Vitamin B1 - Thiamine

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  Thiamine Cl HCl.jpg  

Figure 1. Thiamine chloride hydrochloride, C12H17ClN4OS∙HCl, MW=337,21g/mol


Different forms of thiamine includes thiamine chloride hydrochloride, thiamine nitrate, thiamine pyrophosphate, thiamine monophosphate chloride dihydrate.

Synonyms for vitamin B1:

  • thiamine, thiamin, aneurin, antiberiberi factor

Golden standard

EN 14122:2003 Foodstuffs Determination of vitamin B1 by HPLC

  • corrigendum EN14122:2003/AC (2005)
  • draft prEN 14122:2012 under discussion (March 2012)

Method indicator

  • Name
  • Code


The European Standard EN 14122:2003 specifies a method for the determination of vitamin B1 in foodstuffs by high performance liquid chromatography (HPLC) with enzymatic treatment and pre- or post-column derivatisation. Vitamin B1 is the mass fraction of total thiamine including the phosphorylated derivatives.

This method has been tested in two interlaboratory studies:

  • the first set of tested samples: whole meal flour, milk powder, freeze dried mixed vegetables and pig's liver
    • vitamin B1 concentration: 0,295-0,807 mg/100 g
  • the second set of tested samples: tube feeding solution, baby food with vegetables, meal with fruits, cereal, chocolate powder and food supplement
    • vitamin B1 concentration: 0,11-486 mg/100 g


The method includes acid hydrolysis and extraction of thiamin from food. Dephosphorylation using an enzymatic treatment is carried out after extraction and sample is quantified by HPLC with pre- or post-column derivatisation to thiochrome. The derivatisation to thiochrome enables the use of sensitive and selective fluorescence detection.

Key steps


  • The sample is homogenised before extraction.
  • Acid hydrolysis is carried out by dilute hydrochloric acid or sulphuric acid solution. Acid hydrolysis includes heat treatment.
    • heat treatment: autoclaving in 121 °C for 30 min or heating in 100 °C for 60 min
  • The pH of the sample solution should not be higher than pH=3,0

Enzyme treatment

  • Thiamine pyrophosphate or thiamine monophosphate is added to the test sample to check the enzyme activity .
  • Dephosphorylation can depend on the sample matrix and on the enzyme used. Taka diastase is used currently.
  • The pH of the sample is adjusted to 4,0 before addition of taka diastase.
    • Optimal pH, incubation time and incubation temperature should be checked for each enzyme used
  • When using taka diastase, it should be taken into consideration while calculating the results, that taka diastase may contain thiamin.

HPLC and pre- or post-column oxidation

  • Liquid chromatographic system consisting of a pump, a sample injection device, a fluorescence detector (excitation wavelength e.g. 366 nm, emission wavelength e.g. 435 nm) and a data evaluation system.
  • Pre-column oxidation
    • Some substances (e.g. polyphenols) present in samples can inhibit the oxidative conversion of thiamin to thiochrome. The recovery of the method should be checked. 
    • Stationary phase: reverse phase, particle size 5 µm, diameter 4,0-4,6 mm, length 100-250 mm
    • Mobile phase: methanol and acetate buffer
    • Fluorometric excitation 366 nm and emission 435 nm
  • Post-column oxidation
    • Stationary phase: reverse phase, particle size 5 µm, diameter 4,0-4,6 mm, length 100-250 mm
    • Mobile phase: methanol and phosphate buffer containing tetraethlyammoniumchloride and sodium heptanesulfonate
    • Fluorometric excitation 368 nm and emission 440 nm
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of thiamine from interferences.

Identification and detection

  • After thiamine is oxidised to thiochrome using pre- or post-column oxidation, it can be detected fluorometrically.
  • Identification of thiamine is done by the comparison of the retention time obtained with the standard test solution to that of the sample test solution.
    • Identification can also be done by adding a small amount of the appropriate standard solution to the sample test solution.
  • With some sample matrices two peaks may be seen when using post-column derivatisation; thiamine and its metabolite 2-(1-hydroxyethyl)thiamine (HET).
  • Quantitative determination based on post-column derivatisation is recommended to include the separate determination of thiamine and HET.

Quantification and calculations

  • The external calibration is based on a calibration curve of thiamine chloride hydrochloride.
  • Perform the determination using an external calibration based on the integration of peak areas (preferable) or determination of peak heights (optional), and compare the corresponding values for the thiochrome by the use of a calibration graph.
  • Linearity of the calibration must be checked.
  • The mass fraction of vitamin B1 expressed as thiamine chloride hydrochloride in mg/100g of the sample is calculated. The results are reported as thiamine chloride hydrochloride (Mw=337,28g/mol) in mg/100g if the results are expressed as thiamine (C12H17N4OS, Mw=265,37 g/mol) or thiamine chloride (C12H17ClN4OS, Mw=300,82 g/mol)


  • Thiamine is sensitive to heat and alkaline conditions. Appropriate precautions, like amber glassware, must be undertaken during its analysis.
  • Thiamine is present in foods combined with phosphate and must be hydrolysed and treated with phosphatase before analysis.
  • The purity of thiamine standard substance can vary and therefore the concentration of the stock solution should be checked spectrophotometrically.
  • Since also different vitamers (thiamine hydrochloride, thiamine mononitrate, thiamine monophosphate chloride and thiamine pyrophosphate chloride) can be used in the manufacture of food supplements according to Comission Regulation EC 1170/2009, the mass fraction of vitamin B1 is expressed as thiamine chloride hydrochloride (C12H17ClN4OS•HCl).
  • Alternative HPLC conditions are described
  • Old Datain food composition databases may be derived from
    • microbiological assay
    • fluorometry
    • HPLC
  • Three methods give similar results (Deharveng, 1999)
  • Latest review
    • Blake, C.J.,(2007). Analytical procedures for water-soluble vitamins in foods and dietary supplements:a review. Analytical Bioanalytical Chemistry,389, 63-76
    • Chen P, Wolf WR. 2007LC/UV/MS-MRM for the simultaneous determination of water-soluble vitamins in multi-vitamin dietary supplements. Anal Bioanal Chem387(7):2441-8

Criteria for analytical performance and analytical quality control

Certified Reference Materials/Standard Reference Material


Proficiency testing schemes 

Here are listed some completed, on-going and/or upcoming proficiency testing schemes concerning vitamin B1.

DRRR - Deutsches Referenzbüro für Lebensmittel-Ringversuche und Referenzmaterialien

API - American Proficiency Institute


PTA - Proficiency Testing Australia: Report No. 651 Foor Proficiency Testing Program (Round 31) - Vitamins

 Some upcoming proficiency testing schemes can be found in the EPTIS database.

Other methods available

  • AACC 86-80.01 Thiamine---Thiochrome Method
  • AOAC 938.12 Thiamine hydrochloride (Vitamin B1) in Vitamin Preparations
    • Growth bioassay
  • AOAC 942.23Thiamine (Vitamin B1) in Human and Pet Foods
    • Fluorometric
  • AOAC 953.17 Thiamine (Vitamin B1) in Grain Products
    •  Fluorometric
  • AOAC 957.17 Thiamine (Vitamin B1) in Bread
    • Fluorometric 
  • AOAC 986.27 Thiamine (Vitamin B1) in Milk-Based Infant Formula
    •  Fluorometric


- see separate child page below 

EuroFIR assistance to this method/guidelines