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

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

FOR ERP USE ONLY DO NOT DISTRIBUTE

f) Sequentially add 85 μL of the sample running pre-solution and 5 μL of β-mercaptoethanol to each micro-centrifuge vial. Mix well before heating the vials in a water bath at 100±5 °C for ten minutes. Cool down to room temperature, then centrifuge at room temperature for 1 minute at about 7,000 rpm / 5,000 g. g) Vortex before transferring each sample into corresponding injection vials. E. Sample Analysis a) Set up an optimized separation method for batch analysis including one buffer blank, (10 μL of DI water), a molecular weight size standard, (MW), and a skim milk powder (SMP) sample. b) For each separation cycle (45 minutes), precondition the capillary first with the basic wash solution for 3 minutes, followed by the acidic wash solution for 1 minute, deionized water for 1 minute, and SDS gel buffer for 10 minutes. Set up the separation run for 30 minutes. c) Introduce the samples electrokinetically by applying voltage at -5 kV for 20 seconds. d) Perform the electrophoresis at constant voltage with applied field strength of (-) 497 V/cm and a capillary thermostatted to 25 °C using recirculating liquid coolant. e) The generated current should be approximately 27 μA. f) To avoid reagent depletion, program the system to increment the vial position of all reagents every eight cycles. g) Test the system suitability using the MW marker. The acceptance criteria for the system suitability are as follows: • The migration time of the internal standard should be 12.3±0.5 minutes using the instrument and capillary as per section B (a&b). If using an equivalent capillary or instrument, then define the migration time of the internal standard accordingly. The migration pattern and the migration times of the seven MW markers (10, 20, 35, 50, 100, 150, and 225 kDa) should be completely separated within 30 minutes using this method or when using an equivalent instrument (refer to Figure 2016.15B). • 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