Improving Infant Formula Analysis With Capillary Electrophoresis

Monday, August 23rd, 2021

By Susan Darling, Senior Director, CE and Biopharma Product Management and Marketing, SCIEX

Essential proteins are crucial for infants as they provide the necessary amino acids for growth and development. In human milk, whey and casein proteins are the most abundant, although protein concentration decreases as a mother produces first colostrum, and then transitional milk followed by mature milk.1

Human milk oligosaccharides (HMOs), the third-most abundant ingredient in human milk,1 are also critical for child development.2 The quantities and structures of HMOs differ during the various lactation periods of the mother and are population-specific.3,4

Because of their importance, whey proteins, casein proteins and HMOs are standard components of liquid and powdered infant formulas. It is essential to avoid adulteration and verify the claims of infant formula vendors to ensure the quality, safety and efficacy of infant formulas for targeted populations. This requires accurate methods for measuring the whey, casein and HMO content.

These analyses can be challenging, however. Whey proteins can be difficult to separate from casein proteins,5 and during pasteurization, proteins in infant formula can form complexes with sugars that make effective separation even more difficult.6 Meanwhile, more than 200 HMO structures have been identified,7 many with similar or isomeric structures.8,9

Capillary electrophoresis (CE) solutions, such as CE-sodium-dodecyl sulphate (SDS) and capillary gel electrophoresis (CGE), are practical methods for separating and quantifying whey protein and HMOs, respectively, in infant formula.

CE-SDS performed on a highly resolving, quantitative platform such as the PA 800 Plus Pharmaceutical Analysis System from SCIEX provides excellent separation between whey proteins, casein proteins and immunoglobulins in both protein standard and infant formula samples in under 30 minutes in a reliable and reproducible manner.10

Notably, a separation method developed on the PA 800 Plus System won the Method of the Year Award at the Association of Analytical Chemists (AOAC) International meeting in 2019,11 and it was also chosen to serve as a consensus method for the global trade of infant formula.12

In addition, ultrafast and high-resolution analysis of HMOs is possible using a SCIEX-developed CGE (HR-NCHO and/or SDS-MW gels) method combined with laser-induced fluorescence (LIF) detection.13

Researchers can rapidly and efficiently analyze HMOs from production batches, baby formulas or food additives by simply choosing the appropriate gel-buffer system or mixture that gives the desired resolution. In fact, the same method can be used to decipher the high complexity of HMOs during the entire bioprocessing workflow.

To learn more about how CE can help ensure the quality of infant formula, go to



  1. Ballard, O., Morrow, A.L. Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am. 2013; 60(1): 49-74. doi:10.1016/j.pcl.2012.10.002
  2. Quigley, M., Embleton, N.D., McGuire, W. Formula versus donor breast milk for feeding preterm or low birth weight infants. Cochrane Database Syst Rev. 2019; 7: p. CD002971. doi:10.1002/14651858.CD002971.pub5
  3. Bode, L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology. 2012; 22(9): p. 1147-62. doi:10.1093/glycob/cws074
  4. Coppa, G.V. et al. Oligosaccharides in human milk during different phases of lactation. Acta Paediatr Suppl. 1999; 88(430): p. 89-94. doi:10.1111/j.1651-2227.1999.tb01307.x
  5. Whey protein separation from other proteins difficult Yaguchi, M., Rose, D. Chromatographic separation of milk proteins: a review. J Dairy Sci. 1971; 54(12): 1725-43. doi:10.3168/jds.S0022-0302(71)86106-4
  1. Pasteurization leads to protein-sugar complex formation deWit, J.N., Klarenbeek, G. Effects of Various Heat Treatments on Structure and Solubility of Whey Proteins. J Dairy Sci. 1984; 67(11): 2701-10.  doi:10.3168/jds.S0022-0302(84)81628-8
  1. Ninonuevo, M.R. et al. A strategy for annotating the human milk glycome. J Agric Food Chem. 2006; 54(20): 7471-80. doi:10.1021/jf0615810. PMID: 17002410
  2. Kobata, A. Structures and application of oligosaccharides in human milk. Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86(7): 731-47. doi:10.2183/pjab.86.731
  3. Grabarics, M. et al. Analytical characterization of human milk oligosaccharides – potential applications in pharmaceutical analysis. J Pharm Biomed Anal. 2017; 146: 168-178. doi:10.1016/j.jpba.2017.08.039
  4. Feng, P., Fuerer, C., McMahon, A. Quantification of Whey Protein Content in infant Formulas by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE): Single-Laboratory Validation, First Action 2016.15; J AOAC Int. 2017; 100(2): 510-521. doi:10.5740/jaoacint.16-0344
  5. Lies, M. Study on Quantitation of Whey Protein in Infant Formula Wins AOAC Method of the Year Award. LinkedIn blog, October 10, 2019. “Quantification of Whey Protein Content in Milk-Based Infant Formula Powders by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis (SDS-CGE): Multilaboratory Testing Study, Final Action 2016.15”, P., Fuerer, C., McMahon, A. Quantification of Whey Protein Content in Milk-Based Infant Formula Powders by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE): Multilaboratory Testing Study, Final Action 2016.15. J AOAC Int. 2018; 101(5): 1566–77. doi:10.5740/jaoacint.18-0057
  1. Guttman, A., Szigeti, M., Sarkozi, D. Ultrafast Analysis of Human Milk Oligosaccharides (HMOs) by Capillary Gel Electrophoresis. SCIEX technical note, RUO-MKT-02-11989-B.


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