Niacin

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Niacin.JPG
Figure 1. Niacin, C6H5NO2, MW=123,11 g/mol

Different forms of niacin are niacin (=nicotinic acid, figure 1) and niacinamide (=nicotinamide)

Synonyms for niacin:

  • nicotinic acid, vitamin B3, 3-pyridinecarboxylic acid, 3-carboxypyridine, anti-pellagra vitamin

 Summary



Golden standard

EN 15652:2009 Foodstuffs - Determination of niacin by HPLC

Method indicator

  • Name
  • Code

Scope

The European Standard EN 15652 specifies a method for the determination of the mass fraction of niacin in foodstuffs by high performance liquid chromatography (HPLC). Three alternative extraction phases are described: acid hydrolysis (A), enzymatic hydrolysis (B) and acid/alkaline hydrolysis (C).

The method has been validated in interlaboratory tests:

  • non-fortified and fortified samples ia. breakfast cereal powder, chocolate cereals, cooked ham, green peas, lyophilised green peas with ham, lyophilised soup, nutritive orange juice, milk powder and wheat flour
  • niacin concentration 0,5-24 mg/100 g

In options A and B, the niacin is calculated as a sum of nicotinic acid and nicotinamide, and these options A and B give similar results (expressed as nicotinic acid) in interlaboratory tests. Option C gives similar results for most of the tested samples, non-supplemented cereals being an only exception. In option C, nicotinamide is transformed to nicotinic acid, and the result is calculated and expressed as nicotinic acid.

Principle

Niacin vitamers are extracted from food by acid hydrolysis (A), enzymatic hydrolysis (B) or acid/alkaline hydrolysis (C) and quantified by HPLC. A post-column derivatisation with UV-irradiation followed by a fluorometric detection is used. In options A and B, niacin is calculated as a sum of nicotinic acid and nicotinamide, and it is expressed as nicotinic acid after correction of the molecular weights. In option C, nicotinamide is transformed to nicotinic acid during alkaline hydrolysis, and the result is determined and expressed as nicotinic acid.

Key steps

Extraction

  • Sample is homogenised before extraction.
  • Extraction by acid hydrolysis (option A)
    • Hydrochloric acid solution is added to the sample.
    • The sample solution is heated in a water bath (1 h)
    • pH is adjusted to 4,5.
    • The sample solution is filtered.
  • Extraction by enzymatic hydrolysis  (option B)
    • Sodium acetate solution and NADase solution are added to the sample.
    • The sample solution is incubated  (37 °C, 18 h)
    • The sample solution is filtered.
  • Extraction by acid/alkaline hydrolysis (option C)
    • Hydrochloric acid solution is added to the sample.
    • The sample solution is heated in a water bath (1 h)
    • pH is adjusted to 4,5.
    • The sample solution is filtered.
    • Sodium hydroxide solution is added.
    • The sample solution is autoclaved (120 °C, 1 h).
    • pH is adjusted to 4,5.
    • The sample solution is filtered.
Factors that affects the choice of the extraction method:
  • Option A is the fastest and the cheapest method.
  • If an exact amounts of nicotinic acid and nicotinamide are needed to quantify, option B should be used.  
    • In option A, some of nicotinamide is transformed to nicotinic acid during the acid hydrolysis.
    • Otherwise options A and B give similar results.
  • Option C quantifies the total niacin in foodstuffs.
    • Alkaline hydrolysis may liberate some other forms of niacin that are not normally biologically available.
    • May give higher results than options A and B.

HPLC

  • Liquid chromatographic system consisting of a pump, an injector, a fluorescence detector (excitation wavelength 322 nm and emission wavelength 380 nm) and a data evaluation system.
  • Reverse phase column: particle size 5 µm, diameter 4,0 mm, length 250 mm.
  • Mobile phase: phosphate buffer, hydrogen peroxide, copper sulfate
  • Also other chromatographic systems can be used if equivalent results are guaranteed.
  • The performance criteria is the baseline separation of niacin from interferences.
  • Post-column derivatisation tube and UV lamp
    • The photochemical reaction is carried out using polytetrafluoroethylene (PTFE) tube surrounding a UV black-light-blue (BLB) lamp.


Identification and detection

  • Niacin is detected fluorometrically (excitation wavelength 322 nm, emission wavelength 380 nm)
  • Identification of d-niacin 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 small amount of the appropriate standard solution to the sample test solution.

Quantification and calculations

  • Quantification is done by external standard method by comparing the integrated peak areas or peak heights to the corresponding values of the standard substance. Also calibration curve may be used for quantification.
    • The linearity of the calibration must be checked.
  • Calculations in options A and B:
    • The mass fraction of nicotinic acid and nicotinamide is calculated in mg/100 g of the sample. In options A and B, the result for niacin is reported as nicotinic acid in mg/100 g if the result consists of both nicotinic acid (C6H5NO2, MW=123,11 g/mol) and nicotinamide (C6H6N2O, MW=122,13 g/mol).
  • Calculations in option C:
    • The mass fraction of nicotinic acid is calculated in mg/100 g of the sample



 



Remarks

  • The purity of nicotinic acid and nicotinamide standard substances can vary, and therefore the concentration of the stock solution should be checked spectrophotometrically.
  • Nicotinic acid and nicotinamide are rather stable to atmospheric oxygen, light and heat.
  • Alkaline hydrolysis may liberate some forms of niacin that are not normally biologically available.
  • Niacin can also be measured microbiologically or colorimetrically.
    • toxic cyanogen bromide needed in colorimetric measurement.

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 niacin:

API - American Proficiency Institute

DLA - Dienstleistung Lebensmittel Analytik GbR: 33/2012 Food Supplement II: Biotin, Vitamin C, Niacin and Pantothenic Acid

Livsmedelsverket, National Food Agency of Sweden - Report 17 (2009) Vitamins in Foods - Round V-7

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 86-49.01 Niacin in Enrichment Concentrates
  • AACC 86-50.02 Niacin and Niacinamide in Cereal Products
  • AACC 86-51.01 Niacin---Microbiological Method
  • AOAC 944.13 Niacin and Niacinamide (Nicotinic Acid and Nicotinamide) in Vitamin Preparations
    • microbiological
  • AOAC 961.14 Niacin and Niacinamide in Drugs, Foods and Feeds
    • colorimetric
  • AOAC 968.32 Niacinamide in Multivitamin Preparations
    • spectrophotometric
  • AOAC 975.41 Niacin and Niacinamide in Cereal Products
    • automated
  • AOAC 981.16 Niacin and Niacinamide in Foods, Drugs and Feeds
  • AOAC 985.34 Niacin and Niacinamide (Nicotinic Acid and Nicotinamide) in Ready-To-Feed Milk-Based Infant Formula
    • microbiological, turbidimetric
       

 Literature

  •  see separate child page below

EuroFIR assistance to this method/guidelines