EN 15652:2009
(Main)Foodstuffs - Determination of niacin by HPLC
Foodstuffs - Determination of niacin by HPLC
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.
Lebensmittel - Bestimmung von Niacin mit HPLC
Diese Europäische Norm legt ein Verfahren für die Bestimmung des Massenanteils an Niacin in Lebensmitteln mit Hochleistungsflüssigchromatographie (HPLC) nach drei verschiedenen Hydrolyse¬möglichkeiten fest. Säurehydrolyse (A), enzymatische Hydrolyse (B) oder saure/alkalische Hydrolyse (C).
Das Verfahren wurde in einem Ringversuch an angereicherten und nicht angereicherten Proben wie pulverisierten Frühstückscerealien, Frühstückscerealien mit Schokolade, Kochschinken, grünen Erbsen, grünen Erbsen mit Schinken (lyophilisiert), Suppe (lyophilisiert), an angereichertem Orangensaft, Milchpulver und Weizenmehl validiert, die 0,5 mg/100 g bis 24 mg/100 g an Niacin enthielten. Weitere Informationen, siehe Anhang B.
A und B ergeben ähnliche Ergebnisse für Niacin. Bei den Optionen A und B wird Niacin als die Summe von Nicotinamid und Nicotinsäure berechnet und als Nicotinsäure angegeben [1]. Option C ergibt bei Niacin in nicht angereicherten Cerealien höhere Ergebnisse als A und B, aber bei anderen Lebensmitteln ähnliche Ergebnisse. Bei der Option C wird Nicotinamid in Nicotinsäure umgewandelt und das Niacin als Nicotinsäure berechnet und ausgedrückt [2].
Option A ist schneller und preiswerter als B und C.
Option B wird verwendet, wenn eine genaue Quantifikation von Nicotinamid und Nicotinsäure erforderlich ist. Hierfür ist die Option A nicht geeignet, da während der Säurehydrolyse Nicotinamid in geringem Maße in Nicotinsäure umgewandelt wird.
Bei der Option C wird der Gesamtgehalt an Niacin bestimmt. Die alkalische Hydrolyse kann andere Derivate freisetzen, was zu höheren Befunden von Niacin führt, was in einigen Lebensmitteln wie Mais oder Getreiden üblicherweise nicht biologisch aktiv ist, siehe [3], [4] und [5].
Ein Vergleich der drei verschiedenen Hydrolysewege ist in Anhang C wiedergegeben.
Produits alimentaires - Dosage de la niacine par CLHP
La présente Norme européenne spécifie une méthode de dosage de la fraction massique de niacine dans les produits alimentaires par chromatographie liquide à haute performance (CLHP) suivant trois types différents d'hydrolyse : l'hydrolyse acide (A), l'hydrolyse enzymatique (B) ou l'hydrolyse acide/alcaline (C).
La méthode a été validée par des essais interlaboratoires sur des échantillons enrichis et non enrichis tels que poudre de céréales pour petit déjeuner, céréales au chocolat, jambon cuit, petits pois, petits pois au jambon lyophilisés, soupe lyophilisée, jus d'orange, lait en poudre et farine de blé, à des concentrations allant de 0,5 mg/100 g à 24 mg/100 g. Pour de plus amples informations sur les données de validation, voir l'Annexe B.
A et B donnent des résultats similaires pour la niacine. Dans les options A et B, la teneur en niacine est calculée en tant que somme du nicotinamide et de l'acide nicotinique et elle est exprimée sous forme d'acide nicotinique [1]. L'option C donne des résultats plus élevés que A et B pour la niacine avec des céréales sans ajout de vitamines, mais elle fournit des résultats similaires pour les autres produits. Dans l'option C, la teneur en niacine est calculée et exprimée sous forme d'acide nicotinique après transformation du nicotinamide en acide nicotinique [2].
L'option A est plus rapide et moins onéreuse que B et C.
On utilise l'option B si l'on a besoin d'une quantification exacte du nicotinamide et de l'acide nicotinique. Cela n'est pas réalisable avec l'option A car une petite partie du nicotinamide est transformée en acide nicotinique durant l'hydrolyse acide.
L'option C donne une quantification de la niacine totale. L'hydrolyse alcaline peut libérer d'autres formes donnant des résultats plus élevés pour la niacine, qui ne sont pas normalement disponibles biologiquement dans certains aliments à base de maïs ou de céréales, voir [3], [4] et [5].
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Živila - Določevanje niacina s HPLC
General Information
Overview - EN 15652:2009 (Foodstuffs - Determination of niacin by HPLC)
EN 15652:2009 (CEN) specifies a validated HPLC method for determination of niacin (vitamin B3) in foodstuffs. The standard covers three extraction/hydrolysis options - acid hydrolysis (A), enzymatic hydrolysis (B) and acid/alkaline hydrolysis (C) - followed by HPLC separation with post‑column UV derivatization and fluorimetric detection. The method was validated by interlaboratory trials on a range of matrices (breakfast cereals, chocolate cereals, cooked ham, green peas, soups, orange juice, milk powder, wheat flour) at levels from 0.5 to 24 mg/100 g (see Annex B).
