BS EN 15911-2010 pdf download.Foodstuffs Simultaneous determination of nine sweeteners by high performance liquid chromatography and evaporative light scattering detection.
NOTE In case of cyclamic acid and saccharin, their sodium salts are used, since they are either not available in free form or poorly soluble.
The final concentrations of the individual sweeteners in micrograms per millilitre in the mixed stock solution have to be calculated by using the actually weighed masses.
4.18 Standard solutions.
From the mixed stock solution (4.17) prepare a series of standard solutions containing the sweeteners at levels fitting appropriate limits, e.g. the highest concentration of the calibration shall be at least equivalent to 125 % of the given limits, such as those in Commission Directives (3), [4], [5) (see Table D.1), whilst taking the dilution steps within the procedure into account (see Table 2). For sweeteners not authonsed by the current EU legislation (ALl, DUL and NEO) fictitious maximum usable dosages (MUD) are assumed at approximately 200 mg/I or 200 mglkg.
The user of the standard has to check whether the limits in Table D.1 are still valid. If not, the mass concentration of the standard substance in the calibration solution shall be adjusted to meet the current MUDs.
NOTE The present procedure is simplified by pepaflng one cahbration series for both food matrices. The described calibration series is fitted to canned fruits as the MUDs for canned fruits are in some cases higher than the MUDs for beverages. In case only the latter matrix is analysed the calibration series can be fitted to the MUDs of beverages.
Pipette the following volumes (see Table 2) from the mixed stock solution (4.17) into appropriate volumetric flasks (10 ml to 50 ml) and make up to the mark with buffer solution (4.14) and shake thoroughly.
5.11 SPE Vacuum system, or equivalent.
5.12 Equipment for solvent evaporation.
5.13 pH meter.
5.14 C1SPE cartrIdges.
5.15 Analytical reverse phase column, fully endcapped. allowing sufficient separation of all nine sweeteners.
E.g. with:
an RP C 18 stationary phase of 5 pm; a length of 250 mm;
internal diameter of 3 mm.
5.16 HPLC system. equipped with a binary pump capable of maintaining a flow rate of 0,5 mI/mm, preferably an automatic injection system, and an evaporative light scattering detector.
Other detection systems such as MS as substitute for ELSD or UV and DAD when substances do absorb in the UV region can also be used provided that the equivalent performance characteristics can be obtained.
5.17 Data acquisition and analysis software.
6 Procedure
6.1 General
Comminute the entire Lest sample to give a homogenous suspension (5.8). Liquid samples can be subjected
directly to the extraction procedure.
6.2 PreparatIon of test sample
6.2.1 Step 1
Weigh approximately 5g (M1, recorded to two decimal places) of the homogenised test sample (6.1) into a volumetric flask of 50 ml (V,). Make up to the mark with buffer solution (4.14), mix thoroughly by hand to obtain a homogeneous suspension and sonicate (5.9) for 15 mm.
6.3.1 Step 1
Condition the cartridges (5.14) by applying 3 ml of methanol (412) and let it pass through using a slight vacuum resulting in a flow rate of 1 mI/mm to 2 mI/mm. Make sure that a small portion of methanol remains above the sorbent bed (1 mm).
6.3.2 Step 2
Equilibrate the cartridges (5.14) by applying 2 ml of buffer solution (4.14) and let it pass through using a slight vacuum resulting in a flow rate of 1 mI/mm to 2 mI/mm. Make sure that a small portion of buffer solution remains above the sorbent bed (1 mm). Repeat the procedure two times.
6.3.3 Step 3
Load the cartridges (5.14) with 5 ml of sample extract (V2 first loading), i.e. the supernatant from (6.2.2), and let it pass through using a slight vacuum resulting in a flow rate of 1 mI/mm to 2 mI/mm. Make sure that a small portion remains above the sorbent bed (1 mm). Repeat the procedure once more (V2in total 10 ml).
6.3.4 Step 4
Wash the cartridges (5.14) with 3 ml of buffer solution (4.14) and let it pass through using a slight vacuum resulting in a flow rate of 1 mI/mm to 2 mI/mm. Make sure that a small portion of butter solution remains above the sorbent bed (1 mm).
6.3.5 Step 5
Elute the sweeteners from the cartridges (5.14) by applying 2 ml of methanol (4.12) and collecting the eluate in a graduated 5 ml test tube. Use a slight vacuum to obtain a flow rate of 1 mI/mm. Make sure that a small portion of methanol remains above the sorbent bed (1 mm). Wait 10 mm before applying a second portion of 2 ml of methanol and elute it subsequently to the same 5 ml test tube using the same vacuum conditions but this time letting the cartridges (5.14) run dry.
Avoid in all steps (6.2.1 to 6.3.5) that the sorbent bed runs dry with the only exception of the last step, i.e. second elution of analytes (6.3.5).
6.3.6 Step 6
Evaporate the solvent from the rnethanolic SPE extract to 3 ml under a stream of nitrogen at ambient temperature.
Temperatures above 40 °C have to be avoided, since aspartame can degrade.BS EN 15911-2010 pdf download.