Simultaneous LC determination of tiazofurin,its acetyl and benzoyl esters and their active metabolite thiazole-4-carboxamide adenine dinucleotide in biological samples

A rapid and sensitive HPLC-RP method for simultaneous determination of tiazofurin, its 5'-O acetyl and benzoyl esters and their active metabolite thiazole-4-carboxamide adenine dinucleotide was developed and validated. The method allowed determination and quantification of nanomolar quantities of these substances in cell extracts of treated cells, and was also used in kinetic studies of cellular uptake of tiazofurin and its esters from the cultivation medium. Separation of the analyzed substances from unidentified peaks from both biological materials was achieved by gradient elution, thus reducing the possibility of interference. The mobile phase consisted of a 0.1 M sodium-hydrogen phosphate, pH 5.1 and methanol. Run time was 22 min, with 5 min equilibration time.