[12] High-pressure liquid chromatography of thiamine, thiamine analogs, and their phosphate esters

This chapter describes the high-pressure liquid chromatography of thiamine, their analogs, and their phosphate esters. Of the three thiamine phosphate esters known to occur in biological material, only thiamine diphosphate (ThDP) and thiamine triphosphate (ThTP) appear to have biological activity, free thiamine and thiamine monophosphate (ThMP) being considered only as the hydrolysis products of the more phosphorylated forms of the vitamin. The simultaneous determination of thiamine, oxythiamine (OTh), and their phosphate esters in rat tissue is accomplished by taking advantage of the fact that thiamine compounds give the thiochrome reaction, whereas Oth and its phosphate esters do not form the fluorescent product. The separation of thiamine and its esters after conversion to thiochrome is achieved with conventional column chromatography. High-pressure liquid chromatography (HPLC) has been applied for thiamine determination, generally as a separation method for pharmaceutical application. The chapter describes HPLC methods for the separation of the phosphate esters of thiamine, Oth, or pyrithiamine and the separation of thiamine and Oth.