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Vitamin E ((±)- -Tocopherol) and ß-Carotene in Foodstuffs by HPLC-UV

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Vitamin E ((±)- -Tocopherol) and ß-Carotene in Foodstuffs by HPLC-UV SPSFAM-INGR14 Based from Call for Methods 03-09-2012 File No.: VITA-15 FOR WORKING GROUP USE ONLY No. N03_01ME_engl_v01(VitA,ß -Car,VitE).doc DO NOT DISTRIBUTE Vitamin A, vitamin E, ß-carotene Page: 1 version: 1 of: 25 written: 23.11.2009 printed: 08.02.2010 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 SPSFAM-INGR14 Based from Call for Methods 03-09-2012 File No.: VITA-15 FOR WORKING GROUP USE ONLY No. N03_01ME_engl_v01(VitA,ß -Car,VitE).doc DO NOT DISTRIBUTE Vitamin A, vitamin E, ß-carotene Page: 2 version: 1 of: 25 written: 23.11.2009 printed: 08.02.2010 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 SPSFAM-INGR14 Based from Call for Methods 03-09-2012 File No.: VITA-15 FOR WORKING GROUP USE ONLY No. N03_01ME_engl_v01(VitA,ß -Car,VitE).doc DO NOT DISTRIBUTE Vitamin A, vitamin E, ß-carotene Page: 3 version: 1 of: 25 written: 23.11.2009 printed: 08.02.2010 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) SPSFAM-INGR14 Based from Call for Methods 03-09-2012 File No.: VITA-15 FOR WORKING GROUP USE ONLY No. N03_01ME_engl_v01(VitA,ß -Car,VitE).doc DO NOT DISTRIBUTE Vitamin A, vitamin E, ß-carotene Page: 4 version: 1 of: 25 written: 23.11.2009 printed: 08.02.2010 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%) SPSFAM-INGR14 Based from Call for Methods 03-09-2012 File No.: VITA-15 FOR WORKING GROUP USE ONLY No. N03_01ME_engl_v01(VitA,ß -Car,VitE).doc DO NOT DISTRIBUTE Vitamin A, vitamin E, ß-carotene Page: 5 version: 1 of: 25 written: 23.11.2009 printed: 08.02.2010 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 SPSFAM-INGR14 Based from Call for Methods 03-09-2012 File No.: VITA-15 FOR WORKING GROUP USE ONLY No. N03_01ME_engl_v01(VitA,ß -Car,VitE).doc DO NOT DISTRIBUTE Vitamin A, vitamin E, ß-carotene Page: 6 version: 1 of: 25 written: 23.11.2009 printed: 08.02.2010 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.
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