Protein separation by sodium dodecyl sulfate-capillary

Transcription

1 510 Feng et al.: Journal of AOAC International Vol. 100, No. 2, 2017 INFANT FORMULA AND ADULT NUTRITIONALS Quantification of Whey Protein Content in Infant Formulas by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE): Single-Laboratory Validation, First Action Ping Feng 30F, CITIC Square, 1168 Nan Jing Road (W), Shanghai , 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 % (within day) and % (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 Received October 20, Accepted by SG November 17, 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 methodfeedback@aoac.org. Corresponding author s ping.feng@wyethnutrition.com DOI: /jaoacint 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 A. The samples used during the execution of the validation testing are detailed in Table B.

2 Feng et al.: Journal of AOAC International Vol. 100, No. 2, Table A. Summary of validation characteristics, acceptance criteria, and results Parameter Acceptance criteria (SMPR) Results Applicability Accuracy Repeatability precision Intermediate precision Specificity: Matrix interference LOQ Linearity Range 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). Percentage recovery must be within the theoretical range of %. RSD 3.0% for whey protein g/100 g protein RSD 3.0% for whey protein g/100 g protein Applicable for the determination of whey percentage as the total protein in bovine milkbased infant formula. This method is not applicable to the analysis of hydrolyzed proteinbased infant formulas. Recovery range was %. RSD was % in five different infant formula sample types. RSD was % in five different infant formula sample types. E-grams from injections of No interfering peaks were purified water and processed observed for purified formulation matrix without water or the processed protein ingredients must be formulation matrix. 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 The residuals on the residual plot should be randomly distributed around zero. 20% of total protein in infant formulas Linearity of R 2 of for the area ratio of whey protein to casein Logarithm of R 2 of for whey protein as the percentage of total protein Residuals on the residual plot were randomly distributed around zero. Range of % for whey protein in total protein in infant formulas in the tested linear range AOAC Official Method 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 Table B. Validation test sample description Sample Infant formula 1 Infant formula 2 Infant formula 3 Infant formula 4 Infant formula 5 SMP Sweet whey 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 A. The samples used during the execution of the validation testing are detailed in Table B. 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 ; Beckman Coulter, Inc.). C. Reagents Description First-age infant formula with a manufacturer claim of 60% whey protein, manufactured with sweet whey ingredient First-age infant formula with a manufacturer claim of 60% whey protein, manufactured with sweet whey ingredient First-age infant formula with a manufacturer claim of 65% whey protein, manufactured with α-lac-enriched whey First-age infant formula with a manufacturer claim of 70% whey protein, manufactured with CGMPreduced whey Third-age infant formula with a manufacturer claim of 40% whey protein, manufactured with sweet whey ingredient 20% whey protein Demineralized whey, 13% total protein (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 (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

3 512 Feng et al.: Journal of AOAC International Vol. 100, No. 2, 2017 wash solution (high-purity, 0.1 N HCl), basic wash solution (high-purity 0.1 N NaOH), and an SDS-MW size standard ( kda, 16 mg/ml). (c) Protein IS. 10 kda, Part No. A26487 (Beckman Coulter, Inc.). (d) Water. LC grade. (e) β-mercaptoethanol. Part No. M7154 or M6250 (Sigma). D. Preparation of System Buffer Trays and Standard and Sample Solutions (a) To prepare the system buffer trays, follow the steps in Figure A and load reagents into the system inlet (lower left panel) and outlet (lower right panel). Use 6 6 buffer trays, following the configuration illustrated in the panels. (b) Either weigh 135 ± 5 mg skim milk powder (SMP; protein content around 37%) or 500 ± 20 mg infant formula powder (protein content around 11%) into a 15 ml centrifuge tube. (c) Dissolve the sample and dilute to a 5 ml volume with deionized (DI) water. Mix each tube on a vortex mixer until the samples are homogeneously dissolved. Each final solution should contain about mg/ml protein. (d) Prepare the sample running presolution by mixing 1% SDS sample solution with 10 kda IS peptide using an 84:1 ratio based on the total number of samples to be analyzed in the sample set (90 μl/sample). (e) Pipet 10 μl of each sample solution into separate 2.0 ml microcentrifuge vials. (f) Sequentially add 85 μl sample running presolution and 5 μl β-mercaptoethanol to each microcentrifuge vial. Mix well 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 about 7000 rpm. Figure A. Preparation of system buffer trays. (g) Mix on a vortex mixer before transferring each sample into their corresponding injection vials. E. Sample Analysis (a) Set up an optimized separation method for the batch analysis of up to 24 samples at a time, including a buffer blank (10 μl DI water), an MW size standard, and an SMP sample. (b) For each separation cycle (40 min), precondition the capillary first with basic wash solution, followed by acidic wash solution, DI water, and SDS gel buffer. (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. (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 B. (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 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

