Capillary electrophoresis method for simultaneous determination of neuropeptide GSB-106 and hyaluronan in the preparation of a nasal formulation

Capillary electrophoresis, using capillary electrokinetic chromatography, was used for the simultaneous quantitative determination of the dipeptide GSB-106 and sodium hyaluronan with a molecular weight of 130 kDa in a dosage form developed for intranasal delivery of the peptide to the central nervous system. An optimized leading electrolyte composition was established: 15 mM sodium tetraborate and 15 mM sodium dodecyl sulfate. With positive polarity, the peaks of both components on the electropherogram are located in the anion region. The electropherogram development time is 10 min. The developed method enables the selective determination of both components of the nasal formulation with sensitivity sufficient for spectrophotometric detection (8.8 μg/mL for GSB-106 and 76.2 μg/mL for hyaluronan). The efficiency of determining both components was estimated using the number of theoretical plates and was ~13,000 for GSB-106 and ~5500 for hyaluronan. The practical application of the method was demonstrated using the nasal dosage form of GSB-106, which is based on hyaluronic acid. The method can be successfully applied to the analysis of similar hyaluronic acid-based formulations containing peptides.

1. Gudasheva T. A., Povarnina P. Yu., Seredenin S. B. Dipeptide mimetic of the brain-derived neurotrophic factor prevents impairments of neurogenesis in stressed mice / Bull. Exp. Biol. Med. 2017. Vol. 162. No. 4. P. 454 – 457. DOI: 10.1007/s10517-017-3638-9

2. Garibova T. L., Kraineva V. A., Kotel’nikova S. O., et al. Behavioral effects of dimeric dipeptide BDNF mimetic GSB-106 in a rat model of depressive-like state / Bull. Exp. Biol. Med. 2020. Vol. 169. No. 2. P. 286 – 289. DOI: 10.1007/s10517-020-04869-8

3. Gudasheva T. A., Povarnina P. Yu., Antipova T. A., et al. Neuroregenerative activity of the dipeptide mimetic of brain- derived neurotrophic factor GSB-106 under experimental ischemic stroke / CNS Neurol. Disord. Drug Targets. 2021. Vol. 20. No. 10. P. 954 – 962. DOI: 10.2174/1871527320666210525090904

4. Povarnina P. Yu., Nikiforov D. M., Vorontsova O. N., Gudasheva T. A. Dipeptide BDNF mimetic GSB-106 prevents depression-like behavior and cognitive impairment caused by ischemic stroke in rats / Probl. Biol. Med. Pharm. Chem. 2022. Vol. 25. No. 2. P. 49 – 53 [in Russian]. DOI: 10.29296/25877313-2022-02-08

5. Correale J., Mattera V., Pasquini J. M. Intranasal delivery route for neurodegenerative diseases: recent insights and future directions / Drug Delivery Transl. Res. 2026. Vol. 16. No. 1. P. 76 – 94. DOI: 10.1007/s13346-025-01919-4

6. Kolyvanov G. B., Zherdev V. P., Gribakina O. G., et al. Comparative preclinical pharmacokinetics and bioavailability of antidepressant GSB-106 tablet form / Bull. Exp. Biol. Med. 2019. Vol. 167. No. 5. P. 637 – 640. DOI: 10.1007/s10517-019-04587-w

7. Zherdev V. P., Kolyvanov G. B., Litvin A. A., et al. Pharmacokinetics of dipeptide mimetic BDNF GSB-106 in rats / Pharmacokinet. Pharmacodyn. 2019. No. 1. P. 37 – 43 [in Russian]. DOI: 10.24411/2587-7836-2019-10038

8. Wei S., Zhai Z., Kong X., et al. The review of nasal drug delivery system: The strategies to enhance the efficiency of intranasal drug delivery by improving drug absorption / Int. J. Pharm. 2025. Vol. 676. 125584. DOI: 10.1016/j.ijpharm.2025.125584

