Chromatographic determination of related substances in the original antiepileptic compound GIZH-298 substance: development and validation of a method

Introduction. For the introduction into medical practice of the new original compound codenamed GIZH-298, which exhibits pronounced anticonvulsant activity in in vivo experiments, comprehensive pharmaceutical development is required. One of its key stages ensuring the quality, safety, and efficacy of the future drug product is the development of methods for the related substances control.

Objective. Development and validation of a related substances determination method for the new original anticonvulsant compound GIZH-298 substance by HPLC-UV.

Materials and methods. The studies were performed on pilot samples of the GIZH-298 substance, as well as on the starting, intermediate, and byproducts of its synthesis. Chromatographic separation was carried out on an Infinity 1260 liquid chromatograph (Agilent Technologies, USA) equipped with a variable wavelength spectrophotometric detector, using Agilent OpenLAB CDS ChemStation software. Stress studies to assess specificity were conducted under exposure to 0.1 M HCl, 0.1 M NaOH, 3 % H₂O₂, as well as under light exposure in an ATLAS SUNTEST CPS+ chamber with an ATLAS SunCool cooling system (ATLAS MTT, USA).

Results. A related substances determination method in the GIZH-298 substance by HPLC-UV was developed using a BDS Hypersil C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, USA). Mobile phase: 0.02 M dipotassium hydrogen phosphate solution (pH 7.45±0.05), acetonitrile and methanol (250:100:100). Validated covered specificity, linearity, detection limit, quantitation limit, accuracy, and precision (repeatability). The method detects known process-related impurities at the level of 0.05 % and below, as well as impurities formed during hydrolysis, oxidation, and photodegradation.

Conclusions. The developed method is suitable for assessing related substances in the GIZH-298 pharmaceutical substance and its drug products.

1. Epilepsy: a public health imperative. Summary. Geneva: World Health Organization; 2019 (WHO/MSD/MER/19.2). Licence: CC BY–NC–SA 3.0 IGO.

2. GBD Epilepsy Collaborators. Global, regional, and national burden of epilepsy, 1990-2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet Public Health. 2025 Mar;10(3):e203-e227. doi: 10.1016/S2468-2667(24)00302-5.

3. Durnev AD, Voronina TA, Litvinova SA, et al. Derivatives of 4–benzoylpyridine oximes with anticonvulsant activity as agents for the treatment of epilepsy and paroxysmal states. Patent RF № 2643091; 2018. (In Russ.).

4. Ushkalova EA. Side effects of antiepileptic drugs. Farmateka. 2001;51(9-10):11. (In Russ.).

5. Litvinova SA, Narkevich VB, Gaidukov IO, et al. Analysis of the effect of derivative of pyridine oximes (GIZh-298) on the content of monoamines and their metabolites in the structures of rat brain structures during seizures caused by maximum electric shock. Neyrokhimiya = Neurochemical Journal. 2019;36(3):239-245. (In Russ.). doi: 10.1134/S1027813319020055.

6. Zhmurenko LA, Mokrov GV, Nerobkova LN, et al. Novel 4-benzoylpyridine oxime derivative GIZH-298 with aticonvulsant activity. Farmakokinetika i farmakodinamik = Pharmacokinetics and Pharmacodynamics. 2017;(1):22–26. (In Russ.).

7. Gaydukov IO, Litvinova SA, Voronina TA, et al. Effect of a new 4–benzoylpyridine oxime derivative GIZH–298 and valproic acid on anxiety and depressive–like states, cognitive functions in experiment. Eksperimentalnaya i klinicheskaya farmakologiya = Experimental and Clinical Pharmacology. 2022;85(7):8-12. (In Russ.). doi: 10.30906/0869-2092-2022-85-7-8-12.

8. OFS.1.1.0023. Related impurities in pharmaceutical substances and medicinal products. State Pharmacopoeia of the Russian Federation. 15th ed. (In Russ.).

9. Requirements for conducting research (testing) of medicinal products regarding the assessment and control of impurities. Decision of the EEC Board of October 4, 2022, № 138. (In Russ.).

10. ICH Harmonised Tripartite Guideline: Impurities in new drug substances Q3A(R2). In: Quality guidelines [Internet]. Geneva: ICH; 2006 [cited 2026 May 12]. Available from: https://ich.org/page/quality-guidelines.

11. OFS.1.1.0012. Validation of analytical procedures. State Pharmacopoeia of the Russian Federation. 15th ed. (In Russ.).

12. OFS.1.1.0029. Determination of photostability of medicinal products. State Pharmacopoeia of the Russian Federation. 15th ed. (In Russ.).

13. Requirements for the stability study of medicinal products and pharmaceutical substances. Decision of the EEC Board of May 10, 2018, № 69 (as amended on June 30, 2020, № 86). (In Russ.).

14. OFS.1.1.0013. Statistical processing of the results of physical, physicochemical and chemical tests. State Pharmacopoeia of the Russian Federation. 15th ed. (In Russ.).

15. OFS 2.3.13.0. Statistical processing of the results of physical, physicochemical and chemical tests. Pharmacopoeia of the EAEU, vol. 2. (In Russ.).