Analytical and Bioanalytical Chemistry

, Volume 411, Issue 7, pp 1443–1451 | Cite as

A facile quantification of hyaluronic acid and its crosslinking using gas-phase electrophoresis

  • Hung-Li Wang
  • Chin-Ping Huang
  • Chiu-Hun Su
  • De-Hao TsaiEmail author
Research Paper


We report a facile, high-resolution approach to quantitatively characterize hyaluronic acid (HA) and study its crosslinking reaction using electrospray-differential mobility analysis (ES-DMA). Mobility size distributions, number concentrations, molecular mass distributions, and polydispersity index of HAs were obtained successfully via a rapid analysis by ES-DMA (< 30 min). The limit of detection, the limit of quantification, and the precision of the mobility size measurement achieve 2.5 nm, 4.0 nm, and 0.3 nm, respectively. Size exclusion chromatography (SEC) was employed as an orthogonal approach, showing that the averaged molecular mass and polydispersity index of HA measured by ES-DMA were close to the results of SEC on a semi-quantitative basis. The 1,4-butanediol diglycidyl ether (BDDE)-induced crosslinking of HA was also able to be successfully characterized through a time-dependent study using ES-DMA, which has shown the promise of direct analysis of solution-based reactions. Both the extent and the rate of HA crosslinking (induced by BDDE) were proportional to reaction temperature and concentration ratio of HA to BDDE. The activation energy of the reaction-limited BDDE-induced crosslinking of HA was found to be ≈ 21 kJ/mol. The prototype study demonstrates ES-DMA as a new method for a rapid quantitative characterization of HA and its derivative product and providing a capability of real-time monitoring of the HA crosslinking during formulation process.

Graphical abstract


Hyaluronic acid Mobility Electrospray Molecular mass Concentration Crosslinking 


Funding information

This study received financial support from the Ministry of Science and Technology of the Republic of China (Taiwan) under contract MOST 105-2221-E-007-129-MY3 and MOST 106-3113-E-007-002.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_1584_MOESM1_ESM.pdf (895 kb)
ESM 1 (PDF 894 kb)


