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

peptide indicates that contamination is present and that samples need to be re-prepared using fresh reagents and/or tubes. If not analyzed immediately, the prepared (ready to load) samples can be stored in a refrigerator and analyzed within 48 hours. Replacing the reducing agent β-mercaptoethanol by another agent such as DTT or TCEP is not possible. DTT yields a signal in the high molecular weight whey region and TCEP leads to at least a 10-fold signal reduction.

Replacing the reagent kit with in-house prepared reagents gave equivalent results.

Instrument operation, electropherogram integration and results calculation

Since the quantification of whey protein is based on area normalization, a stable and consistent baseline is very important for reproducibility and accuracy of the data. Among the analytical data for all individual samples, the large RSDr and RSDR are almost all caused by baseline shifting in different directions (up or down). All the formulas for infants and young children contain considerable amounts of fat that reacts with SDS, migrates into the capillary, and forms a big shallow peak in front of the 10 kDa peptide. As a consequence, the signal baseline in the protein region will gradually drift downwards. An upward drifting of the baseline is an abnormal condition of the system. However, statistical analysis of all data including those with slight baseline drift in different directions demonstrates that the results can still meet the AOAC SMPR [4] . The integration has to strictly follow the integration instructions in the method. The instructions include integrating the areas of the low molecular weight whey proteins as well as the casein proteins using automatic integration from the beginning of the first protein peak until the valley between the κ-casein peak and the peak of Ig heavy chain and BSA (two components merged together as one peak). This is then followed by manual integration of the remaining peaks in order to accommodate baseline drifting. Seven different models of CE instruments from four different manufacturers were used by the 19 laboratories. Since the four instrument manufacturers use different effective length of capillaries, one of the system suitability criteria (the migration time of the 10 kDa internal standard) was omitted. Other than that, all other integration parameters and system suitability checks were kept the same. The test results indicated that as long as the results of the QC sample are within the required range, all results from the test samples are acceptable. When raw data is analyzed, even though the Ig heavy chain and BSA peak is the last peak on the electropherogram in most formulas, a full scale of the electropherogram should be examined in order to ensure that larger proteins are not omitted from the integration. Conclusion