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et al
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AOAC I
nternational
V
ol
.
100, N
o
.
2, 2017
511
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
standards must be used while performing
this analytical method. Laboratory personnel
handling solvents, acids, and reagents should be
knowledgeable of their potential hazards. Consult
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.
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
.
A. Principle
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.
B. Apparatus
(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.).
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
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
Table 2016.15A. Summary of validation characteristics,
acceptance criteria, and results
Parameter
Acceptance criteria (SMPR)
Results
Applicability
Determination of total whey
proteins, including hydrolyzed
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).
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.
Accuracy
Percentage recovery must be
within the theoretical range of
95–105%.
Recovery range was
97.4–106.4%.
Repeatability
precision
RSD ≤ 3.0% for whey protein
g/100 g protein
RSD was 0.3–1.2%
in five different infant
formula sample types.
Intermediate
precision
RSD ≤ 3.0% for whey protein
g/100 g protein
RSD was 0.5–1.1%
in five different infant
formula sample types.
Specificity:
Matrix
interference
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.
No interfering peaks were
observed for purified
water or the processed
formulation matrix.
LOQ
≤10 whey protein g/100 g
protein
20% of total protein in
infant formulas
Linearity
R
2
must be ≥0.99. The
residuals on the residual
plot should be randomly
distributed around zero.
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
Range of 20–100% for
whey protein in total
protein in infant formulas
in the tested linear range
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