Confirmation of the order of elution of the products of the interaction of gentamicin with ortho-phthalic aldehyde by the rate of the derivatisation reaction
Abstract
The aim of this study was the confirmation of the order of elution of the products of interaction of the components of gentamicin with ortho-phthalic aldehyde (OPA) according to the rate of the derivatisation reaction in the presence of DMSO, as well as to assess the effect of the content of DMSO in the mixture on the reaction rate.
Gentamicin is a broad-spectrum antibiotic widely used in medicine. The structural components of gentamicin differ in the number and position of methyl radicals substituting the hydrogen atoms of one of the amino groups.
Chromatographic analysis was carried out on a Prominence LC-20 liquid chromatograph with an automatic dispenser and a diode-matrix detector and a pH meter with a combined glass electrode.
The separation of the products of interaction of the components of gentamicin with OPA in a reversed-phase system was optimized using a mixture of acetonitrile - water - perchloric acid - ammonia as a mobile phase. When optimizing the conditions for the separation of derivatives of gentamicin and OPA, it was shown that the separation of peaks improved when the pH of the salt component of the mobile phase changed from 2.5 to 6.1. In addition, a gradient profile that provided the minimum analysis duration with the complete separation of the components was selected.
The effect of the DMSO content on the rate of derivatisation and the stability of the reaction products was studied. It was shown that the content of organic solvent in the range of 20-30 vol.% provides the maximum stability of the resulting products.
It was found that the rate of the derivatisation reaction is maximum for the first product eluted, minimum for the last product, and has an intermediate value for components with intermediate retention times. The reaction rate depends on the number of methyl substituents of the amino group and their position, which has a steric effect on the interaction of the components with the reagent. The hydrophobicity of the separated products also depends on these factors, which explains the correlation of the order of elution with the rate of the derivatisation reaction.
The obtained patterns can be used in the development of methods for the analysis of organic substances and their mixtures by HPLC with precolumn derivatisation.
Downloads
References
European Pharmacopoeia. 8th edition (EP 8.0), 2013, Vol. 2, pp. 2326-2328.
The United States Pharmacopoeia. The National Formulary (USP36 – NF31), 2013, Vol. 2, pp. 3720-3721.
European Pharmacopoeia Reference Standard. Information Leaflet. Gentamicin for Peak Identification Crs batch 3. 2017, 3 p.
Moldoveanu S., David V., J. Chroma-tography Library, 2002, Vol. 65, pp. 341-401.
Abdulsalam I. Al-Amoud, Brian J. Clark, Henry Chrystyn, J. Chromatogr. B, 2002, Vol. 769, pp. 89-95.
Cabanes A., Cajal Y., Haro I., Garcia Anton J.M. et al., J. Liq. Chromatogr., 1991, Vol. 14, No 10, pp. 1989-2010.
Walker S.E., Coates P-E., J. Chroma-togr., 1981, Vol. 223, pp. 131-138.
Larsen N-E., Marinelli E., J. Chroma-togr. B, 1980, Vol. 221, pp. 182-187.
Win L. Chiou Roger L. Nation Geoffrey W. Peng Shiew-Mei Huang, Clin. Chem, 1978, Vol. 24, No 10, pp. 1846-1847.
GOST 31789-2012. Fish, marine in-vertebrates and products of their processing. Quantitative determination of biogenic amines by high-performance chromatog-raphy, M., Standardinform, 2014, 11 p.
Gyarmati B., Hegyesi N., Pukánszky B., Szilágyi A., Express Polymer Letters, 2015, Vol. 9, No 2, pp 154-164.
Barends D. M., Zwaan C. L., Hulshoff A., J. Chromatogr. B, 1981, Vol. 222, pp. 316-323.
Golubitsky G.B., Vladimirova E.V., Ostrovsky K.P., Gelperina S.E., Sorptsionnye I khromatograficheskie protsessy, 2017, Vol. 17, No 5, pp. 772-780.
Golubitsky G.B., Ostrovsky K.P., Gelperina S.E., Sorptsionnye I khromato-graficheskie protsessy, 2018, Vol. 18, No 4, pp. 543-553.