9. Tarasyuk A. V., Sazonova N. M., Shumskii A. N., et al. Physicochemical properties of the original antidepressant GSB-106 and development of a method for the analysis of GSB-106 substance / Pharm. Chem. J. 2019. Vol. 53. No. 1. P. 71 – 78. DOI: 10.1007/s11094-019-01958-5

10. Gribakina O. G., Bochkov P. O., Shevchenko R. V., Zherdev V. P. Validation of the HPLC/MS technique for GSB-106 determination in rats’ blood plasma / Éksp. Klin. Farmakol. 2018. Vol. 81. No. S. P. 60 – 61 [in Russian].

11. Stefanik O., Majerova P., Kovac A., et al. Capillary electrophoresis in the analysis of therapeutic peptides — A review / Electrophoresis. 2024. Vol. 45. Nos. 1 – 2. P. 120 – 164. DOI: 10.1002/elps.202300141

12. Alkrad J. A., Merstani Y., Neubert R. H. H. New approaches for quantifying hyaluronic acid in pharmaceutical semisolid formulations using HPLC and CZE / J. Pharm. Biomed. Anal. 2002. Vol. 30. No. 4. P. 913 – 919. DOI: 10.1016/s0731-7085(02)00329-1

13. Kakehi K., Ueda M., Suzuki S., Honda S. Determination of hyaluronic acid by high-performance liquid chromatography of the oligosaccharides derived therefrom as 1-(4-methoxy)phenyl-3-methyl-5-pyrazolone derivatives / J. Chromatogr. 1993. Vol. 630. Nos. 1 – 2. P. 141 – 146. DOI: 10.1016/0021-9673(93)80449-i

14. Kühn A. V., Raith K., Sauerland V., Neubert R. H. H. Quantification of hyaluronic acid fragments in pharmaceutical formulations using LC-ESI-MS / J. Pharm. Biomed. Anal. 2003. Vol. 30. No. 5. P. 1531 – 1537. DOI: 10.1016/s0731-7085(02)00544-7

15. Rothenhöfer M., Grundman M., Bernhardt G., et al. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the sensitive determination of hyaluronan oligosaccharides / J. Chromatogr. B. 2015. Vol. 988. P. 106 – 115. DOI: 10.1016/j.jchromb.2015.02.027

16. Ruckmani K., Shaikh S. Z., Khalil P., et al. Determination of sodium hyaluronate in pharmaceutical formulations by HPLC-UV / J. Pharm. Anal. 2013. Vol. 3. No. 5. P. 324 – 329. DOI: 10.1016/j.jpha.2013.02.001

17. Çağlar E. Ş., Üstündağ Okur N., Karasulu H. Y. Development and validation of an HPLC method for the determination of hyaluronic acid active substance in pharmaceutical formulations / J. Res. Pharm. 2022. Vol. 26. No. 1. P. 112 – 122. DOI: 10.29228/jrp.108

18. Ermolenko Yu., Antonova M., Polivanova A., Gelperina S. The method of capillary electrophoresis for quantitative determination of hydrophobized hyaluronic acid in its micellar forms / Anal. Biochem. 2025. Vol. 696. 115684. DOI: 10.1016/j.ab.2024.115684

19. ICH Q2(R2). Validation of analytical procedures — Scientific guideline. https://www.ema.europa.eu/en/ich-q2r2-validation-analytical-procedures-scientific-guideline (accessed April 7, 2026).

20. OFS.1.1.0012. Validation of analytical procedures / State Pharmacopoeia of the Russian Federation (XV ed.). https://meganorm.ru/mega_doc/norm/gosudarstvennaya-farmakopeya/1/ofs_1_1_0012_obshchaya_farmakopeynaya_statya_validatsiya.html?ysclid=mqvbeyrla6278756045 (accessed April 7, 2026) [in Russian].

