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Working Group on Anthocyanins

Chair, Dana Krueger, KruŜger Food Laboratories, IncInc. 

What Are Anthocyanin Pigments?Secondary Metabolites of many plants, particularly in fruits and flowers

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What Are Anthocyanin Pigments?Responsible for the red to purple color of many plants

Anthocyanin Pigments

Flavanoid Phenolic Compounds with a particular oxonium ion ring structure

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Anthocyanidins (Anthocyanin Aglycones)

Anthocyanidin R1 R2

Pelargonidin H H

Cyanidin OH H

Delphinidin OH OH

Peonidin OCH3 H

R3, R4=OH

Petunidin OCH3 OH

Malvidin OCH3 OCH3

Anthocyanins Structures

• R3 Substitutions:

– Simple Sugars (Glucoside, Arabinoside, Galactoside)

– Oligosaccharides (Rutinoside, Sambubioside, Sophoroside, Glucosylrutinoside)

– Glycoside Acylation – Coumarate, Acetate, Oxalate

• R4 Substitutions:

– OH

– Glucoside

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Why Do We Care?

• Anthocyanins are:• Anthocyanins are:

– Dietary Antioxidants

– Important Quality Assurance Parameters for Fruit Juices, Wines

– An Identity and Authenticity Index for fruit juices d h h i i i f dand other anthocyanin containing foods

– Natural Color Additives which need Methods for Detection and Control 

Challenges of Anthocyanin Analysis

• Multiple Analytes – > 500 anthocyanin knownMultiple Analytes > 500 anthocyanin known pigments, >50 in common foods

• Lack of Standards – Many compounds are not available as commercial standards.  Those that are available are expensive and of uncertain purity

• Stability of pigments particularly for quantitative• Stability of pigments, particularly for quantitative analysis

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Approaches to Anthocyanin Analysis

• Proximate Methods based on Color Intensity– Rapid, but of uncertain accuracy in many cases– Only measures anthocyanins as a class

• Qualitative HPLC Profile Methods– Widely used for authenticity and identity studies

• Quantitative Methods– HPLC with external etandards– Normalization of HPLC peak area to Proximate analysis total anthocyanin content

Current AOAC Methods

• AOAC 967.17 ‐ Paper Chromatography

– Qualitative profile method

– Old technology, largely obsolete

• AOAC 2005.02 ‐ Spectrophotometry, pH DifferentialDifferential

– Quantitative proximate method

– Fast and convenient, but not useful for identity testing

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Other Official or Validated Methods

IFU M th d 71 (HPLC UV t it il / t /f i id l t)• IFU Method 71 (HPLC‐UV, acetonitrile/water/formic acid solvent)

• USP 37/ NF 32 Bilberry Extract Method (HPLC‐UV, acetonitrile/methanol/water/formic solvent)

• USP Draft Pomegranate Method? (HPLC‐UV, methanol/water/formic acid solvent)

• European Pharmacopoeia (Essentially the same as AOAC 2005 02)European Pharmacopoeia (Essentially the same as AOAC 2005.02)

• 7 CFR §52.2462 (1993) – USDA spectrophotometric method for color in red grape juice

• IFU Method 80 ‐ spectrophotometric method for color in clear and hazy juices

Variations of HPLC Anthocyanin Methods

Primary Solvents: Acetonitrile Methanol mixturesPrimary Solvents:  Acetonitrile, Methanol, mixtures

Solvent Acidulants:  Formic Acid, Acetic Acid, Phosphoric Acid, Perchloric Acid, Trifluroacetic Acid

Other Solvent Modifiers: Buffers (Acetate, Phosphate), Ion Pair Reagents, THF as selectivity modifier

Columns: RP18 Columns, UHPLC, Mixed Mode Columns

Detectors:  UV‐Vis Absorbance, Photodiode Array, Mass Spectrometer

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QUESTIONS