EN 15652:2009 – Foodstuffs – Determination of niacin by HPLC

1 Scope
This European Standard specifies a method for the determination of the mass fraction of niacin in foodstuffs by high performance liquid chromatography (HPLC) by three different ways of hydrolysis, acid hydrolysis (A), enzymatic hydrolysis (B) or acid/alkaline hydrolysis (C). The method has been validated in interlaboratory tests on fortified and non-fortified samples such as breakfast cereal powder, chocolate cereals, cooked ham, green peas, lyophilized green peas with ham, lyophilized soup, nutritive orange juice, milk powder and wheat flour, at levels from 0,5 mg/100 g to 24 mg/100 g.
For further information on the validation data, see Annex B. A and B give similar results for niacin. In options A and B niacin is calculated as the sum of nicotinamide and nicotinic acid, and expressed as nicotinic acid [1]. Option C gives higher results than A and B for niacin with non-supplemented cereals, but similar results for other products. In option C, niacin is calculated and expressed as nicotinic acid after transformation of nicotinamide into nicotinic acid [2].
Option A is faster and cheaper than B and C. Option B is used if an exact quantification of nicotinamide and nicotinic acid is needed. This cannot be done with option A, because there is a slight transformation of nicotinamide into nicotinic acid during the acid hydrolysis. Option C quantifies total niacin.
The alkaline hydrolysis is able to liberate other forms giving higher results for niacin, which in some foods such as maize and cereals are not normally biologically available, see [3], [4] and [5]. Information on a comparison between the three different ways of hydrolysis is given in Annex C.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies.
For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use – Specification and test methods (ISO 3696:1987)
3 Principle Niacin vitamers are extracted from food by an acid (option A), an enzymatic (option B) or an acid/alkaline (option C) treatment and quantified by HPLC with a fluorimetric detection after a post-column derivatization with UV irradiation, see [1] and [2].
For option A and option B, niacin is determined as the sum of nicotinamide and nicotinic acid. Niacin is expressed as nicotinic acid after correction of the molecular weights. For option C, niacin is determined and expressed as nicotinic acid.
The alkaline treatment transforms all nicotinamide into nicotinic acid.
4 Reagents
4.1 General During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 1 according to EN ISO 3696:1995.
EN 15652:2009 – Foodstuffs – Determination of niacin by HPLC