Optimization of the thin layer chromatography method conditions for the detection of related dioxidine impurities

Abstract

When developing methods for analysing active pharmaceutical substances (API), it is necessary to take into account modern requirements for the content of impurities in them. At the same time, for some drugs that have been on the pharmaceutical market for a long time, analysis methods are still used that are unable to provide the necessary sensitivity and specificity. One of these API is 2,3-bis(hydroxymethyl)quinoxaline-1,4-dioxide (dioxidin), which has high bactericidal activity against a wide range of microorganisms. According to the requirements of the State Pharmacopoeia of the Russian Federation XIV edition, related impurities of this API are analysed by thin layer chromatography (TLC). However, this method is semi-quantitative and relative. The purpose of this work is to determination of impurities in the dioxydine substance using high-performance liquid and thin-layer chromatography methods. In accordance with the requirements of the monograph of the State Pharmacopoeia of the Russian Federation, the total content of all impurities in the API is assessed and only the content of quinoxidine is determined separately, which is often absent from the substance. For the first time, three typical dioxidine related impurities were revealed using HPLC. Next, we selected the optimal conditions for their detection by TLC. Methanol/acetonitrile (75/25) and dichloromethane/acetonitrile (50/50) mixtures were the most suitable as a solvent for impurities and the TLC elution system, respectively. Under the selected conditions, satisfactory separation of the chromatographic zones of all four impurities and the dioxidine substance was observed. The corresponding zones were clearly visible at a substance concentration of 1%. However, reducing their concentration to the required 0.1% made it difficult to visually detect impurities. Thus, even when optimizing TLC conditions, it is impossible to obtain reliable data on the content of related impurities, which is due to the limitations of the method. On the contrary, the analysis of dioxidine substances by HPLC allows fairly accurate quantitative determination of individual related impurities.