ISO 23349:2020 pdf free download – Animal and vegetable fats and oils一 Determination of sterols and stanols in foods and dietary supplements containing added phytosterols.
Plant sterols and plant stanols, collectively referred to as phytosterols, are increasingly recognized for their role in reducing the risk of coronary heart disease by lowering serum total and low-density lipoprotein cholesterol. Because of these potential health benefits, health claim regulations authorizing the addition of phytosterols and phytosterol esters to foods and dietary supplements now exist in countries throughout Europe, North and South America, Asia, New Zealand and Australia.
This document was developed in response to the worldwide demand for a reference method for quantifying total and individual phytosterols in foods and dietary supplements containing added phytosterols, and in the phytosterol food additive concentrates used to prepare such products. This reference method is based on the single-laboratory validated methods of Clement et al.[1] and Srigley and Haile[2] for the preparation and gas chromatographic separation of phytosterol trimethylsilyl ether derivatives, respectively.
In 2016 to 2017, an international collaborative study co-organized by the United States Food and Drug Administration (FDA), Cargill (USA) and the American Oil Chemists’ Society (AOCS) was carried out to evaluate the performance of this method for the determination of total and individual phytosterols in foods, dietary supplements and phytosterol concentrates.[3] A total of 14 laboratories from 6 countries successfully completed the analysis of 18 test materials, upon which the method was approved as AOCS Official Method Ce 12-16[4].
This reference method is appropriate for the determination of the five major phytosterols (i.e. campesterol, campestanol, stigmasterol, -sitosterol and sitostanol) that are the subject of FDA’s health claim regulation for phytosterols and the reduced risk of coronary heart disease.
d) Transfer 300 iii of derivatized sample solution, 1 ml of toluene or ethyl acetate (5.3) and 25 mg of sodium sulfate (5.12) to an autosampler vial (6.17). Cap tightly and vortex (6.18) to mix. The sample is now ready for gas chromatographic separation (see Clause 10).
9.3 15 mm and 120 mm alkaline protocols
Use this protocol for most foods, dietary supplements and steryl/stanol ester concentrates.
a) Accurately weight the test portion into a tarred round bottom flask (6.7.1). Record the exact weight.
b) Add a few boiling chips (6.19) and place a PTFE sleeve (6.7.2) in the neck of the flask.
c) Add 5,00 ml of the appropriate epicoprostanol IS solution (5.9 or 5.10).
d) Add 5 ml of sodium hydroxide solution.
e) Boil the sample at 100 °C for 15 mm or 120 mm (see 91.).
f) Remove the flask from the heat source, insert a stopper (6.7.3) and let cool to room temperature.
g) Add 7 ml of hydrochloric acid, insert a stopper (6.7.3) and shake to mix.
h) Add 40 ml of sodium chloride solution (5.14), insert a stopper (6.7.3) and shake to mix. Allow the two phases to separate. Wait until the cloudy organic phase is clear before proceeding.
i) Transfer 300 p.1 of organic phase and 25 mg of sodium sulfate (5.12) to an autosampler vial (6.17).
j) Add 0,5 ml of pyridine 5JJ and 1 ml of BSTFA (5.2). Cap tightly and vortex (6.18) to mix. The sample is now ready for gas chromatographic separation (see Clause 10).
9.4 45 mm acid/iS mill alkaline protocol
Use this protocol for some foods and dietary supplements containing added phytosterol esters. Acid hydrolysis prior to saponification is needed for the complete liberation of phytosterol esters from certain matrices, such as some cereals.
a) Accurately weight the test portion into a tarred round bottom flask (6.7.1). Record the exact weight.
b) Add a few boiling chips (6.19) and place a PTFE sleeve (6.7.2) in the neck of the flask.
c) Add 5 ml of hydrochloric acid (52).
d) Add 5,00 ml of the appropriate epicoprostanol IS solution (5.9 or 5.10).
e) Boil the sample at 100 °C for 45 mm.
f) Remove the flask from the heat source, insert a stopper (6.7.3) and let cool to room temperature.
g) Add 40 ml of sodium chloride solution (5.14), insert a stopper (6.7.3) and shake to mix. Allow the two phases to separate.
h) Transfer the organic phase (as much as possible without disrupting the solids on the surface) to a new round bottom flask (6.7.1).
i) Add 5 ml of sodium hydroxide solution (5.4).