AOAC SPIFAN WPC ERP-Final Action Review (Nov. 2017)

2016.15 (Nov. 2017) - WPC-31 Publications-Final Action Review

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• The acceptance criteria for the separation cycle are: the migration time of the internal standard should be 12.3±0.5 minutes (if using an equivalent instrument, the migration time should be as defined beforehand). The degree of baseline drop from the migration time of internal standard to the peak valley between the end of casein and the peak of Ig H and BSA should be no more than 25% of height of the internal standard in the sample. h) Integration of the electropherograms (eGrams): Integrate eGrams of both SMP (Figure 2016.15C) and infant formulas (Figure 2016.15D) by setting the baseline from 0.4 minutes before the internal standard peak to the valley between the end of κ-casein peak and the peak of Ig H and BSA (no negative peaks should be generated by the integration); perform a manual integration from the peak of Ig H and BSA to the end of the last peak in the eGrams (integrate all peaks after the Ig H and BSA peak). i) Determination of the casein region: For SMP, set the casein integration start time just before the β-casein peak (about 3.1 minutes after the peak of 10 kDa internal standard). For infant formulas, identify the β-casein peak according to the SMP eGram and set the casein integration start time just before this peak. For all samples, set the end time at the valley between the end of the κ-casein peak and the Ig H and BSA peak (about 7.0 minutes after the 10 kDa internal standard). j) If using 32 Karat software from Beckman, the data processing steps are described in the Appendix. F. Calculations a) Separately sum peaks in three regions: two at each end of the eGram (lower molecular weight and higher molecular weight whey proteins), and one in the middle. The middle region corresponds to casein proteins (A cn ), and the two others are summed together to obtain whey proteins (A w ). b) Calculation of whey protein content: The whey protein content is calculated using the following equations: Whey protein % = A w,c A w,c + A cn ×100 (1) A w,c = A w ×1.4 (2) Where: A w. = total integrated areas of whey components A w, c = corrected integrated area of whey components A cn = integrated area of casein components 1.4 = correction factor to account for the difference between the mass-to- area ratio of whey and casein proteins Note: the peaks in the egrams may shift backwards gradually from the beginning of the run to the end of the run. The internal standard peak is used to correct the migration times for all the peaks in the egram.