DEVELOPMENT OF METHODS OF PRE-COLUMNAR DERIVATIZATION OF GLUTATHIONES RECOVERED BY 4-METHOXY-2-NITROPHENYLISOTHIOTOCIONATE FOR DETERMINATION BY METHOD OF HIGH-EFFECTIVE LIQUID CHROMATOGRAPHY


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Nowadays the pharmacological role of glutathione in the therapy of carcinogenesis, neurodegenerative and ocular diseases, heart diseases, the immune system and aging of the organism is being actively investigated. Therefore, for the development of pharmaceutical medical forms on its basis, it is necessary to create an optimal analytical base. The aim of this study is to develop a methodology for the analysis of glutathione recovered by pre-columnar derivatization of 4-methoxy-2-nitrophenyl isothiocyanate. Materials and methods. Since glutathione does not have the necessary spectral characteristics for its direct analysis, a methodology for the determination of glutathione with the use of pre-columnar derivatization of 4-methoxy-2-nitrophenyl-isothiocyanate by reversed-phase high-performance chromatography (RP HPLC) has been developed on that basis. Detection of the resulting derivative has been carried out by absorption in UV light using a diode array detector. Results and discussion. In the course of the experiment described, chromatograms of a glulathione derivative with 4-methoxy-2-nitrophenyl isothiocyanate were obtained. This technique was also evaluated for the possibility of quantitative determination of glutathione. The sensitivity of the methods was 0.01% or 3.1*10-1 mol. The linear relationship between the analytical signal (peak area) and concentration was observed within the range of 0.01–0.08% and the correlation coefficient of 0.995. Conclusion. In the course of the studies, a methodology for the determination of glutathione has been developed with the use of pre-columnarderivatization of 4-methoxy-2-nitrophenyl-isothiocyanate by RP HPLC. In this case, the derivative is formed with the retention time of 22.3 minutes and the absorption maximum of 398 nm. This method also allows estimating the quantitative content of the object under study.