Key topics and technical requirements
- Three extraction routes
- Option A - acid hydrolysis (HCl, boiling 1 h): faster and cheaper; slight conversion of nicotinamide to nicotinic acid.
- Option B - enzymatic hydrolysis (NADase, 37 °C, ~18 h): used when separate quantification of nicotinamide and nicotinic acid is required.
- Option C - acid + alkaline hydrolysis (including autoclave step): measures total niacin (all forms converted to nicotinic acid), often higher for cereals due to liberation of bound, non‑bioavailable forms.
- Quantification
- Niacin is reported as nicotinic acid after molecular weight correction.
- Options A and B sum nicotinamide + nicotinic acid; Option C converts nicotinamide to nicotinic acid for total niacin.
- Chromatography & detection
- Reverse‑phase HPLC (recommended RP‑18 column, 250 × 4 mm, 5 µm) with mobile phase containing phosphate buffer + H2O2 + Cu2+.
- Post‑column UV irradiation (BLB lamp) with fluorescence detection (excitation 322 nm, emission 380 nm).
- Sample prep & validation
- Homogenization, filtration (0.45 µm) and matrix‑specific extraction steps are defined.
- Precision and comparison data are provided in Annexes B and C.
Applications - who uses EN 15652
- Food testing laboratories and quality control units verifying niacin content for:
- Nutritional labeling and compliance
- Fortification checks in cereals and processed foods
- Regulatory testing and official control
- Food manufacturers, cereal processors and nutrition researchers assessing:
- Total vs. bioavailable niacin
- Effectiveness of fortification processes
Related standards and references
- EN ISO 3696:1995 - water for analytical laboratory use (referenced for reagent grade water).
- Annex B - interlaboratory validation data; Annex C - comparison of hydrolysis options.
Keywords: EN 15652, niacin determination, HPLC, nicotinic acid, nicotinamide, acid hydrolysis, enzymatic hydrolysis, alkaline hydrolysis, post‑column derivatization, fluorimetric detection.
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Niacin mit HPLCProduits alimentaires - Dosage de la niacine par CLHPFoodstuffs - Determination of niacin by HPLC67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 15652:2009SIST EN 15652:2009en,fr,de01-september-2009SIST EN 15652:2009SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15652May 2009ICS 67.050 English VersionFoodstuffs - Determination of niacin by HPLCProduits alimentaires - Dosage de la niacine par CLHPLebensmittel - Bestimmung von Niacin mit HPLCThis European Standard was approved by CEN on 23 April 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15652:2009: ESIST EN 15652:2009
Typical chromatogram . 15Annex B (informative)
Precision data for acid-, enzymatic- and acid/alkaline hydrolysis . 16Annex C (informative)
Comparison between three different ways of hydrolysis . 19Bibliography . 21 SIST EN 15652:2009
-18 °C. 4.2.15 Hydrochloric acid solution (options A and C), c(HCl) = 0,1 mol/l
1 This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15652:2009
WARNING 1
— Harmful UV light could come out of the metal box containing the lamp. WARNING 2 — If bubble formation occurs in the tube due to overheating, the tube should be efficiently cooled by air circulation, for example by lifting the box.
2 LiChrospher® 60 RP-18 Select B endcapped and VL-120 BLB are examples of suitable products available commercially. This information is given for the convenience of users of ths European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15652:2009
Key 1 lamp tube 2 from column 3 to detector Figure 1 — Schematic representation and dimensions (mm) of the lamp, lamp housing (in upside down position) and placement of the lamp housing on bench (in operating position)
Dimensions in millimetres
Key 1 reflector 2 lamp tube Figure 2 — Cross section of the lamp housing (in upside down position) with tube lamp and dimensions SIST EN 15652:2009
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Frequently Asked Questions
EN 15652:2009 is a standard published by the European Committee for Standardization (CEN). Its full title is "Foodstuffs - Determination of niacin by HPLC". This standard covers: 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.
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.
EN 15652:2009 is classified under the following ICS (International Classification for Standards) categories: 67.050 - General methods of tests and analysis for food products. The ICS classification helps identify the subject area and facilitates finding related standards.
