This note describes a determination method for Vitamin B1 (chemical name: thiamine) using HPLC coupled with post-column derivatization.
Vitamin B1 exists not only as free thiamine itself but also as its phosphate derivatives. In this note, to determine the total amount of vitamin B1, thiamine phosphate esters were converted to thiamine by utilizing enzymatic reaction. After purification procedures, the sample solutions were injected into the HPLC system.
Thiamine in injected solution is separated from other compounds in the ODS column, reacted with potassium ferricyanide under alkaline condition, and determined by fluorescence detector with high sensitivity and selectivity.
Sample pretreatment was performed with solid-phase extraction (SPE) instead of permutit (activated zeolite resin) method, which is established by Japanese Food Sanitation Inspection Guidelines. SPE is also described as reference method in the guidelines and offered easy way to remove impurities.