ISO 23349:2020 pdf download – Animal and vegetable fats and oils – Determination of sterols and stanols in foods and dietary supplements containing added phytosterols
9.1 Selection of analysis protocol
For new formulations (phytosterol type and matrix), determine the optimal protocol by triplicate analysis according to the following protocols: a) 120 min alkaline, b) 15 min alkaline and c) 45 min acid/15 min alkaline. Select the protocol that produces the highest phytosterol recovery and lowest variability. Further refine this protocol, as needed, by decreasing the saponification time from 120 min to 60 min to 30 min, or the acid hydrolysis time from 45 min to 30 min to 15 min, with triplicate analyses for each. The optimal protocol will show a repeatability coefficient of variation (C V,r ) of less than 5 % for triplicate analyses over a period of five days of testing. The 120 min alkaline protocol is appropriate for the analysis of most foods and dietary supplements containing added phytosterols. The 45 min acid/15 min alkaline protocol can be required for complete liberation of phytosterol esters from certain matrices, such as some cereals.
9.3 15 min and 120 min 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 (5.6).
e) Boil the sample at 100 °C for 15 min or 120 min (see 9.1).
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 (5.7), 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 μl of organic phase and 25 mg of sodium sulfate (5.12) to an autosampler vial (6.17).
j) Add 0,5 ml of pyridine (5.1) 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 min acid/15 min 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) 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 (5.7).
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 min.
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 (5.6).
j) Boil the sample at 100 °C for 15 min.
k) Remove the flask from the heat source, insert a stopper (6.7.3) and let cool to room temperature.
l) Add 7 ml of hydrochloric acid (5.7), insert a stopper (6.7.3) and shake to mix.