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The standard EN 15652:2009 focuses on the determination of niacin in foodstuffs through high-performance liquid chromatography (HPLC), employing three distinct methods of hydrolysis: acid hydrolysis (A), enzymatic hydrolysis (B), and acid/alkaline hydrolysis (C). This comprehensive scope encompasses a broad range of food products, including 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, with niacin levels measured from 0.5 mg/100 g to 24 mg/100 g. One of the significant strengths of this standard is its validation through interlaboratory tests that ensure reliability in various food matrices. Furthermore, the findings indicate that options A and B yield comparable results for niacin, both of which measure niacin as a sum of nicotinamide and nicotinic acid and express it as nicotinic acid. Option A stands out for its efficiency and cost-effectiveness in terms of time and resources, making it accessible for routine analysis. Option B is particularly useful for precise quantification needs as it allows for accurate differentiation between nicotinamide and nicotinic acid. This level of specificity is crucial for research and quality control in foodstuffs. In contrast, option C provides a broader quantification by encompassing total niacin, utilizing alkaline hydrolysis to release additional forms of niacin, though this may include amounts not typically bioavailable in certain cereals. The inclusion of comparative analysis in Annex C enhances the document's relevance, allowing users to make informed decisions about which hydrolysis method to employ based on their specific analytical requirements. This standard is essential for food quality assessment, nutritional labeling, and regulatory compliance in the food industry, thereby supporting public health initiatives through accurate dietary assessment.
EN 15652:2009 표준은 고성능 액체 크로마토그래피(HPLC)를 사용하여 식품에서 니아신을 측정하는 방법에 대한 구체적인 지침을 제공합니다. 이 표준은 산가수분해(A), 효소가수분해(B), 알칼리/산가수분해(C) 등 세 가지 방법을 통해 니아신의 농도를 결정하는 범위를 포함하고 있습니다. 이 표준의 강점 중 하나는 다양한 식품 샘플에 대한 유효성을 검증했다는 점입니다. 아침 시리얼 가루, 초콜릿 시리얼, 조리된 햄, 녹색 완두콩, 햄과 함께 동결건조된 녹색 완두콩, 동결건조된 수프, 영양 주스, 분유, 밀가루 등에 대한 검증 데이터가 제공되며, 이들 샘플은 0.5 mg/100 g에서 24 mg/100 g의 농도 범위에서 테스트되었습니다. 검증 데이터에 대한 추가 정보는 부록 B에서 확인할 수 있습니다. A 및 B 옵션은 니아신에 대한 유사한 결과를 나타내며, 이 두 가지 방법에서는 니코티나미드와 니코틴산의 총합으로 계산되어 니코틴산으로 표현됩니다. 반면 C 옵션은 비보충 시리얼에서 A 및 B보다 높은 니아신 값을 제공하지만, 다른 제품에서는 유사한 결과를 보여줍니다. C 옵션에서는 니코티나미드를 니코틴산으로 전환하여 총 니아신이 계산되고 표현됩니다. A 옵션은 B 및 C 보기보다 빠르고 저렴하여 자원 관리 효율성을 높이는 데 기여합니다. B 옵션은 니코티나미드와 니코틴산의 정확한 정량이 필요할 때 사용되는데, A 옵션에서는 산가수분해 과정에서 니코티나미드가 조금 변화하기 때문에 정확한 정량이 불가능합니다. C 옵션은 총 니아신을 정량화하며, 알칼리 가수분해는 비생물학적으로 이용 가능한 다른 형태를 방출할 수 있어, 옥수수와 시리얼과 같은 일부 식품에서 니아신 측정 시 결과가 높아질 수 있습니다. 세 가지 가수분해 방법의 비교 정보는 부록 C에서 제공되며, 식품 분석 분야에서 니아신 측정의 신뢰성과 정확성을 높이는 데 중요한 기준을 제공합니다. 이러한 측면에서 EN 15652:2009 표준은 식품 과학 및 품질 관리 분야에서 유용하고 필수적인 도구로 여겨진다.
標準EN 15652:2009は、食品中のナイアシンをHPLC（高性能液体クロマトグラフィー）によって測定する方法を定めた重要な文書です。この標準は、酸加水分解（A）、酵素加水分解（B）、および酸/アルカリ加水分解（C）の3つの異なる加水分解方法を使用しており、その幅広い適用範囲が魅力的です。 この標準の強みは、様々な食品サンプル、例えば朝食シリアルパウダーや調理ハム、冷凍グリーンエンドウなど、フォーティファイドおよびノンフォーティファイドの試料において、ナイアシンの精度ある測定を実現している点です。特に、この方法は0.5 mg/100 gから24 mg/100 gの濃度範囲で検証されており、実際の食品分析においても、適切な信頼性を提供しています。また、付録Bには、検証データに関する詳細情報が載せられており、実験室間のテスト結果を参照できることから、ユーザーにとって実用的です。 オプションAでは、ナイアシンの測定が迅速かつコスト効率良く行えるため、特にスピードが求められる分析において強力な選択肢です。一方、オプションBは、ナイアシンの成分であるニコチンアミドとニコチン酸の正確な定量が必要な場合に有効であるため、高い精度が求められる分析で活躍します。オプションCは、ナイアシンの全体量を定量化できる特徴があり、特定の食品の中で生物的に利用可能なナイアシンの測定にも適しています。 このように、EN 15652:2009は、食品中のナイアシンを測定する上で非常に関連性の高い標準であり、異なる加水分解方法の比較に関する情報も附属書Cに提供されています。これにより、分析者は目的に応じた方法を選択し、食品分析の信頼性を確保することができます。
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