4 Feng et al.: Journal of AOAC International Vol. 100, No. 2, Figure B. Separation of the protein MW size standard. 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 (A cn ), and the two others are summed together to obtain the whey proteins (A w ). (b) Whey protein content is calculated using the following equations: Aw,c Percentage of whey protein = (1) A + A w,c A = A 1.4 (2) w,c w where A w = total integrated areas of whey components; A w,c = corrected integrated area of whey components; A cn = 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. cn Results and Discussion Specificity (a) Reagent blank. Each sample sequence was started with purified water as a blank. The blank e-gram is shown in Figure 1 (gray line). No peaks were detected after the 10 kda peptide internal standard (IS). There was no significant interference from other components in the protein region. (b) Placebo test. To test for the presence of interference from nonprotein components in infant formulas, a placebo infant formula trial sample that contained all of the ingredients that are typical first-age formulas, except protein (vitamins, minerals, fat, and carbohydrates), was manufactured. SDS-CGE did not detect significant peaks at any of the protein regions in the e-gram (Figure 1, black line). (c) Specific protein migration time and migration pattern of whey proteins and caseins. The SDS-CGE method can separate individual whey and casein protein standards very well, as demonstrated with standard solutions containing five major whey proteins (Figures 2 and 3) or four casein proteins (Figure 4), as well as with fresh raw milk (Figure 5). Protein phosphorylation and glycosylation delay casein migration times relative to their molecular sizes (Figure 6). Protein glycation the nonenzymatic sugar modification of amines and the early stage of a Maillard reaction occurs during the mixing and heating of milk proteins with lactose (3), which results in the splitting of several individual milk proteins into several peaks representing the modified protein glycoforms. This was seen for α-lac and β-lg, where splitting was observed in a commercial sweet whey protein ingredient (Figure 7) and by comparing the casein peaks in fresh milk and in an SMP sample (Figures 5 and 8). Although glycation prevents the complete separation of all proteins individually, whey proteins

5 514 Feng et al.: Journal of AOAC International Vol. 100, No. 2, 2017 Figure 1. E-grams of pure water blank (gray line) and placebo (processed; black line). Compared with the reagent blank (gray line), there was no significant interference from nonprotein components in the protein range. Figure 2. E-gram of the five major whey protein standards mixed (group 1): α-lac, β-lg, bovine immunoglobulin G [IgG light (L) and heavy (H) chains], BSA, and LF. All standards were from Sigma. Figure 3. E-gram of the five major whey protein components standards mixed (group 2): α-lac, β-lg, CGMP, immunoglobulin G [IgG light (L) and heavy (H) chains], and BSA. All standards were from Sigma, except for CGMP, which was from Arla Ingredients, Inc. Figure 4. E-gram of the casein protein standard from Sigma.

6 Feng et al.: Journal of AOAC International Vol. 100, No. 2, Figure 5. E-gram of raw milk. Figure 6. E-gram of major whey proteins and casein proteins (black) compared with the MW marker (circled kda values). Figure 7. Typical e-gram of sweet whey ingredients.

7 516 Feng et al.: Journal of AOAC International Vol. 100, No. 2, 2017 Figure 8. Typical e-gram of SMP. still migrate as two groups either before or after the caseins. Caseins were eluted as a single group between light and heavy chains of immunoglobulins, where almost no major whey proteins were found (Figure 8). Linearity Linearity data were obtained by spiking seven different levels of whey protein ingredients into a fixed level of SMP. Whey Table 1. Testing scheme for linearity and accuracy (whey protein ranging from 20 to 80%) Protein Ingredients a Weight, mg DI water, ml b % mg/ml Lot No. SMP DY19 WPC C22JUL15J1 Experiment c SMP, μl WPC35, μl DI water, μl Total, μl Whey content, % WPCL WPC35L WPC35L WPC35L WPC35L WPC35L WPC35L a WPC = Whey protein concentrate. b DI = Deionized. c L0 6 = Levels 0 6. protein content was designed to range from 20% (no added whey) to about 80% in the mixtures of whey and skim milk. The typical spiking scheme is presented in Table 1. Analyses were performed in duplicate on a single day, as well as in single analyses on 3 separate days. The relationship between the amount of added whey protein and the whey protein-to-casein ratio proved to be linear, and the coefficients of determination (R 2 ) were all higher than (Table 2). The relationship between the amount of added whey protein and the percentage of added whey in the total protein proved to be logarithmic; the R 2 were also all higher than (Table 2). Typical linearity relationships are shown in Figure 9. Table 2. Summary of R 2 and the equations for seven levels of whey protein in total proteins for the whey-to-casein ratio and added whey protein as the percentage of total protein Day 1: Avg. for two replicates Day 2: Single Whey/casein Day 3: Single Day 4: Single R Equation y = x y = x Percentage whey added y = x y = 0.044x R Equation y = ln(x) y = ln(x) y = ln(x) y = ln(x) Figure 9. Typical linearity relationships between the area ratio of whey to casein and measured whey percentage versus whey protein amount added.

