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510 Feng et al.: Journal of AOAC International Vol. 100, No . 2, 2017
Quantification of Whey Protein Content in Infant Formulas by
Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis
(SDS-CGE): Single-Laboratory Validation, First Action 2016.15
Ping Feng
30F, CITIC Square, 1168 Nan Jing Road (W), Shanghai 200041, P.R.China
Christophe Fuerer
Nestlé Research Centre, Vers-Chez-Les-Blanc, PO Box 44, 1000 Lausanne 26, Switzerland
Adrienne McMahon
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
in close agreement with declared values, falling
within 5% of the declared value in 76% of samples
and within 10% in 95% of samples.
Protein 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.
SLV
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.
INFANT FORMULA AND ADULT NUTRITIONALS
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
Corresponding author’s e-mail:
DOI: 10.5740/jaoacint.16-0344
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