manual of methods for determining micronutrients in fortified foods

B. Determination of thiamin in flours by high-performance liquid
chromatography
I. References
Rettenmaier R, Vuilleumier JP and Muller-Mullot W. Determination of Vitamin B1 in Complete
Feeds. In: Keller, H.E. Analytical Methods for Vitamins and Carotenoids in Feeds. Animal Nutrition
and Health Vitamins and Fine Chemicals Division, Roche. Switzerland. pp 23-26
II. Principle
Thiamin is extracted in an autoclave with diluted sulfuric acid. After enzyme hydrolysis, thiamin
is oxidized with potassium ferricyanide in sodium hydroxide to form the thiochrome, which is
fluorescent. The extract is injected into a HPLC onto a reverse phase column (C18) with fluorescence
detection: excitation at 370 nm and emmision at 430 nm.
Recovery of Thiamin in Infant Formula 1849 (NIST) was 95.8 % as measured in the CPHL Laboratory.
III. Critical points and precautions
Thiamin is labile to light and heat. In acid medium, below pH 5.5, thiamin is more heat-resistant,
whereas is unstable in alkaline and neutral medium. Samples and standard solutions must be protected
from light and a thin layer of liquid paraffin is added during autoclaving to protect from heat.
The oxidizing agent must be prepared freshly, right before the reaction is carried out. The reaction to
form the thiochrome must be done a few minutes before injecting in the HPLC to avoid underestimating
thiamine content due to losses caused by the basic pH of the oxidizing agent.
Prior to injecting standards and samples in the injection valve, the column must be stabilized with the
mobile phase. At the end of the run, rinse the column thoroughly with HPLC-water to eliminate all
salt residues from the mobile phase. Then, wash it with methanol. NEVER leave the mobile phase
in the column. Follow the washing protocol recommended for the column used.
IV. Equipment and materials
• Autoclave 121-123°C
• Agitator Vortex type
• Analytical balance (± 0.0001 g)
• Water bath (40°C)
• HPLC system
− Pump operating continuously at 1.0-2.0 mL/min with a flow precision of ± 1% or better
− Injector. A manual injector or auto sampling injector with a 100 μL fixed loop having a typical
sampling precision of ±0.25% or better
− Reverse-phase C18 column, 5µm (4.6x150 mm)
− Fluorescent detector: Excitation wavelength: 370 nm; emission wavelength: 430 nm.
• Volumetric flasks (25, 100 and 1000 mL)
• Beakers (25, 100 and 1000 mL)
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