ISO 23293:2020 pdf free.Milk-based infant formula powders – Quantification of whey protein content by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE).
7 Procedure
7.1 Sample preparation
7.1.1 Weigh 500 mg ± 20 mg of infant formula powder into a 10 ml centrifuge tube. A skim milk powder
(SMP) sample is used as a reference to align the whey and casein regions in the infant formula samples.
Prepare the SMP using a sample mass of 135 mg ± 5 mg.
7.1.2 Disperse the sample in S ml of water. Vortex each tube until the solution appears homogeneous. Each final solution contains between 10 mg/ml and 15 mg/ml of protein.
7.1.3 Pipette 10 il of each sample solution into separate microcentrifuge vials.
7.1.4 Sequentially add 85 il of the sample running pre-solution (5) and 5 iii of 3-mercaptoethanol
(5.S) to each micro-centrifuge vial. Mix well before heating the vials in a water bath or heating block at 95 °C ± 5 °C for 10 mm. Cool down to room temperature. Centrifuge at room temperature for 1 mm at about S 000g (approximately 7 000 r/min in a table-top centrifuge).
7.1.5 Vortex before transferring each sample into corresponding injection vials.
7.2 CGE analysis
7.2.1 The separation and the quantification have proven to be satisfactory if the following experimental conditions are followed.
7.2.2 Load the reagents according to the instructions of the capillary electrophoresis instrument manufacturer (see Figure A.1 for an example).
7.2.3 Set up an optimized batch analysis separation method including a reagent blank (10 tl of water replacing the sample solution), a molecular weight size standard (Si) and a SMP sample.
To prepare the reagent blank, replace the sample solution (see 7.1.3) by 10 uI water. Its function is to monitor any contamination in the buffer or reagents (there should be no peaks in the migration range of milk protein as defined from the 10 kDa internal standard protein). SMP works as a reference to verify the migration times for whey proteins and caseins.
7.2.4 For each separation cycle (45 mm), precondition the capillary first with the basic wash solution (SA) for 3 mm, followed by the acidic wash solution (5) for 1 mm and water for 1 mm. Fill the capillary with the SDS-MW gel separation buffer (51) for 10 mm. Set the separation run time to 30 mm.
7.2.5 Introduce the samples electrokinetically by applying voltage at —5 kV for 20 s.
7.3 Electropherogram processing
7.3.1 Electropherogram integration
Automatically integrate the electropherograms from the valley located at 0,5 mm to 1 mm before the 10 kDa internal protein standard peak (include any peak immediately adjacent to the 10 kDa internal protein standard) to the valley between the end of K-caseln peak and the peak of Ig H and BSA (no negative peaks should be generated by the integration). Then, perform a manual integration from the valley before the peak of Ig H and BSA until the end of the last peak in the electropherogram (integrate all peaks including those after the peak of Ig H and BSA if present). Examples of electropherograms including the automated and manual integration regions are given in Figures A.2 and A.3 for SMP and infant formulas, respectively. Examples of integration parameters are listed in Table A.1.
7.3.2 Identification of the casein region
In SMP, the casein integration area starts just before the 13-casein peak (the highest peak of the electropherogram) and stops at the valley between the end of the K-casein peak and the peak of Ig H and BSA. For infant formulas, identify the 3-casein peak and the valley by comparison with the SMP electropherogram.ISO 23293 pdf download.