A simplified approach to the determination of thiamine and riboflavin in meats using reverse phase HPLC

A simplified procedure for the determination of thiamine and riboflavin contents in meat products is proposed. The vitamins were extracted from the meats by digestion with 0.1 M hydrochloric acid followed by an enzymatic hydrolysis step in which phosphorylated forms of both vitamins were hydrolysed using an acid phosphatase enzyme of defined activity (0.5 U/mg) derived from wheat germ. Riboflavin in the filtered hydrolysates was determined directly by high performance liquid chromatography (HPLC) on a reverse phase (C18) column using a methanol:water (40:60) mobile phase and a fluorescence detector (excitation wavelength, 450 nm; emission wavelength, 510 nm). After conversion to thiochrome and partitioning into isobutanol, thiamine was determined on the same column by fluorescence (excitation wavelength, 366 nm; emission wavelength, 434 nm) using 80:20 methanol:water as the eluent. Measured levels of both vitamins were found to be very close to certified values provided with a lyophilized reference liver sample, while the contents determined for a range of meats were also in good agreement with published values.