Abstract
The developing human brain requires high amounts of sialic acids. While human milk is very rich in sialic acids, cow’s milk based infant formulas provide lower amounts of sialic acids, and sialic acids are absent in soy milk based formulas. This has prompted interest in the supplementation of formulas with sialic acids, either free or bound to glycoconjugates. In order for fortification of infant formulas with sialic acids to be appropriate for the developmental needs of the infant, an accurate quantitation of sialic acid content of infant formulas through a reliable and easy-to-use method is, therefore, of great interest to industry. In the present method, we describe the application of one of the most widely used analytical techniques to the quantitation of sialic acids in infant formulas. Briefly, sialic acids are hydrolyzed from glycoconjugates, derivatized using 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB), and separated using reversed-phase high-performance liquid chromatography. The method fulfilled the established criteria for validation, with an interassay standard deviation of less than 5%, accuracy greater than 97%, and surrogate recovery between 98 and 104%. An investigation of the ruggedness of the method identified two key criteria: both standards and samples must be subjected to the same temperature and pH conditions for an accurate quantitation; and prolonged storage (more than 2 days) of the DMB reagent and derivatives must be avoided. In conclusion, this method is specific, straightforward, and accurate and can be easily performed in a quality-assurance laboratory to track the level of sialic acid in formulas that contain both inherent and fortified amounts of sialic acids.
Similar content being viewed by others
References
Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth J (eds) (1999) Essentials of glycobiology, chap 15, 1st edn. Cold Spring Harbor Laboratory Press, Plainville
Wang B, Brand-Miller J (2003) Eur J Clin Nutr 57:1351–1369
Colombaioni L, Garcia-Gil M (2004) Brain Res Rev 46:328–355
Durbec P, Cremer H (2001) Mol Neurobiol 24:53–64
Wang B, Brand-Miller J, McVeagh P, Petocz P (2001) Am J Clin Nutr 74:510–515
Martin MJ, Martin-Sosa, S, Garcia-Pardo LA, Hueso P (2001) J Dairy Sci 84:995–1000
Wang B, Brand-Miller J, McVeagh P, Petocz P (2001) Asia Pac J Clin Nutr 10(Suppl):S33
Svennerholm L (1957) Biochim Biophys Acta 24:604–611
Warren L (1959) J Biol Chem 234:1971–1975
Lamari FN, Karamanos NK (2002) J Chromatogr B 381:3–19
Zanetta JP, Pons A, Iwersen M, Mariller C, Leroy Y, Timmerman, Schauer R (2001) Glycobiology 11:663–676
Rohrer JS (2000) Anal Biochem 283:3–9
Siskos PA, Spyridaki MH (1999) J Chromatogr B 724:205–212
Makatsori E, Karamanos NK, Anastassiou ED, Hjerpe A, Tsegenidis T (1998) J Liq Chromatogr Relat Technol 21:3031–3045
Honda S, Akao E, Suzuki S, Okuda M, Kakehi K, Nakamura J (1989) Anal Biochem 180:351–357
Hara S, Takemori Y, Yamaguchi M, Nakamura M, Ohkura Y (1987) Anal Biochem 164:138–145
Powell LD, Hart GW (1986) Anal Biochem 157:179–185
Anumula KR (1995) Anal Biochem 230:24–30
Sanchez-Diaz A, Ruano MJ, Lorente F, Hueso P (1997) J Ped Gastroenterol Nutr 24:405–410
Martin-Sosa S, Martin MJ, Garcia-Pardo LA, Hueso P (2004) J Ped Gastroenterol Nutr 39:499–503
ICH (1996) ICH harmonised tripartite guidelines Q2A. Validation of analytical procedures: methodology. ICH, Geneva
Feinberg M, Boulanger B, Dewé W, Hubert P (2004) Anal Bioanal Chem 380:502–514
Thompson M, Ellison SLR, Fajgelj A, Willetts P, Wood R (1999) Pure Appl Chem 71:337–348
Neeser JR, Golliard M, Del Vedovo S (1991) J Dairy Sci 74:2860–2871
Lönnerdal B, Atkinson S (1995) In: Jensen RG (ed) Handbook of milk composition. Academic, San Diego
Acknowledgements
This work was partially supported by the Programa Torres Quevedo, from the Spanish Ministry of Education and Science and the European Social Fund. The authors would like to thank M.H. Dohnalek and P.A. Prieto for the critical review of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Martín, M.J., Vázquez, E. & Rueda, R. Application of a sensitive fluorometric HPLC assay to determine the sialic acid content of infant formulas. Anal Bioanal Chem 387, 2943–2949 (2007). https://doi.org/10.1007/s00216-007-1160-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-007-1160-z