Feng et al.: Journal of AOAC International Vol. 100, No.

4, 2017  1177

OFFICIAL METHODS

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

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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

Stakeholder Panel on Infant Formula and AOAC Official Method 2016.15

Adult Nutritionals Quantification of Whey Protein Content in Infant
Formulas by S­ odium Dodecyl Sulfate-Capillary Gel
Expert Review Panel for Nutrient Methods Electrophoresis (SDS-CGE)
First Action 2016
Darryl Sullivan, Chair, Covance Labs
John Austad, Covance Labs [Applicable for the determination of the whey-to-casein
Sneh Bhandari, Silliker Labs protein ratio, ranging from 20:80 to 80:20, in bovine
Esther Campos-Gimenez, Nestlé milk-based infant formula powders. This method is not applicable
Scott Christiansen Perrigo to the analysis of hydrolyzed protein-based infant formulas.]
Hans Cruijsen, FrieslandCampina Caution: Correct personal and environmental safety
Jon DeVries, General Mills/Medallion Labs standards must be used while performing
Brendon Gill, Fonterra this analytical method. Laboratory personnel
Don Gilliland, Abbott Nutrition handling solvents, acids, and reagents should be
Min Huang, Frontage Labs knowledgeable of their potential hazards. Consult
Estela Kneeteman, INTI the Material Safety Data Sheets for information
Adrienne McMahon, Nestlé on hazards and how to take proper precautions.
Bill Mindak, FDA Only transfer solvents and acids inside efficient
Maria Ofitserova, Pickering Lab fume hoods and extractors. Ensure all glassware
Melissa Phillips, NIST is free from chipping and hairline cracks.
Shay Phillips, Mead Johnson A summary of all validation experiments and results can be
Karen Schimpf, Abbott Nutrition found in Table 2016.15A. The samples used during the execution
Guenther Raffler, CLF-Eurofins of the validation testing are detailed in Table 2016.15B.
Kate Rimmer, NIST
Wil van Loon, FrieslandCampina A. Principle
Jinchuan Yang, Waters Corp.
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 nm2. 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.

Submitted for publication October 2016.
Adopted as a First Action Official Method by the Expert Review
Panel on Nutrient Methods
Corresponding author’s e-mail: ping.feng@wyethnutrition.com
DOI: 10.5740/jaoacint.2016_15
(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.).
1178  Feng et al.: Journal of AOAC International Vol. 100, No. 4, 2017

Table 2016.15A.  Summary of validation characteristics, Table 2016.15B.  Validation test sample description
acceptance criteria, and results
Sample Description
Parameter Acceptance criteria (SMPR) Results Infant formula 1 First-age infant formula with a manufacturer claim of
Applicability Determination of total whey Applicable for the 60% whey protein, manufactured with sweet whey
proteins, ­including hydrolyzed ­determination of whey ingredient
forms, as the percentage percentage as the total
Infant formula 2 First-age infant formula with a manufacturer claim of
of protein content (protein protein in bovine ­milk-
60% whey protein, manufactured with sweet whey
content as defined by the based infant ­formula.
ingredient
­appropriate ­regulatory This method is not
agencies). To be ­applicable ­applicable to the analysis Infant formula 3 First-age infant formula with a manufacturer claim of
to milk-based infant formula of hydrolyzed protein- 65% whey protein, manufactured with α-Lac-enriched

