The use of reversed-phase chromatography methods for the determination of di-tert-butylphenols in biological fluids
Abstract
The objects of the study were biologically active polyalkylphenols with di-tert-butyl radicals in positions "2" and "4" (2,4-di-tert-butylphenol (2,4-DTBP), 2,6-di-tert-butylphenol (2,6-DTBP) and 2,6-di-tert-butyl-4-methylphenol (2,6-DTB-4-MP) toxic to humans. TLC (a thin layer of CTX-1A silica gel modified with hydrocarbons with a chain length of C14-C15), conventional pressure liquid column chromatography (Silasorb C-18 sorbent column 150×10 mm) and HPLC (250×4.6 mm Discovery® C18 column with precolumn 20×4.0 mm) were used as analysis methods.
The features of the chromatographic activity of a selected class of polyalkylphenols in a thin layer and columns of reversed phases were studied using polar eluents, including various (5-100 vol. %) of the organic component, which could be acetone or acetonitrile. The main parameters of the chromatographic mobility of analytes were calculated as criteria for identifying di-tert-butylphenols using the group of considered methods. It was shown that the most acceptable conditions for the separation of considered compounds are provided as a result of the use of mobile phases water-acetonitrile (40:60) and buffer solution (pH 5.5) – acetonitrile (30:70) (TLC), water - acetonitrile (20:80 ) (column chromatography of ordinary pressure), acetate buffer solution (pH 5.5) - acetonitrile (30:70) (HPLC). The chromatographic mobility of the analysed compounds under the proposed conditions depends on the number and mutual arrangement of alkyl radicals in their molecules and increases with the increase of their hydrophilicity in the series 2,4-DTB-4-MP˂2,6-DTBP ˂2,4-DTBP.
Based on the complex of performed experiments, methods for determination of the investigated substances in liquid biomatrices (blood and plasma) have been developed. According to the main validation criteria, these methods correspond to the requirements existing in the practice of biological and chemical-toxicological analysis. Limiting minimum quantities of di-tert-butylphenols detected by the developed methods in 1 g of blood are (1.8-8.0) 10-6 g (for TLC) and (1.0-5.0) 10-6 g (for HPLC), in 1 g of plasma – (1.6-5.0) 10-6 g (for TLC) and (1.0-3.0) 10-6 g (for HPLC).
The methods developed based on the results of studies after the validation assessment can be used for chemical and toxicological analysis of di-tert-butyl derivatives of phenol for evidence of the facts of poisoning by these biologically active substances.
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