Maria Ofitserova, PhD, and Sareeta Nerkar, PhD, Pickering Laboratories Inc.
Figure 1: Chromatogram of 1 μg/mL calibration standard of Thiamine and its phosphorylated derivatives.
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Thiamine (vitamin B1) plays an important role in many cellular processes and its deficiency can quickly lead to serious health
problems. Since humans and animals can't synthesize vitamin B1, they must obtain a sufficient amount through their diet. The
requirements of nutritional labeling have led to increased demand for methods to analyze vitamin B1 in different matrices.
Figure 2: Chromatogram of Vitamin B complex supplement.
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This application note describes a sensitive and accurate HPLC method capable of measuring Thiamine and its biologically active
phosphorylated derivatives in foods and dietary supplements. Thiamine and its derivatives are separated on reversed phase
column and converted using post-column derivatization into highly fluorescent compounds. To determine total vitamin B1 content
in foods, an enzymatic reaction with Taka-diastase was employed to convert all Thiamine esters to free Thiamine.
Method
Sample Preparation
Figure 3: Chromatogram of cereal sample.
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Food samples:
To 5 g of sample add 60 mL of 0.1 N HCl, blend at high speed for 2–3 min and heat the mixture at 100 °C for 1 h. Cool the
mixture to room temperature and adjust pH to 4.0–4.5 using 2.5 M Sodium Acetate solution. Add 200 mg of Taka-diastase, shake
well and incubate for 18 h at 45 °C. After enzymatic hydrolysis is complete add 2 mL of 50% Trichloroacetic acid solution
in water and heat at 100 °C for 15 min to precipitate proteins. Adjust pH to 2.6–2.8 with Sodium Acetate and bring the volume
to 100 mL with DI water. Filter through 0.45 μm filter.
Table I: Thiamine analysis in foods
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Dietary supplements:
Mix the contents of at least 10 capsules. Take a 250 mg portion and dissolve in 100 mL of DI water acidified to pH 2.6-2.8
with 0.1 N HCl. Dilute the solution further with acidified water to fit the calibration curve as needed. Filter through 0.45
μm filter
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