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Table 2016.15A. Summary of validation characteristics, acceptance criteria, and results Parameter Acceptance criteria (SMPR) Results Applicability Determination of total whey proteins, including hydrolyzed

Table 2016.15B. Validation test sample description Sample Description Infant formula 1 First-age infant formula with a manufacturer claim of 60% whey protein, manufactured with sweet whey ingredient Infant formula 2 First-age infant formula with a manufacturer claim of 60% whey protein, manufactured with sweet whey ingredient Infant formula 3 First-age infant formula with a manufacturer claim of 65% whey protein, manufactured with α-Lac-enriched whey Infant formula 4 First-age infant formula with a manufacturer claim of 70% whey protein, manufactured with CGMP- reduced whey Infant formula 5 Third-age infant formula with a manufacturer claim of 40% whey protein, manufactured with sweet whey ingredient SMP 20% whey protein Sweet whey Demineralized whey, 13% total protein

Applicable for the determination of whey percentage as the total protein in bovine milk- based infant formula. This method is not applicable to the analysis of hydrolyzed protein- based infant formulas.

forms, as the percentage of protein content (protein content as defined by the appropriate regulatory agencies). To be applicable to milk-based infant formula products (including those from bovine milk and, if possible, milk of other species and products containing hydrolyzed casein). Percentage recovery must be within the theoretical range of 95–105%. RSD ≤ 3.0% for whey protein g/100 g protein E-grams from injections of purified water and processed formulation matrix without protein ingredients must be evaluated for the presence of peaks at the migration times corresponding to analyte protein-related peaks. ≤10 whey protein g/100 g protein R 2 must be ≥0.99. The residuals on the residual plot should be randomly distributed around zero. RSD ≤ 3.0% for whey protein g/100 g protein

Recovery range was 97.4–106.4%.

Accuracy

RSD was 0.3–1.2% in five different infant formula sample types. RSD was 0.5–1.1% in five different infant formula sample types. No interfering peaks were observed for purified water or the processed formulation matrix. 20% of total protein in infant formulas Linearity of R 2 of 0.993–0.999 for the area ratio of whey protein to casein Logarithm of R 2 of 0.993–0.996 for whey protein as the percentage of total protein Residuals on the residual plot were randomly distributed around zero. Range of 20–100% for whey protein in total protein in infant formulas in the tested linear range

Repeatability precision

Intermediate precision

the Material Safety Data Sheets for information on hazards and how to take proper precautions. Only transfer solvents and acids inside efficient fume hoods and extractors. Ensure all glassware is free from chipping and hairline cracks.

Specificity: Matrix

interference

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 .

LOQ

A. Principle

Linearity

In sodium dodecyl sulfate-capillary gel electrophoresis (SDS- CGE), proteins in infant formula samples are denatured by anionic surfactant SDS and reduced by β-mercaptoethanol. The SDS-bonded electrically charged proteins migrate in an electrical field filled with a separation gel and are detected by UV at 220 nm 2 . 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. (a)  ProteomeLab PA 800 Plus .—Beckman Coulter, Inc. (Fullerton, CA) or equivalent, equipped with a UV detector set at 220 nm. Peak area integration can be achieved by using any suitable software (e.g., Waters Empower, Beckman 32 Karat, or equivalent). (b)  Bare fused-silica capillaries .—50 μm id × 20 cm (e.g., Model 338451; Beckman Coulter, Inc.). B. Apparatus

Range

AOAC Official Method 2016.15 Quantification of Whey Protein Content in Infant Formulas by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE) First Action 2016 [Applicable for the determination of the whey-to-casein protein ratio, ranging from 20:80 to 80:20, in bovine milk-based infant formula powders. This method is not applicable to the analysis of hydrolyzed protein-based infant formulas.] Caution : Correct personal and environmental safety

C. Reagents

(a)  SDS-MW gel buffer .—Part No. A30341 (Beckman Coulter, Inc.); recipe readily supplied by the vendor. (b)  SDS-MW analysis kit (2 ).—Part No. 390953 (Beckman Coulter, Inc.), including bare fused-silica capillaries (50 μm id × 20 cm), SDS-MW sample buffer (100 mM Tris–HCl, pH 9.0; with 1% SDS), 10 kDa protein internal standard (IS), acidic

standards must be used while performing this analytical method. Laboratory personnel handling solvents, acids, and reagents should be knowledgeable of their potential hazards. Consult