21. Safarov R., Fedotova O., Uvarova A., et al. Review of intranasal active pharmaceutical ingredient delivery systems / Pharmaceuticals. 2024. Vol. 17. No. 9. 1180. DOI: 10.3390/ph17091180

22. Tai J., Han M., Lee D., et al. Different methods and formulations of drugs and vaccines for nasal administration / Pharmaceutics. 2022. Vol. 14. No. 5. 1073. DOI: 10.3390/pharmaceutics14051073

23. Daku Abubakar B., Rosdan B. S. M., Noratigah M., Nurul A. A. Advances in polysaccharide-based formulations for intranasal drug delivery: A review / J. Drug Delivery Sci. Technol. 2024. Vol. 101. No. 12. 106266. DOI: 10.1016/j.jddst.2024.106266

24. Jin Y.-J., Ubonvan T., Kim D.-D. Hyaluronic acid in drug delivery systems / J. Pharm. Invest. 2010. Vol. 40(spc). P. 33 – 43. DOI: 10.4333/kps.2010.40.S.033

25. Di Cicco M., Alicandro G., Claut L., et al. Efficacy and tolerability of a new nasal spray formulation containing hyaluronate and tobramycin in cystic fibrosis patients with bacterial rhinosinusitis / J. Cystic Fibrosis. 2014. Vol. 13. No. 4. P. 455 – 460. DOI: 10.1016/j.jcf.2014.02.006

26. Vlasenko I. V., Menshutina N. V. Development of nasal pharmaceutical products for systemic exposure: use of permeation enhancers to achieve target product profile / Probl. Biol. Med. Pharm. Chem. 2024. Vol. 27. No. 4. P. 3 – 10 [in Russian]. DOI: 10.29296/25877313-2024-04-01

27. Gudasheva T. A., Tarasyuk A. V., Pomogaibo S. V., et al. Design and synthesis of dipeptide mimetics of the brain-derived neurotrophic factor / Russ. J. Bioorg. Chem. 2012. Vol. 38. P. 243 – 252. DOI: 10.1134/s1068162012030053

28. Tallerova A. V., Povarnina P. Yu., Blynskaya E. V., et al. Antidepressant effect of a dipeptide brain-derived-neurotrophic-factor mimetic (GSB-106) in a generic oral dosage form / Pharm. Chem. J. 2018. Vol. 52. No. 5. P. 397 – 399. DOI: 10.1007/s11094-018-1828-z

29. Pharmaceutical quality of inhalation and nasal products — Scientific guideline / European Medicines Agency (EMA). https:// www.ema.europa.eu/en/pharmaceutical-quality-inhalation-nasal-products-scientific-guideline (accessed April 7, 2026).

30. Illum L. Nasal drug delivery — Recent developments and future prospects / J. Controlled Release. 2012. Vol. 161. No. 2. P. 254 – 263. DOI: 10.1016/j.jconrel.2012.01.024

31. Peer D., Florentin A., Margalit R. Hyaluronan is a key component in cryoprotection and formulation of targeted unilamellar liposomes / Biochim. Biophys. Acta. 2003. Vol. 1612. No. 1. P. 76 – 82. DOI: 10.1016/s0005-2736(03)00106-8

32. Bui V. D., Duong V. H., You D. G., et al. Improving wound healing function and storage stability of stem cell-derived extracellular vesicles via lyophilized hyaluronic acid formulation / J. Nanobiotechnol. 2026. Vol. 24. No. 1. 283. DOI: 10.1186/s12951-026-04201-y

33. Maleki A., Kjøniksen A. L., Nyström B. Effect of pH on the behavior of hyaluronic acid in dilute and semidilute aqueous solutions / Macromol. Symp. 2008. Vol. 274. No. 1. P. 131 – 140. DOI: 10.1002/masy.200851418

34. Ward C. N., LeBlanc P. E., Burrell R. E. Effects of composition and pH on the degradation of hyaluronate and carboxymethyl cellulose gels and release of nanocrystalline silver / J. Appl. Biomater. Funct. Mater. 2024. Vol. 22. 91. DOI: 10.1177/22808000241257124