  1. 1.
    Burdick JA, Prestwich GD. Hyaluronic acid hydrogels for biomedical applications. Adv Mater. 2011;23:H41–56.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Highley CB, Prestwich GD, Burdick JA. Recent advances in hyaluronic acid hydrogels for biomedical applications. Curr Opin Biotechnol. 2016;40:35–40.CrossRefPubMedGoogle Scholar
  3. 3.
    Zhang Z, Christopher GF. The nonlinear viscoelasticity of hyaluronic acid and its role in joint lubrication. Soft Matter. 2015;11:2596–603.CrossRefPubMedGoogle Scholar
  4. 4.
    Neuman MG, Nanau RM, Oruna-Sanchez L, Coto G. Hyaluronic acid and wound healing. J Pharm Pharm Sci. 2015;18:53–60.CrossRefPubMedGoogle Scholar
  5. 5.
    De Boulle K, Glogau R, Kono T, Nathan M, Tezel A, Roca-Martinez JX, et al. A review of the metabolism of 1,4-butanediol diglycidyl ether-crosslinked hyaluronic acid dermal fillers. Dermatol Surg. 2013;39:1758–66.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Volpi N. On-line HPLC/ESI-MS separation and characterization of hyaluronan oligosaccharides from 2-mers to 40-mers. Anal Chem. 2007;79:6390–7.CrossRefPubMedGoogle Scholar
  7. 7.
    Kenne L, Gohil S, Nilsson EM, Karlsson A, Ericsson D, Helander Kenne A, et al. Modification and cross-linking parameters in hyaluronic acid hydrogels--definitions and analytical methods. Carbohydr Polym. 2013;91:410–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Kim B, Woo S, Park YS, Hwang E, Moon MH. Ionic strength effect on molecular structure of hyaluronic acid investigated by flow field-flow fractionation and multiangle light scattering. Anal Bioanal Chem. 2015;407:1327–34.CrossRefPubMedGoogle Scholar
  9. 9.
    Shanmuga Doss S, Bhatt NP, Jayaraman G. Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography. J Chromatogr B. 2017;1060:255–61.CrossRefGoogle Scholar
  10. 10.
    Schante CE, Zuber G, Herlin C, Vandamme TF. Chemical modifications of hyaluronic acid for the synthesis of derivatives for a broad range of biomedical applications. Carbohyd Polym. 2011;85:469–89.CrossRefGoogle Scholar
  11. 11.
    Gaborieau M, Castignolles P. Size-exclusion chromatography (SEC) of branched polymers and polysaccharides. Anal Bioanal Chem. 2011;399:1413–23.CrossRefPubMedGoogle Scholar
  12. 12.
    Mendichi R, Schieroni AG, Grassi C, Re A. Characterization of ultra-high molar mass hyaluronan: 1. Off-line static methods. Polymer. 1998;39:6611–20.CrossRefGoogle Scholar
  13. 13.
    Bacher G, Szymanski WW, Kaufman SL, Zollner P, Blaas D, Allmaier G. Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses. J Mass Spectrom. 2001;36:1038–52.CrossRefPubMedGoogle Scholar
  14. 14.
    Tsai DH, Elzey S, Delrio FW, Keene AM, Tyner KM, Clogston JD, et al. Tumor necrosis factor interaction with gold nanoparticles. Nanoscale. 2012;4:3208–17.CrossRefPubMedGoogle Scholar
  15. 15.
    Pease LF 3rd, Tsai DH, Brorson KA, Guha S, Zachariah MR, Tarlov MJ. Physical characterization of icosahedral virus ultra structure, stability, and integrity using electrospray differential mobility analysis. Anal Chem. 2011;83:1753–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Pease LF 3rd, Elliott JT, Tsai DH, Zachariah MR, Tarlov MJ. Determination of protein aggregation with differential mobility analysis: application to IgG antibody. Biotechnol Bioeng. 2008;101:1214–22.CrossRefPubMedGoogle Scholar
  17. 17.
    Malm L, Hellman U, Larsson G. Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis. Glycobiology. 2012;22:7–11.CrossRefPubMedGoogle Scholar
  18. 18.
    Weiss VU, Golesne M, Friedbacher G, Alban S, Szymanski WW, Marchetti-Deschmann M, et al. Size and molecular weight determination of polysaccharides by means of nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA). Electrophoresis. 2018;39:1142–50.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wasiak I, Kulikowska A, Janczewska M, Michalak M, Cymerman IA, Nagalski A, et al. Dextran nanoparticle synthesis and properties. PLoS One. 2016;11:e0146237.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Szymanski WW, Bacher G, Allmaier G. In: Szymanski WW, Wagner P, Itoh M, Ohachi T, editors. Nanostructured materials and their applications. Vienna: Facultas; 2004.Google Scholar
  21. 21.
    Hong M, Sudor J, Stefansson M, Novotny MV. High-resolution studies of hyaluronic acid mixtures through capillary gel electrophoresis. Anal Chem. 1998;70:568–73.CrossRefPubMedGoogle Scholar
  22. 22.
    Tsai DH, DelRio FW, Keene AM, Tyner KM, MacCuspie RI, Cho TJ, et al. Adsorption and conformation of serum albumin protein on gold nanoparticles investigated using dimensional measurements and in situ spectroscopic methods. Langmuir. 2011;27:2464–77.CrossRefPubMedGoogle Scholar
  23. 23.
    Tai JT, Lai CS, Ho HC, Yeh YS, Wang HF, Ho RM, et al. Protein-silver nanoparticle interactions to colloidal stability in acidic environments. Langmuir. 2014;30:12755–64.CrossRefPubMedGoogle Scholar
  24. 24.
    Chang WC, Tai JT, Wang HF, Ho RM, Hsiao TC, Tsai DH. Surface PEGylation of silver nanoparticles: kinetics of simultaneous surface dissolution and molecular desorption. Langmuir. 2016;32:9807–15.CrossRefPubMedGoogle Scholar
  25. 25.
    Mälson T, Lindqvist BL. Gel of crosslinked hyaluronic acid for use as a vitreous humor substitute. WO/1986/000079; 1986.Google Scholar
  26. 26.
    Elzey S, Tsai DH, Yu LL, Winchester MR, Kelley ME, Hackley VA. Real-time size discrimination and elemental analysis of gold nanoparticles using ES-DMA coupled to ICP-MS. Anal Bioanal Chem. 2013;405:2279–88.CrossRefPubMedGoogle Scholar
  27. 27.
    Mulholland GW, Donnelly MK, Hagwood CR, Kukuck SR, Hackley VA, Pui DYH. Measurement of 100 nm and 60 nm particle standards by differential mobility analysis. J Res Natl Inst Stand Technol. 2006;111:257–312.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Tsai DH, Lu YF, DelRio FW, Cho TJ, Guha S, Zachariah MR, et al. Orthogonal analysis of functional gold nanoparticles for biomedical applications. Anal Bioanal Chem. 2015;407:8411–22.CrossRefPubMedGoogle Scholar
  29. 29.
    Nguyen TP, Chang WC, Lai YC, Hsiao TC, Tsai DH. Quantitative characterization of colloidal assembly of graphene oxide-silver nanoparticle hybrids using aerosol differential mobility-coupled mass analyses. Anal Bioanal Chem. 2017;409:5933–41.CrossRefPubMedGoogle Scholar
  30. 30.
    Tai JT, Lai YC, Yang JH, Ho HC, Wang HF, Ho RM, et al. Quantifying nanosheet graphene oxide using electrospray-differential mobility analysis. Anal Chem. 2015;87:3884–9.CrossRefPubMedGoogle Scholar
  31. 31.
    Tsai DH, Cho TJ, DelRio FW, Gorham JM, Zheng J, Tan J, et al. Controlled formation and characterization of dithiothreitol-conjugated gold nanoparticle clusters. Langmuir. 2014;30:3397–405.CrossRefPubMedGoogle Scholar
  32. 32.
    Berg JC. An introduction to interfaces & colloids : the bridge to nanoscience. Singapore: World Scientific; 2010.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Hung-Li Wang
    • 1
  • Chin-Ping Huang
    • 2
  • Chiu-Hun Su
    • 2
  • De-Hao Tsai
    • 1
    Email author
  1. 1.Department of Chemical EngineeringNational Tsing Hua UniversityHsinchuRepublic of China
  2. 2.Material and Chemical Research LaboratoriesIndustrial Technology Research InstituteHsinchuRepublic of China

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