Analysis of Thiamin by Reverse Phase C18 Open Column Chromatography

Abstract

A modification of the AOAC method for thiamin determination utilizing reversed phase (RP) C18 packing material was developed for analysis of milk, infant formula and breakfast cereal products. Thiamin was extracted from food samples and hydrolyzed following a similar Association of official Analytical Chemists (AOAC) (1984) procedure. The sample filtrate was purified by passing through the reversed phase C18 column. An isocratic mobile phase consisting of 3% KCl and methanol (70 : 30) was used to elute thiamin from the reversed phase C18 column. The sample eluate was oxidized to thiochrome and quantified on a fluorometer using similar AOAC procedure. The experimental technique was compared to the existing AOAC (1984) method, which employs a Bio-Rex 70 resin in the purification step of thiamin analysis. There was no significant difference in the thiamin content of the samples determined by the two methods. However, there was significant difference in the recovery of thiamin, when taken through the method or added to food samples before extraction. The recoveries of the added thiamin were 97.97 ± 0.69% and 92.67 ± 1.25% for RP C18 and AOAC (1984), respectively, the coefficient of variation was 0.70% and 1.34% for RP c18 and AOAC (1984) methods respectively. (1984.), respectively, the coefficient of variation was 0.70% and 1.34% for RP C18 and AOAC (1984) methods respectively. The RP C18 method was found to be better than the AOAC (1984) method in terms of accuracy, precision, and reproduciblity. And the RP C18 proved to be faster than the AOAC ((1984) method. There was no difference in the cost of the two methods. Analysis for thiamin in the two infant formulas used in this study, gave values that were 132.37% and 202.90% of the declared levels, while thiamin content of breakfast cereal was 210.33% of the declared level.