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products (including those based infant formulas. whey
from ­bovine milk and, if Infant formula 4 First-age infant formula with a manufacturer claim
­possible, milk of other species of 70% whey protein, manufactured with CGMP-
and products containing reduced whey
hydrolyzed casein).
Infant formula 5 Third-age infant formula with a manufacturer claim
Accuracy Percentage recovery must be Recovery range was of 40% whey protein, manufactured with sweet whey
within the ­theoretical range of 97.4–106.4%. ingredient
95–105%.
SMP 20% whey protein
Repeatability RSD ≤ 3.0% for whey protein RSD was 0.3–1.2%
­precision g/100 g protein in five different infant Sweet whey Demineralized whey, 13% total protein
­formula sample types.
Intermediate RSD ≤ 3.0% for whey protein RSD was 0.5–1.1%
­precision g/100 g protein in five different infant (e)  β-Mercaptoethanol.—Part No. M7154 or M6250
­formula sample types. (Sigma).
­Specificity: E-grams from injections of No interfering peaks were
­Matrix purified water and ­processed observed for purified
D. Preparation of System Buffer Trays and
­interference formulation matrix without water or the processed
­protein ­ingredients must be formulation matrix. Standard and Sample Solutions
­evaluated for the presence of
peaks at the migration times (a)  To prepare the system buffer trays, follow the steps in
­corresponding to analyte
protein-related peaks.
Figure 2016.15A and load reagents into the system inlet (lower
left panel) and outlet (lower right panel). Use 6 × 6 buffer trays,
LOQ ≤10 whey protein g/100 g 20% of total protein in
protein infant formulas
following the configuration illustrated in the panels.
(b)  Either weigh 135 ± 5 mg skim milk powder
Linearity R2 must be ≥0.99. The Linearity of R2 of
residuals on the residual 0.993–0.999 for the area
(SMP; protein content around 37%) or 500 ± 20 mg infant
plot should be randomly ratio of whey protein to formula powder (protein content around 11%) into a 15 mL
distributed around zero. casein centrifuge tube.
Logarithm of R2 of (c)  Dissolve the sample and dilute to a 5 mL volume with
0.993–0.996 for whey deionized (DI) water. Mix each tube on a vortex mixer until
protein as the percentage
the samples are homogeneously dissolved. Each final solution
of total protein
should contain about 10–15 mg/mL protein.
Residuals on the ­
(d)  Prepare the sample running presolution by mixing
residual plot were
­randomly ­distributed 1% SDS sample solution with 10 kDa IS peptide using an
around zero. 84:1 ratio based on the total number of samples to be analyzed
Range Range of 20–100% for in the sample set (90 μL/sample).
whey protein in total (e)  Pipet 10 μL of each sample solution into separate 2.0 mL
­protein in infant formulas microcentrifuge vials.
in the tested linear range
(f)  Sequentially add 85 μL sample running presolution and
5 μL β-mercaptoethanol to each microcentrifuge vial. Mix well
C. Reagents before heating the vials in a water bath at 100 ± 5°C for 10 min.
Cool down to room temperature, then centrifuge for 1 min at
(a)  SDS-MW gel buffer.—Part No. A30341 (Beckman about 7000 rpm.
Coulter, Inc.); recipe readily supplied by the vendor. (g)  Mix on a vortex mixer before transferring each sample
(b)  SDS-MW analysis kit (2).—Part No. 390953 (Beckman into their corresponding injection vials.
Coulter, Inc.), including bare fused-silica capillaries (50 μm
id × 20 cm), SDS-MW sample buffer (100 mM Tris–HCl, pH E. Sample Analysis
9.0; with 1% SDS), 10 kDa protein internal standard (IS), acidic
wash solution (high-purity, 0.1  N HCl), basic wash solution (a)  Set up an optimized separation method for the batch
(high-purity 0.1  N NaOH), and an SDS-MW size standard analysis of up to 24 samples at a time, including a buffer blank
(10–225 kDa, 16 mg/mL). (10 μL DI water), an MW size standard, and an SMP sample.
(c)  Protein IS.—10 kDa, Part No. A26487 (Beckman (b)  For each separation cycle (40 min), precondition the
Coulter, Inc.). capillary first with basic wash solution, followed by acidic wash
(d)  Water.—LC grade. solution, DI water, and SDS gel buffer.
 Feng et al.: Journal of AOAC International Vol. 100, No. 4, 2017  1179
Figure 2016.15A.  Preparation of system buffer trays.
(c)  Introduce the samples electrokinetically by applying
voltage at –5 kV for 20 s.
(d)  Perform electrophoresis at constant voltage with
an applied field strength of –497 V/cm and the capillary
thermostatted to 25°C using recirculating liquid coolant.
(e)  The current generated should be approximately 27 μA.
(f)  Program the system to automatically replenish all
reagents through incremental increases in buffer array after
every eight cycles.
Figure 2016.15B.  Separation of the protein MW size standard.
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(g)  Test system suitability using the MW marker.
Acceptance criteria for the system suitability are as follows:
The migration time of the IS should be 12.3 ± 0.5 min, and
the migration pattern and migration times of the seven MW
markers (10, 20, 35, 50, 100, 150, and 225 kDa) should
completely separate within 30 min using this method. See
Figure 2016.15B.
(h)  Acceptance criteria for the separation cycle are as follows:
The migration time of the IS should be 12.3 ± 0.5 min, the degree
1180  Feng et al.: Journal of AOAC International Vol. 100, No. 4, 2017
of baseline drop from the migration time of the IS to the peak
valley between the end of casein and the peak of immunoglobulin
heavy chain (Ig H) and bovine serum albumin (BSA) should be no
more than 25% of the height of the IS of the sample.
(i)  To integrate SMP and infant formula electrophoregrams
(e-grams), set the baseline at 0.4 min before the IS peak to
the valley between the end of the κ-casein peak and the Ig H
and BSA peak; perform a manual integration from the valley
between the end of the κ-casein peak and the peak of Ig H and
BSA to the end of the last peak in the e-grams (at least 9 min
after the peak of the 10 kDa IS).
(j)  To determine the casein region, set the start time for
casein integration just before the β-casein peak in the e-gram
of the SMP (about 3.1 min after the peak of the 10 kDa IS).
Referencing the SMP, identify the β-casein peak in the infant
formula samples, then set the start time of the casein region
in the infant formula to just before the β-casein peak. Set
the end  time at the valley between the end of the κ-casein
peak and the Ig H and BSA peak (about 7.0  min after the
10 kDa IS).
F. Calculations
(a)  To calculate whey protein content, separately sum
the peaks in the following three regions: two at each end of
the e-gram (smaller and larger whey proteins) and one in the
middle. The middle region corresponds to casein proteins (Acn),
and the two others are summed together to obtain the whey
proteins (Aw).
(b)  Whey protein content is calculated using the following
equations:
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A w,c
Percentage of whey protein = (1)
A w,c + A cn
A w,c = A w × 1.4 (2)
where Aw = total integrated areas of whey components;
Aw,c = corrected integrated area of whey components; Acn
= integrated area of casein components; and 1.4 = CF to account
for the difference between the mass-to-area ratio of whey
and  casein proteins.