BS ISO 20633:2015 pdf free.Infant formula and adult nutritionals – Determination of vitamin E and vitamin A by normal phase high performance liquid chromatography.
BS ISO 20633 specifies a method for the simultaneous quantitative determination of vitamin E (a-tocopherol and a-tocopheryl acetate) and vitamin A (13-cis and all-trans isomers of retinyl palmitate and retinyl acetate) present in all forms of infant and adult formulas (powders, ready-to-feed liquids and liquid concentrates).
Retinol is not used for fortification purposes and therefore is not addressed in this method. The innate amount in products is insignificant.
Stereoisomers of vitamin E, a-tocopherol and a-tocopheryl acetate, are not differentiated in this method.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1 adult nutritional
nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment, made from any combination of milk, soy, rice, whey, hydrolysed protein, starch and amino acids, with and without intact protein
2.2 infant formula
breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feeding [SOURCE: Codex Standard 72-19811
3 Principle
This procedure utilizes the proteolytic enzyme, papain, to hydrolyze the hydrophilic protein coating of fat micelles in milk or soy-based infant formulations in an aqueous solution. The hydrophobic contents of the micelles are then extracted quantitatively into iso-octane in a single extraction. The extract is analysed by normal phase IIPLC using an analytical column with gradient elution. Quantification of a-tocopherol and a-tocopheryl acetate is done using fluorescence detection with excitation and emission wavelengths at 280 nrn and 310 nm. Retinyl palmitate (cis and trans) and retinyl acetate (cis and trans are quantified using UV detection at 325 nm.
4 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and
distilled or demineralized water or water of equivalent purity.
4.1 Methyl-t-butyl ether, also known as tert-butylmethylether, HPLC grade.
4.2 n-Hexane, HPLC grade.
4.3 Ethanol, HPLC grade.
4.4 Methanol, IIPLC grade.
4.5 Iso-octane (2,2,4-trimethylpentane), IIPLC grade.
4.6 Papain (from Carica papaya),  3 U/mg. Sigma 762201) or equivalent.
4.7 Hydroquinone, Sigma H9003’) or equivalent.
4.8 Glacial acetic acid, analytical reagent grade.
4.9 Anhydrous sodium acetate.
4.10 Dilute hydrochloric acid solution.
Dilute 100 ml of a hydrochloric acid solution with a mass fraction of 36 % to 200 ml with water.
4.11 Papain solution, mass concentration p = 20 g/l.
Dissolve 100 mg hydroquinone and 4 g anhydrous sodium acetate in approximately 80 ml of water in a 100 ml one-mark volumetric flask (iil). Adjust the pH to 5,0 with dilute hydrochloric acid solution (4J.O). Add 2 g of papain and make up to volume. Prepare fresh prior to use.
4.12 Acidified methanol solution.
Add 20 ml of glacial acetic acid to 11 of methanol and mix. Prepare fresh on the day of use.BS ISO 20633 pdf download.