6. AOACSPIFANMethods-2018Awards

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510 F eng et al .: J ournal of AOAC I nternational V ol . 100, N o . 2, 2017

INFANT FORMULA AND ADULT NUTRITIONALS

Quantification of Whey Protein Content in Infant Formulas by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE): Single-Laboratory Validation, First Action 2016.15 P ing F eng 30F, CITIC Square, 1168 Nan Jing Road (W), Shanghai 200041, P.R.China C hristophe F uerer Nestlé Research Centre, Vers-Chez-Les-Blanc, PO Box 44, 1000 Lausanne 26, Switzerland A drienne M c M ahon Wyeth Nutrition Ireland, Askeaton, Co. Limerick, Ireland

Protein separation by sodium dodecyl sulfate-capillary gel electrophoresis, followed by UV absorption at 220 nm, allows for the quantification of major proteins in raw milk. In processed dairy samples such as skim milk powder (SMP) and infant formulas, signals from individual proteins are less resolved, but caseins still migrate as one family between two groups of whey proteins. In the first group, α-lactalbumin and β-lactoglobulin migrate as two distinct peaks. Lactosylated adducts show delayed migration times and interfere with peak separation, but both native and modified forms as well as other low-MW whey proteins still elute before the caseins. The second group contains high-MW whey proteins (including bovine serum albumin, lactoferrin, and immunoglobulins) and elutes after the caseins. Caseins and whey proteins can thus be considered two distinct nonoverlapping families whose ratio can be established based on integrated areas without the need for a calibration curve. Because mass-to-area response factors for whey proteins and caseins are different, an area correction factor was determined from experimental measurement using SMP. Method performance assessed on five infant formulas showed RSDs of 0.2–1.2% (within day) and 0.5–1.1% (multiple days), with average recoveries between 97.4 and 106.4% of added whey protein. Forty-three different infant formulas and milk powders were analyzed. Of the 41 samples with manufacturer claims, the measured whey protein content was Received October 20, 2016. Accepted by SG November 17, 2016. This method was approved by the AOAC Expert Review Panel for SPIFAN Nutrient Methods as First Action. The Expert Review Panel for SPIFAN Nutrient Methods invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. Corresponding author’s e-mail: ping.feng@wyethnutrition.com DOI: 10.5740/jaoacint.16-0344

in close agreement with declared values, falling within 5% of the declared value in 76% of samples and within 10% in 95% of samples. P rotein separation by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE), followed by UV absorption at 220 nm, allows for the quantification of major proteins in raw milk. In processed dairy samples such as skim milk powder (SMP) and infant formulas, signals from individual proteins are less resolved, but caseins still migrate as one family between two groups of whey proteins. In the first group, α-lactalbumin (α-Lac) and β-lactoglobulin (β- Lg) migrate as two distinct peaks. Lactosylated adducts show delayed migration times and interfere with peak separation, but both native and modified forms as well as other low-MW whey proteins still elute before caseins. The second group contains high-MW whey proteins [including bovine serum albumin (BSA), lactoferrin (LF), and immunoglobulins] and elutes after the caseins. Caseins and whey proteins can thus be considered as two distinct, nonoverlapping families whose ratio can be established based on integrated areas without the need for a calibration curve. The mass-to-area response factors are different for whey proteins and caseins, and the distinct area correction factor (CF) was determined from experimental measurements using SMP samples. This single-laboratory validation (SLV) report summarizes the results of the experiments performed to validate the Quantification of Whey Protein Content in Infant Formulas by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE) method following AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN)-recommended guidelines for the completion of an SLV study with reference to SPIFAN Standard Method Performance Requirements (SMPRs ® ) for whey protein-to-casein ratios. The validation experiments, designed per SPIFAN guidelines for SLV studies (1), have demonstrated that the method is accurate, precise, specific, and linear in the analytical range, and that the method is suitable for its intended purpose. A summary of all validation experiments and results can be found in Table 2016.15A . The samples used during the execution of the validation testing are detailed in Table 2016.15B . SLV