作者简介

K. Alexeeva

FSAEI HE Belgorod National State Research University of the Russian Federation

Email: 740890@bsu.edu.ru

D. Pisarev

FSAEI HE Belgorod National State Research University of the Russian Federation

Email: pisarev@bsu.edu.ru

O. Novikov

FSAEI HE Belgorod National State Research University of the Russian Federation

Email: novikov@bsu.edu.ru

A. Malyutina

FSAEI HE Belgorod National State Research University of the Russian Federation

Email: malyutina_a@bsu.edu.ru

参考

  1. Dickinson D.A., Forman H.J. Cellular glutathione and thiols metabolism // Biochem. Pharmacol. 2012. Vol. 64.P. 1019–1026.
  2. Forman H.J., Dickinson D.A. Oxidative signaling and glutathione synthesis // Biofactors. 2003. Vol. 17. P. 1–12.
  3. Liu H., Wang H., Shenvis S., Hagen T.M., Liu R.M. Glutathione metabolism during aging and in Alzheimer disease // Ann.N.Y.Acad.Sci. 2004. Vol. 1019. P. 346–349.
  4. Wang H., Liu H., Liu R.M. Gender difference in glutathione metabolism during aging in mice, Exp. Gerontology. 2003. Vol. 38. P. 507–517.
  5. De Leve L., Kaplowitz N. Importance and regulation of hepatic GSH // Sem.Liver.Dis. 1990. Vol. 10. P. 251–266.
  6. Meister A., Anderson M. E. Glutathione // Ann.Rev.Biochem. 1983. Vol. 52. P. 711–760.
  7. Hogg N. The biochemistry and physiology of S-nitrosothiols // Ann.Rev. Pharmacol.Toxicol. 2002. Vol. 42. Vol. 585–600.
  8. Oja S.S., Janaky R., Varga V., Saranasaari P. Modulation of glutamate receptor functions by glutathione //Neurochem.Int. 2000. Vol. 37. P. 299–306.
  9. Pompella A., Visvikis A., Paolicchi A., De Tata V., Casini A.F. The changing faces of glutathione, a cellularprotagonist // Biochem Pharmacol. 2003. Vol. 66. P. 1499–1503.
  10. Forman H.J., Zhang H., Rinn A. Glutathione: Overview of its protective roles, measurement, and biosynthesis // Mol.Aspects.Med. 2009. Vol. 30. P. 1–12.
  11. Woodbridge J.E. Glutathione reagent and test method. United States Patent: 3864085A. 1975.
  12. Hissin P.J., Hilf R.A fluorometric method or determination of oxidized and reducedglutathione in tissues // Anal. Biochem. 1976. Vol. 74. P. 214-226.
  13. Agrawal S.B., Agrawal M., Lee E.H., Kramert G.F. Changes in polyamine andglutathione contents of a green alga, Chlorogonium elongatum (dang) france exposedto mercury // Environ.Exp.Botany. 1992. Vol. 32. P. 145–151.
  14. Ellman G.L. Tissue sulfhydryl groups // Arch.Biochem.Biophys. 1959. Vol. 82. P. 70–81.
  15. Sutariya V., Wehrung D., Geldenhuys W.J. Development and validation of a novel RP-HPLC method for the analysis of reduced glutathione // J. Chromatogr.Sci. 2012. Vol. 50. Is. 3. P. 271–276.
  16. Iwasaki Y., Saito Y., Nakano Y., Mochizuki K., Sakata O., Ito R., Saito K., Nakazawa H. Chromatographic and mass spectrometric analysis of glutathione in biological samples // J.Chromatogr. B. Analyt. Technol. Biomed. LifeSci. 2009. Vol. 877. Is. 28. P. 3309–3317.
  17. Di Pietra A.M., Gotti R., Bonazzi D., Andrisano V., Carvini V. HPLC determination of glutathione and L-cysteine in pharmaceuticals after derivatization with ethacrynic acid // J.Pharm.Biomed.Anal. 2013. Vol. 12. Is. 1. P. 91–98.
  18. Giustarini, D., Dally-Donne I., Milzani A., Fanty P., Rossi R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide // Nat.Protoc. 2013. Vol. 8. P. 1660-1669.
  19. Lee S., Yim J., Lim K., Kim J. Validation of a liquid chromatography tandem mass spectrometry method to measure oxidized and reduced forms of glutathione in whole blood and verification in a mouse model as an indicator of oxidative stress // J.Chromatogr.B. 2016. Vol. 1019. P. 45–50.
  20. Gawlik M., Krzyżanowska W., Gawlik M.B., Filip M. Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization // Acta Chromatographica. 2014. Vol. 36. P. 335–345.
  21. Bald E., Głowacki R. Analysis of saliva for glutathione and metabolically related thiols by liquid chromatography with ultraviolet detection // Amino Acids. 2005. Vol. 28. Is. 4. P. 431–433.
  22. Yan M., G.-B. Shi G., Sui Y., Guo T., Zhang J.-W., Fan S.-J. Determination of reduced glutathione in human plasma by RP-HPLC // Pharmaceutical Journal of Chinese People’s Liberation Army. 2008. Vol. 3. P. 251–253.
  23. Safavi A., Maleki N., Farjami E., Aghakhani Mahyari F. Simultaneous Electrochemical Determination of Glutathione and Glutathione Disulfide at a Nanoscale Copper Hydroxide Composite Carbon Ionic Liquid Electrode, Analytical Chemistry. 2009. Vol. 81. Is. 18. P. 7538–7543.
  24. Raoof J., Ojani R., Karimi-Maleh H. Electrocatalytic oxidation of glutathione at carbon paste electrode modified with 2,7-bis (ferrocenyl ethyl) fluoren-9-one: application as a voltammetric sensor // Journal of Applied Electrochemistry. 2009. Vol. 39. Is. 8. P. 1169–1175.
  25. European Pharmacopoeia. 8th ed. – European Directorate for Quality of Medicines and Health care. Strasbourg, France; 2014. 2727 p.

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