ISO 23349:2020 download free.Animal and vegetable fats and oils 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.
ISO 23349 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 al111 and Srigley and Haile[21 for the preparation and gas chromatographic separation of phytosterol trimethylsilyl ether derivatives, respectively.
Tn 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.[l 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 12-16141.
This reference method is appropriate for the determination of the five major phytosterols (i.e. campesterol, campestanol, stigmasterol, f3-sitosterol and sitostanol) that are the subject of FDA’s health claim regulation for phytosterols and the reduced risk of coronary heart diseasell.
1 Scope
ISO 23349 specifies a reference method for the determination of free sterols/stanols and steryl/ stanol esters (0.1 % to 97 % mass fraction) in foods and dietary supplements containing added phytosterols and in phytosterol food additive concentrates.
Milk and milk products (or fat coming from milk and milk products) are excluded from the scope of this document. This method does not apply to matrices that contain rice bran oil at concentrations of more than 20 % due to possible interferences in the gas chromatogram.
2 Normative references
There are iio ourmative rderences in ISO 23349.
4 Principle
Free sterols/stanols and saponified steryl/stanol esters are derivatized to phytosterol trimethylsilyl
(TMS) ethers and separated on a capillary gas chromatography column.
5 Reagents
Use only reagents of recognized analytical grade.
WARNING — Attention is drawn to regulations that specify the handling of hazardous substances. Technical, organizational and personal safety measures shall be followed. A chemical fume hood shall be used for the work.
5.1 Pyridine.
5.2 N,O-Bis(trimethylsilyl) trifluoroacetamide + 1 % trimethyichiorosilane (BSTFA).
5.3 Internal standard dissolving solvent, toluene (recommended) or ethyl acetate.
5.4 Methanol.
5.5 Sodium hydroxide, pellets, of purity  97 %.
d) Transfer 300 p1 of derivatized sample solution, I ml of toluene or ethyl acetate (5.3) and 25 mg of sodium sulfate (5.1.2) to an autosampler vial (6.12). Cap tightly and vortex (6.1UJ to mix. The sample is now ready for gas chromatographic separation (see Oause 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 (6.2.1). Record the exact weight.
b) Add a few boiling chips (6.19) and place a PTFE sleeve (612) in the neck of the flask.
c) Add 5,00 ml of the appropriate epicoprostanol IS solution (5,9 or 5.1Q).
d) Add 5 ml olsodlum hydroxide solution (5.6).
e) Boil the sample at 100°C for 15 minor 120 mm (see 9.1).
f) Remove the flask from the hear source, insert a stopper (6.7.3) and let cool to room temperature.
g) Add 7 ml of hydrochloric acid (5.2). insert a stopper (62.3) and shake to mix.
h) Add 40 ml of sodium chloride solution (5.3.4), 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 p1 of organic phase and 25mg of sodium sulfate (5.12) to an autosampler vial (6.17).
J) Add 0.5 ml of pyridlne (5.1) and I ml of BSTFA (5.2) Cap tightly and vortex (6.10) to mix. The sample Is now ready for gas diromatographic separation (see Clause 10).
9.4 45 mm acid/iS mm 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 (see 9.1).
a) Accuratelyweight the test portion into a tarred round bottom flask (6.2.1). Record the exact weight.
b) Add a few boiling chips (6.1.9) and place a PTFE sleeve (622) in the neck of the flask.
c) Add S ml of hydrochloric acId (51).
d) Add 5.00 ml of the appropriate epicoprostanol IS solution (5,9 or £10).
e) Boil the sample at 100°C for 45 mm.
fJ 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 (5.1.4). 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 (6.11).
I) Add 5 ml of sodium hydroxide solution (5.6).
) Boll the sample at 100°C for IS mm.
k) Remove the flask from the heat source, insert a stopper (613) and let cool to room temperature.
I) Add 7 ml of hydrochloric acId (51). insert a stopper (623) and shake to mix.
13.1 Verify acceptable chromatographic separations by analysing the phytosterol reference standard. Ensure that a baseline resolution is achieved between peaks corresponding to campesterol and campestanol, and 13-sitosterol and sitostanol. Decrease the flow rate of the Il2 carrier gas (e.g. by 0,05 ml/ mm to 0,1 mI/mm) if a greater resolution is needed.
13.2 Perform a spike-recovery experiment to verify the accuracy of the method for determining phytosterols in the subject sample matrix. Prepare the spiked sample by adding a known amount of stigmasterol to the subject sample and analyse according to the appropriate sample preparation protocol. Calculate the recovery of stigmasterol by determining the analysed content, taken as a proportion of the spiked content and corrected for any incurred amount in the sample.
13.3 Analyse a blank matrix sample to verify the absence of interferences in the analysis due to matrix, reagents or equipment used. Confirm that the chrornatogram of the blank matrix sample is devoid of peaks eluting in the region in which phytosterols elute (i.e. 23 mm to 60 mm).
13.4 A standard reference material has yet to be created for food and dietary supplement samples containing added phytosterols.