Сapreomycin IA and IB Identification and Their Content Ratio Quantification in Capreomycin Pharmaceutical Preparations Using NMR Spectroscopic Methods

Background. Capreomycin is a multicomponent natural antibiotic and is a mixture of 4 related compounds (capreomycins IA, IB, IIA, and IIB). Quality control of capreomycin medicinal preparations by active substance is carried out according to the «capreomycin I» indicator, not taking into account the individual contributions of capreomycins IA and IB. With this approach, no correlation is observed between the total content of capreomycin IA and IB components determined by chromatographic methods and capreomycin activity determined by microbiological methods. WHO recommended studying the feasibility of introducing the ratio between capreomycin IA and IB in the specifications for capreomycin preparations.

The aim of the study was to develop a technique for quantitative estimation of the capreomycin IA and IB content ratio in the drug preparation Capreomycin sulphate using NMR spectroscopy.

Objects of the study. Capreomycin sulphate standard samples, pharmaceutical preparations Capreomycin, powder for preparation of solutions for intravenous and intramuscular administration from various manufacturers. Material and methods: NMR spectroscopy, HPLC.

Results. Structural interpretation of 1H and 13C spectra of capreomycin sulfate in D₂O solution was performed. 13C spectra revealed characteristic signals of capreomycin IA and IB, which did not overlap with the signals of minor capreomycin components and related impurities. On the basis of normalized integral intensities, the ratio of mole fractions of capreomycin IA and IB (IA/IB) was calculated. The results of IA/IB measurement using 13C NMR and HPLC practically coincide when the chromatographic conditions allow separating the signals of the major and impurity components of capreomycin on the chromatogram.

1. Laughlin Z. T., Conn G. L. Tuberactinomycin antibiotics: Biosynthesis, anti-mycobacterial action, and mechanisms of resistance. Front Microbiol. 2022; 13: 961921. doi: 10.3389/fmicb.2022.961921.

2. Wang M., Gould S. J. Biosynthesis of capreomycin. 2. Incorporation of L-serine, L-alanine, and L-2,3-diaminopropionic acid. J Org Chem. 1993; 58 (19): 5176–5180.

3. Lin Y., Li Y., Zhu N., Han Y., Jiang W., Wang Y., Si S., Jiang J. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10. Antimicrob Agents Chemother. 2014; 58 (4): 2038–2044. doi: 10.1128/aac.02394-13.

4. Herr Jr E. B., Redstone M. O. Chemical and physical characterization of capreomycin. Ann N Y Acad Sci. 1966; 135 (2): 940–6.

5. Monograph Capreomycin sulfate. United States Pharmacopeia. USP43-NF38. Rockville, MD; 2023.

6. Monograph Capreomycin sulfate. Indian Pharmacopoeia. IX ed. 2022; II: 1726.

7. Monograph Capreomycin sulfate. Pharmacopoeia of the peoples republic of China. 2020; II: 287–288.

8. Bycroft B. W., Cameron D., Croft L. R., Hassanali-Walji A., Johnson A. W., Webb T. Total structure of capreomycin IB, a tuberculostatic peptide antibiotic. Nature. 1971; 231 (5301): 301–302.

9. Lightbown J. W., De Rossi P., Isaacson P. International standards and international reference preparations. Bull World Heath Organ. 1972; 47 (3): 343–356.

10. Nomoto S., Teshima T., Wakamiya T., Shiba T. The revised structure of capreomycin. J Antibiot. 1977; 30 (11): 955–959. doi: 10.7164/antibiotics.30.955.

11. Lea M. L. A physiological study of streptomyces capreolus and factors governing growth and capreomycin biosynthesis [dissertation]. Liverpool John Moores University; 2007. doi: researchonline.ljmu.ac.uk/view/theses/rg=5Fpha.html.

12. Fifty-first report of the WHO Expert Committee on specifications for pharmaceutical preparations. Geneva: World Health Organization; 2017 (WHO technical report series; No. 1003).

13. WHO Expert Committee on Biological Standardization, sixty-seventh report. Geneva: World Health Organization; 2017 (WHO technical report series; No. 1004).

14. Yakupov I. Yu., Kuleshova S. I., Simonova E. P., Demidov A. S. Ionpair chromatography for the determination of capreomycin sulfate components and related substances. Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products. Regulatory Research and Medicine Evaluation. 2023; 13 (2–1): 271–282. doi: https://doi.org/10.30895/1991-2919-2023-451. (in Russian)

15. Liua G., Luan B., Liang G Xing L., Huang L., Wangc C. et al. Isolation and identification of four major impurities in capreomycin sulfate. J Chromatogr. A. 2018; 1571: 155–164. doi: 10.1016/j.chroma.2018.08.015.

16. Malz F., Jancke H. Validation of quantitative NMR. J Pharm Biomed Anal. 2005; 38 (5): 813–823. doi: 10.1016/j.jpba.2005.01.043.

17. General monograph.1.2.1.1.1.007 Nuclear magnetic resonance spectroscopy. State Pharmacopoeia of the Russian Federation X. V. ed. https://pharmacopoeia.regmed.ru/pharmacopoeia/izdanie-15/1/1-2/1-2-1/1-2-1-1-metody-spektralnogo-analiza/spektroskopiya-yadernogo-magnitnogo-rezonansa/ (in Russian)