AOAC Final Action Methods in 2018

with a separation gel and are detected by UV at 220 nm. Caseins and whey proteins are separated as two distinct, nonoverlapping groups of peaks whose ratio can be established based on integrated areas without the need for a calibration curve. A mass-to-area correction factor (CF) of 1.4 was used for whey proteins versus caseins in the calculation of whey protein content. B. Apparatus (a)  ProteomeLab PA 800 Plus .—Beckman Coulter, Inc. (Fullerton, CA, USA) or equivalent, equipped with UV detector set at 220 nm. Peak area can be integrated using any suitable software (Waters Empower, Beckman 32 Karat, or equivalent). (b)  Bare, fused-silica capillaries .—50 μm id × 30 cm with effective length of 20 cm (Model 338451; Beckman Coulter, Inc., or equivalent). ( c )  Analytical balance .—Four decimal places. ( d )  Temperature-controlled water bath .—95 ± 5 ° C. ( e )  Microcentrifuge.— For 1.5 mL vials. ( f )  Vortex mixer . ( g )  Adjustable pipets .—20, 100, and 1000 μL (Eppendorf or equivalent). ( h )  Safe-lock centrifuge tubes.— 2.0 mL (e.g., Eppendorf 022363344). ( i )  Round-bottom centrifuge tube.— 10 mL (e.g., Eppendorf 022822179). C. Reagents (a)  SDS-MW gel buffer .—Part No. A30341 (Beckman Coulter, Inc.); recipe readily supplied by vendor. (b)  SDS-MW analysis kit .—Part No. 390953 (Beckman Coulter, Inc.), including bare, fused-silica capillaries (50 μm id × 30 cm); SDS-MW gel buffer; SDS-MW sample buffer (100 mM Tris–HCl,

AOAC Official Method 2016.15 Quantification of Whey Protein Content in Milk-Based Infant Formula Powders Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE) [Applicable for determination of whey-to-casein protein ratio, ranging from 20:80 to 80:20, in bovine milk-based infant formula powders. The method is not applicable to the analysis of hydrolyzed or plant protein-based infant formulas.] Caution : Correct personal and environmental safety standards must be used while performing the method. Laboratory personnel handling solvents, acids, and reagents should be knowledgeable of their potential hazards. Consult Material Safety Data Sheets (MSDS) for information on hazards and take proper precautions. Transfer solvents, including β-mercaptoethanol and acids, inside efficient fume hoods and extractors. Because of β-mercaptoethanol buildup, perform measurements in a well-vented room. Ideally, install the instrument directly in a fume hood. Ensure all glassware is free from chipping and hairline cracks. See Table 2016.15 for results of the interlaboratory study supporting acceptance of the method. A. Principle In SDS-CGE, proteins in infant formula samples are denatured by anionic surfactant SDS and reduced by β-mercaptoethanol. The SDS- bonded, electrically charged proteinsmigrate in an electrical field filled First Action 2016 Final Action 2018

Table 2016.15. Interlaboratory study results for quantification of whey protein content in milk-based infant formula powders a Matrix Level b (whey %) No. labs Mean (whey %) Recovery, % RSD r , % RSD R , % HorRat Whey dominant infant formula 53 19 56.71 106 1.67 3.14 1.44 Third age formula 40 19 41.69 104 2.11 4.22 1.85 α-Lac enriched whey formula 65 19 65.51 100 1.44 2.18 1.02 NIST SRM 1849a c 32 19 43.11 134 3.51 5.94 2.62 Whey dominant infant formula 60 19 60.69 101 2.11 2.37 1.11 Third age formula 50 19 49.99 100 1.63 3.57 1.61 Formula with added LF ≥60 18 63.63 106 0.92 2.37 1.11 Formula with added α-Lac 70 19 73.24 105 0.83 2.30 1.10 Formula with added α-Lac 70 19 68.92 98 1.67 2.59 1.22 Formula with added α-Lac 70 19 69.26 99 1.82 3.20 1.51 Formula with raw milk ≥60 19 63.40 106 2.09 3.33 1.55 Formula with reduced CGMP d 70 19 69.41 99 1.57 2.89 1.37 Organic formula 60 19 64.92 108 1.01 2.43 1.14 Formula with EDW e 60 19 60.56 101 1.47 2.61 1.21 Formula with raw milk 60 19 62.36 104 1.61 2.76 1.29 a  The first five samples came from the SPIFAN test kit, and the next 10 samples were sampled from the Chinese market. b  Claims based on the formulation/label. c  Based on the formulation and the calculation results from 2012.07 ( see 50.5.01). d  CGMP = Casein GlycoMacroPeptide. e  EDW = Electrodialysis demineralized whey.

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