Standard DIN EN 12823-2 Determination of Vitamin A by high performance liquid chromatography Part 2: Measurement of ß-carotene (July 2000)

3. European Standard prEn 12822 Determination of Vitamin E by high performance liquid chromatography Measurements of -, -, - and -tocopherol, Final draft, September 1999 4. Official collection of methods for sampling and examining foods pursuant to § 64 of the Foods and Other Commodities Act (LMBG) Determination of vitamin A in dietary food products L49.00-3, May 1985; Determination of vitamin A in food products by HPLC Part 1: Measurements of all- trans retinol and 13-cis retinol L00.00-63/1; Part 2: Measurement of ß-carotene L00.00-63/2, July 2001; Determination of vitamin E in food products by HPLC, L00.00-62, July 2001 5. VA504_06_V01 Method validation

10 Attachments Attachement 1 Form for the calibration and purity assessment of retinol and d,l- -tocopherol Attachement 2 Form for the calibration and purity assessment of retinol palmitate Attachement 3 Form for the calibration and purity assessment of ß-carotene Attachement 4 Chromatogram of a retinol and tocopherol standard Attachement 5 Chromatogram of a ß-carotene standard