BS ISO 20633:2015 pdf download – Infant formula and adult nutritionals — Determination of vitamin E and vitamin A by normal phase high performance liquid chromatography.
6.1.3 Wet blended powder samples For wet blended homogenous powder samples, accurately weigh approximately 0,525 g into a screw- top 50 ml centrifuge tube. Add 5 ml warm water of approximately 40 °C and shake to dissolve. 6.1.4 Liquid samples For ready-to-feed samples or concentrated liquid products, accurately weigh approximately 5,0 g (m 3 ) of thoroughly homogenized sample into a screw-top 50 ml centrifuge tube. 6.1.5 Sample extraction To the above weighed solutions, add 5 ml of papain solution (4.11). Mix to disperse each sample, cap, and place the tubes in a 37 °C ± 2 °C water bath for 20 min to 25 min. Remove the samples from the water bath and cool. Place in a freezer for about 5 min or refrigerate for about 20 min. Add 20 ml acidified methanol (4.12) to each sample tube and shake the tubes for 10 min preferably with a mechanical shaker. Accurately pipet 10 ml of iso-octane to each sample tube. Close tightly to avoid leakage and shake the tubes for 10 min preferably with a mechanical shaker. Centrifuge for 10 min at 4 000 rpm to obtain a clear iso-octane layer. Transfer an aliquot of the clear iso-octane layer into amber vials for HPLC analysis. 6.2 HPLC analysis 6.2.1 General The separation and the quantification have proven to be satisfactory if the following experimental conditions are followed: Column: Agilent Zorbax® NH 2 3) (5 µm, 150 mm × 4,6 mm); Mobile phase A: n-hexane; Mobile phase B: mixture of 750 ml of n-hexane, 250 ml of methyl-t-butyl ether and 3 ml of methanol;
For the purposes of this method there is no differentiation of the varying contributions of cis- and trans- isomers to the total retinyl palmitate/acetate activity. For vitamin A peak integration, sum the area of the 13-cis and all trans isomers of retinyl palmitate/acetate and calculate against the trans isomer. Convert vitamin A results to retinol using stoichiometric calculations in accordance with Formula (12): Vitamin A as retinol (µg/100 g) = (retinyl palmitate in µg/100 g × 0,55) + (retinyl acetate µg/ 100 g × 0,87) (12) Convert vitamin E results to α-tocopherol using stoichiometric calculations: — 1 mg of α-tocopheryl acetate is equal to 1,10 α-tocopherol, and — 1 mg = 1,10 DL α-tocopherol (synthetic vitamin E; all racemic α-tocopherol).