The assay of Vitamin B1 (thiamine) by fluorimetry involves measuring the fluorescence emitted by thiamine or its derivatives after a specific chemical reaction that enhances its fluorescence. Here's an overview of the steps involved in this assay:


1. Principle:

Vitamin B1 (thiamine) itself is not strongly fluorescent. However, when it is oxidized to thiochrome, a highly fluorescent compound, the fluorescence can be measured using a fluorimeter. The intensity of the fluorescence is directly proportional to the concentration of thiamine in the sample.

2. Preparation of Sample:

  • Extraction: Thiamine is extracted from the sample (biological fluids, food, etc.) by hydrolysis under acidic conditions.
  • Neutralization: After extraction, the sample is neutralized to prevent further degradation of thiamine.

3. Oxidation to Thiochrome:

  • Oxidation Reaction: Thiamine is treated with an oxidizing agent, typically alkaline potassium ferricyanide. This converts thiamine into its fluorescent derivative, thiochrome.
  • The reaction occurs in alkaline conditions (pH around 12-13).

4. Fluorescence Measurement:

  • The fluorescence of thiochrome is measured using a fluorimeter.
  • Excitation wavelength: Thiochrome is excited at around 365 nm.
  • Emission wavelength: The emitted fluorescence is measured at around 435-440 nm.

5. Quantification:

  • A calibration curve is generated using known concentrations of thiamine standards.
  • The fluorescence intensity of the sample is compared to the calibration curve to determine the concentration of Vitamin B1 in the sample.

6. Advantages of Fluorimetry:

  • Sensitivity: Fluorimetry is highly sensitive, making it suitable for detecting low concentrations of Vitamin B1.
  • Specificity: The assay is relatively specific to Vitamin B1, provided the extraction and oxidation steps are performed carefully.

7. Limitations:

  • The method requires careful handling during oxidation to avoid over-oxidation or degradation of thiamine.
  • Fluorescence measurements are sensitive to environmental factors like light and temperature, so proper control measures are necessary.

In conclusion, fluorimetry provides a sensitive and reliable method to assay Vitamin B1 by converting it into its fluorescent form, thiochrome, and measuring the fluorescence intensity.