8 Feng et al.: Journal of AOAC International Vol. 100, No. 2, Table 3. Spike-recovery data obtained in duplicate for different levels on 1 day Spike level Integrated area Measured Theoretical Total whey Casein Whey, % Whey, % spiked Whey, % spiked Recovery, % Table 4. Spike-recovery data obtained in singlet for different levels on 3 different days Level Accuracy (Spike Recovery) Accuracy was evaluated in samples where four levels of sweet whey ingredient were spiked into the same level of SMP by comparing the theoretically calculated percentage whey values with the measured values obtained using equations 1 and 2 (for skim milk, where no whey proteins were added, the CF was set to 1.29). The results are listed in Tables 3 and 4. The SMPs and whey protein ingredients used in Tables 3 and 4 are from different lot numbers. Precision Two independent sample preparations were tested on each day for 6 separate days for the following: three infant formula samples (shown in Table 5), one SMP sample, and one sweet Day Total whey Area Measured Theoretical Casein Whey, % Whey, % spiked Whey, % spiked Recovery, % Table 5. Repeatability and intermediate precision for three infant formulas Formula/Replicate Day 1/1 1/2 2/1 2/2 3/1 3/ Avg RSD r, % RSD R, % whey (demineralized) sample (shown in Table 6). Two other infant formula samples were tested by preparing and analyzing six replicates each day on 3 different days using this method (shown in Table 7). The percentage of whey protein was reported to one decimal place for individual and averaged replicates. The SD and percentage RSDs were calculated and also reported to one decimal place. Data are presented in Tables 5 7. The typical e-grams for each infant formula sample are presented in Figures The molar attenuation (or molar extinction) coefficient, reflected as the mass-to-area ratio at 220 nm, is an intrinsic Table 6. Repeatability and intermediate precision for SMP and demineralized sweet whey ingredient Day SMP a SMP a DW b DW b a b Avg RSD r, % RSD R, % Because SMP is not processed like infant formula, a CF of 1.29 was used. Because whey protein contains almost no caseins, no CF was used. Table 7. Repeatability and intermediate precision for two infant formulas Rep. 1 Rep. 2 Rep. 3 Rep. 4 Rep. 5 Rep. 6 Avg. SD RSD Day Formula Total Formula Total

9 518 Feng et al.: Journal of AOAC International Vol. 100, No. 2, 2017 Figure 10. E-gram of infant formula 1 with sweet whey ingredient (60% whey claim). Figure 11. E-gram of infant formula 2 with sweet whey ingredient (60% whey claim). property of proteins and depends on the proteins amino acid sequence and molecular structure status. Unfortunately, no literature is currently available regarding whey protein and casein ratios under SDS-CGE conditions, nor for proteins after infant formula processing. In contrast, the mass ratio of whey proteins to caseins is well established (4, 5; Table 8) and can be calculated as 26.9% (whey proteins versus caseins) in bovine milk and SMP. Figure 12. To correct for the difference between the mass-to-area ratios of whey proteins and caseins, 13 SMP samples from different batches and suppliers were analyzed by SDS-CGE with 50 measurements. The two whey protein areas and the one casein area were integrated, and the area percentage ratio of whey proteins to caseins was established at 20.8% (Table 9). The mass-to-area CF for whey proteins relative to caseins was obtained by comparing the whey-to-casein mass percentage E-gram of infant formula 3 with α-lac-enriched whey ingredient (65% whey claim).

