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Grape seed extract is a heterogeneous mixture of gallic acid, monomers, dimers, trimers, tetramers, polymers and other oligomers. The basic building blocks are molecules of catechin, epicatechin, epicatechin gallate, gallic acid esters, glycosides and peptides. Due to the high degree of heterogeneity, several analytical techniques are required to characterize grape seed extract.

TLC (Thin Layer Chromatography)

What is measured:qualitative separation of phenolics, used to determine if the extract is grape based

Type of method:chromatographic, based on size and polarity of phenolics

Standard:gallic acid, catechin, epicatechin and a commercially available grape seed extract

Issues: qualitative only, can not distinguish between grape skin and grape seed

lack of a non-partisan "commercial" grape seed extract standard

Grape Seed Method Evaluation Committee has adopted this method "industry wide" to qualitatively assess if an extract is grape based

This procedure was proffered by ESA Laboratories and includes normal phase TLC separation of preacetylated powder extracts

In 1998 three independent laboratories (Alpha Chemical Laboratory, Industrial Laboratory, and PhytoChem) validated the TLC method by testing 14 grape seed extracts, bilberry, green tea, pycnogenol and cranberry extracts as well as 13 finished products. This is a qualitative test designed to identify grape seed extract and differentiate it from botanicals that may have similar components.

Procyanidolic Value and Porter Value

The Procyanidolic assay is also referred to as the Bate-Smith assay.

What is measured:phenolic dimers and larger, qualitative value that indicates relative presence
Type of method:spectrophotometric, based on acid hydrolysis and color formation
Standard:none
Issues:poor reproducibility (procyanidolic is the least reproducible)

larger molecules give higher values

Grape Seed Method Evaluation Committee recommends that these methods are not used for quantification of total phenols or OPC’s in grape seed extracts

The "Procyanidolic Index" (also called the Bates-Smith Assay) involves a testing method that measures the change in color when the product is mixed with certain chemicals; the greater the color in change, the higher the OPCs. It must be noted, however, that the Procyanidolic Index is a relative value that can measure well over 100. Unfortunately, a Procyanidolic Index of 95 was erroneously taken to mean 95% OPC by some and began appearing on the labels of finished products. . All current methods of analysis suggest that the actual OPC content of these products is much lower than 95%.

The Porter and Procyanidolic value assays are colorimetric tests based on acid hydrolysis. Dimer and larger molecules are converted to anthocyanidins by acid hydrolysis. A standard curve is not incorporated and thus results can only be reported as values, not as percentages. Another artifact of these two assays is that on a molar basis the values increase with molecular size, i.e. if a grape seed extract is comprised primarily of larger polymers, it will have higher Procyanidolic and Porter values than an extract comprised of the same molar concentration of dimers.

It is also difficult to obtain reproducible results with the Procyanidolic value assay, as the results are very dependent on the testing conditions and cyanidin (the anthocyanidin formed during the acid hydrolysis step) is relatively unstable. Dr. V.L. Singleton, a world-renowned authority on grape phenols commented on the Procyanidolic assay "values obtained are necessarily somewhat arbitrary". Three independent laboratories conducted a study on both the Porter and Procyanidolic value assays in early 1997 (Industrial Laboratories, Alpha Chemical and Biomedical Laboratories and Hauser) and found large lab-to-lab variation in the Procyanidolic assay. Some companies utilize a modified Bate-Smith assay to increase the reproducibility of results. The Porter assay contains ammonium iron (III) sulfate, which stabilizes the reaction better than in the Procyanidolic assay and renders the results less dependent on conditions.

Procyanidolic assay

proanthocyanidins (dimers & larger) + HCl/isopropanol + heat = cyanidin, Abs 550 nm

Porter assay

proanthocyanidins (dimers & larger) + NH₄Fe(SO₄)₂ · 12 H₂O + HCl/n-butanol + heat = cyanidin, Abs 550 nm

The chemical reaction to the right depicts both the Porter value and Procyanidolic Value assays.

Total phenols (Folin-Ciocalteau)

Total phenols (Folin-Ciocalteau)

What is measured:concentration of total phenols

Type of method:spectrophotometric, based on a colorimetric oxidation/reduction reaction

Standard:gallic acid

Issues:all phenolic groups are quantitated (e.g. quercetin, anthocyanins, green tea phenolicscan not tell if an extract is adulterated
can not differentiate types of phenol present (e.g. monomer vs. dimer vs. trimer)

the presence of proteins, nucleic acids and ascorbic acid could alter the response factor

Method has been used in the wine industry for over 30 years

Grape Seed Method Evaluation Committee has agreed to examine this method as possible "industry wide" method

This is a colorimetric oxidation/reduction assay that measures all phenolic molecules with no differentiation between gallic acid, monomers, dimers and larger phenolic compounds. A gallic acid standard curve is used and results are typically expressed as gallic acid equivalents (GAE). This method has been used in the wine industry for over 30 years. The first paper on this method was published in 1927 and in 1965 Singleton and Rossi improved the reproducibility of the assay. Swain and Goldstein considered this the method of choice for estimating total phenol content in complex plant products. This is a sensitive and quantitative method, independent of the degree of polymerization.

phenolics + alkaline + FC reagent + heat = blue colored product, Abs 755 nm

FC reagent is an oxidizing agent comprised of heteropolyphosphotungstate-molybdate. The blue colored product is a mixture of the 1-, 2-, 4-, and 6-electron reduction products in the tungstate series P₂W₁₈O₆₂^-7 to H₄P₂W₁₈O₆₂^-8 and the 2-, 4-, and 6-electron reduction products in the molybdate series H₂P₂Mo₁₈O₆₂^-6 to H₆P₂Mo₁₈O₆₂^-6.