10 Feng et al.: Journal of AOAC International Vol. 100, No. 2, Figure 13. E-gram of infant formula 4 with CGMP-reduced sweet whey ingredient (70% whey claim). Figure 14. E-gram of infant formula 5 with sweet whey ingredient (40% whey claim). Table 8. Protein profile of bovine milk and calculated whey protein as the mass percentage of casein (4, 5) Whole milk content Protein MW, kda g/l (5) % as1-casein as2-casein b-casein k-casein g-casein a-lac BSA Immunoglobulin Peptone Peptone LF Sum Casein Whey Whey as percentage of casein Milk fat globule membrane b-lg CGMP 9.15 ratio from the literature with the area percentage ratio obtained with the SDS-CGE method. Based on the literature mass percentage ratio (26.9%) and the experimental area percentage ratio (20.8%), a CF of 1.29 should be applied to the integrated signal of whey proteins. To evaluate the impact of the infant formula manufacturing process on the area CF of whey proteins to caseins, a whey protein-dominant infant formula was manufactured. Two samples were taken; one before processing and one after. The test results are listed in Table 10 and indicate that processing further increased this ratio 1.11-fold. Therefore, a final CF of 1.4 for whey protein-to-casein area for infant formulas was chosen (Table 9). Forty-three infant formulas manufactured by both Chinese and international manufacturers with different whey ingredients, including regular sweet whey, α-lac-enriched whey, LF-added whey, and casein glycomacropeptide (CGMP)-reduced whey, were analyzed and compared with manufacturers claims (Table 11). The results show that among the 41 samples with manufacturers claims, measured whey content was in close agreement with declared value: within 5% of the declared value for 31 (76%) samples and within 10% for 37 (90%) samples. Two infant formulas did not contain added whey protein; hence, a factor of 1.29, not 1.4, should be used. Taking this into account, 39 (95%) samples were within 10% of the declared value.

11 520 Feng et al.: Journal of AOAC International Vol. 100, No. 2, 2017 Table 9. Measured results of whey protein as the area percentage of caseins for different batches of SMP samples from different suppliers by SDS-CGE and the calculated area CF of whey proteins to caseins Whey as percentage of casein (mass) Literature (4, 5) 26.9 Whey as percentage of casein (area) Lot No. n a Avg. SD EY CY DY DY DY DY29 b M M M MSK SMP DN SMP DN SMP DN Avg. c SD d 0.99 CV, % 4.78 CF 1.29 Process impact e 1.11 Final CF 1.4 a n = Number of measurement. b Four different batches of capillaries with two different sets of reagent kits on 12 different days. c Avg. = Average. d SD = Standard deviation. e Obtained by evaluating processed and finished infant formula product (Table 10). Table 10. Comparison of the area percentage of whey protein between the times after compounding and after spray-drying during the processing of formula trial samples Before processing After processing Area Whey Casein Whey/casein Conclusions and Recommendations The SDS-CGE method is capable of accurately determining the ratio of whey to casein in infant formulas manufactured using different whey ingredients. Because whey and casein proteins, as groups, have distinct migration times, the measurements will not miss individual proteins. As a consequence, absolute quantification of individual or total proteins is not necessary. It was recommended that the method be further validated by conducting a multilaboratory study. This would generate valuable method performance data, including RSD R, further enhancing the potential of this method for use in a routine QC environment. CF Table 11. Measured whey protein content in 43 different infant formulas made by both local and international manufacturers Manufacturer Measured whey, % Product whey claim, % a n Avg. SD % of claim b (62.5) c b d N/L e c >60 (65) Pass f N/L L1 g L2 g a Numbers in parentheses represent value considered. b CGMP-reduced sweet whey formula. c α-lac-enriched formula. d LF-enriched formula. e N/L = Not labeled. f Conform to claim. g The claim of 21.2% comes from the value for SMP, not a real claim.

12 Feng et al.: Journal of AOAC International Vol. 100, No. 2, Acknowledgments We thank Hongxu Chen and Jianhua Wu (AB Sciex) for providing the PA 800 Plus instrument and instrument maintenance, Chao Wu (Hilmar Ingredients) for helping us understand the meaning of split peaks for infant formula proteins, Longfei Wang (Nestlé Food Safety Institute, Beijing) for providing the laboratory facilities, and Wyeth Nutrition Suzhou for supplying pilot-scale placebo infant formula samples and samples with different protein ingredients. References (1) Official Methods of Analysis (2012) 19th Ed., AOAC INTERNATIONAL, Rockville, MD, Appendix L: AOAC Recommended Guidelines for Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Single-Laboratory Validation (2) (January 2014) Application Guide, PA 800 Plus Pharmaceutical Analysis System, Part No. A51970AD, Beckman Coulter, Inc., Fullerton, CA (3) Rudloff, S., & Lönnerdal, B. (1992) J. Pediatr. Gastroenterol. Nutr. 15, doi: / (4) Walstra, P., Wouters, J.T.M., & Geurts, T.J. (2005) Dairy Science and Technology, 2nd Ed., CRC Press, Boca Raton, FL (5) Koletzko, B., Baker, S., Cleghorn, G., Neto, U.F., Gopalan, S., Hernell, O., Hock, Q.S., Jirapinyo, P., Lonnerdal, B., Pencharz, P., Pzyrembel, H., Ramirez- Mayans, J., Shamir, R., Turck, D., Yamashiro, Y., & Zong-Yi, D. (2005) J. Ped. Gastroenterol. Nutr. 41, , Table 3