Some work has been initiated to determine if monomers react differently than larger molecules in this assay. Preliminary results show that the standard F-C assay may be biased in favor of grape seed extracts with high monomer contents since monomers and OPCs in grape seed extracts have different response factors. (See Addendum A: Calculation of Monomer Bias Correction Factor.) Label claims based on these results will be relative to the response factor of the compounds being tested. There is no study that we are presently aware of that has made a definitive 1:1 correlation between the mass of OPCs (monomers, oligomers or polymers) and an equal quantity of gallic acid. The method can be used for standardization, but not direct quantitation. Work is continuing in this area.

This method has been cited as being the Association of Official Agricultural Chemists’ method for determining total polyphenol content in wines.

Vanillin assay^,

with methanol as the solvent

What is measured:flavan-3-ols

Type of method:spectrophotometric

Standard:catechin

Issues:color production is dependent on many factors

kinetics of the reaction are complicated

a pure sample is required

Grape Seed Method Evaluation Committee has agreed to move away from this method

with glacial acetic acid as the solvent

What is measured:flavan-3-ols

Type of method:spectrophotometric, color formation is directly related to the concentration offlavan-3-ol end groups

Standard:catechin

Issues:concentration of phenol affects the degree of polymerization a pure sample is required

Grape Seed Method Evaluation Committee has agreed to move away from this method

The vanillin assay has been utilized in the cereal industry since the 1950’s. Dr. Larry Butler (Purdue University) has done many comparative studies with sorghum. The kinetics of the reaction are different with the two solvents (methanol and glacial acetic acid), with methanol yielding the more complicated kinetics. With glacial acetic acid the reaction kinetics are less complex (i.e. monomers and polymers react similarly), the reaction only occurs at the end groups, and the colored product which is formed and measured is more stable. Obtaining reproducible results with the vanillin assay with methanol may be difficult. The person running the analysis must be well-trained and the results are dependent on such techniques as the angle the methanol is added to the vial.

The article by Butler et. al contains the statement "for convenience, catechin, a monomeric flavan-3-ol unit of condensed tannins, is often used to standardize the assay rather than purified condensed tannin, although this leads to a considerable overestimation of tannin content". Further in the article it is also stated that "methanol affects the reaction with flavan-3-ol monomers and their oligomeric and polymers quite differently, producing complex kinetic patterns that make standardization of tannin analysis with monomers such as catechin tenuous at best."

Reverse Phase HPLC (High Pressure Liquid Chromatography)

What is measured:relative % monomers, oligomers and polymers percent of gallic acid, catechin and epicatechin by weight
Type of method:chromatographic, based on polarity of phenolics

Standard:monomer standards are readily available - gallic acid, catechin, epicatechin, epicatechin gallate

Grape Seed Method Evaluation Committee has agreed to adopt this method "industry wide" to determine % monomers in grape seed extract

With this method the percent by weight of gallic acid and phenolic monomers such as catechin, epicatechin and epicatechin gallate can be determined by using readily available standards from such companies as Sigma/Aldrich. Unfortunately, except for one dimer, no large molecule phenolics are commercially available.

In past experiences grape seed manufacturers have found that there is no standard RP-HPLC procedure among independent laboratories and that a simple request for % monomers will yield different results as well as different compounds. If this analysis is to be performed by an independent laboratory the monomers of interest (i.e. those typically found in grape seed extract) must be stated: catechin, epicatechin, and epicatechin gallate, as well as gallic acid (a phenolic acid). Depending on the solvents used and the length of the run some of the monomer peaks may co-elute with other phenolics. Care needs to be given to ensure that peaks of interest are pure peaks (i.e. no co-elution) and that the larger phenolic molecules are flushed from the column prior to subsequent runs.

GPC (Gel Permeation Chromatography)

What is measured:concentration of total phenols

Type of method:chromatographic, based on size of phenolics

Standard:"a well characterized" reference material, attempts have been made to test catechin or

epicatechin but these two monomers have different absorptions at 280nm.

Issues:short life span of column

not everyone has the necessary instrumentation

use of a non-partisan standard

difficult to differentiate types of phenol present (e.g. monomer vs. dimer vs. trimer)

some have noted that larger molecules seem to "stick on the column"

Grape Seed Method Evaluation Committee has agreed to examine this method as possible "industry wide" method

The basic principle of gel permeation chromatography is that molecules are separated by size on a specific column and when they elute the intensity of absorbency is measured at a specific wavelength, 280nm for phenols. All of the molecules tend to elute in a hump and the peak area of the hump is directly proportional to the concentration of phenols in the product, relative to the "reference material". An internal standard is used to improve the accuracy of the results. Fuzzati et. al. identified three separate regions of the "hump" as 1) monomers, 2) monomers, dimers and their gallates and 3) trimers through heptemers and their gallates based on a "well characterized reference material" and mass spectroscopy.