Quality Focus: The Laboratory Notebook

Answering important questions about quality.

By Robert Green

This month we discuss some sampling issues, the difference between anthocyanins and anthocyanidins (which with spring in the air is particularly appropriate) and the perennial favorite Folin-Ciocalteau assay.

Q. I had a pre-purchase sample of soy tested (the specification was for 20% isoflavones) and it passed. Based on that I purchased 50 kilos of the material. When the material arrived I tested it again, and this time it failed. The manufacturer said I already approved the material and the second test was not binding. What should I do?

A. We limit this column to analytical, and not legal or business advice, so we cannot tell you what to do. We will, however, explore the analytical implications.

The question raises one of the most critical, and perhaps most overlooked, issues in analytical testing—sampling. We wrote the industry treatise on this several years ago which you can download from our website, so we will not repeat that material here. Nevertheless, the subject is important enough to briefly explore.

An analytical lab can only test the sample of material in its possession, so great care must be taken in insuring that sample is representative of the actual material in question. As in all industries, scoundrels exist who send a first rate sample for testing but then ship inferior material once a purchase order is issued. But even with the best of intentions the material can differ. Different batches of the same material made in the same plant using the same procedure can vary. These variances can be caused by varying raw materials, peculiarities in the manufacturing process (e.g., a delay in the production process or an item of equipment out of specification) or even the storage of the material post-production. That’s why lot numbers are assigned, so different batches can be identified.

You also must take great care to ensure the sample you send to the lab is representative of the material at issue. You mentioned you took delivery of 50 kilos of material. Scooping a bit of material from one spot will not provide a sample, which is representative of all 50 kilos. Procedures have been developed to insure an analysis of the sample you send accurately reflects the state of the entire shipment.

You should be commended for testing the delivered material to confirm (or in your case deny) that the material meets specifications. While no one likes to spend money on analytical testing, your situation shows that spending a bit now may save you from a financial and legal nightmare later. Analytical testing by qualified personnel is relatively inexpensive insurance.

Q. I received an analytical report on my cranberry product and am confused by the terms “anthocyanidins” and “anthocyanins.” Please explain the difference?

A. Let’s start at the top. Flavonoids are plant metabolites with various roles, including attracting pollinators. They are of great interest because they are antioxidants, inhibit cell proliferation and have demonstrated anti-inflammatory properties, among other things.

Anthocyanins are a subclass of flavonoids. They have sugar moieties attached to a carbon skeleton and modified electrochemical properties. These modified properties cause the variations of reds and blues in flowers and fruits. In fact, the word “anthocyanin” is said to be derived from two Greek words meaning plant and blue. These colors attract insects and birds, which in turn causes pollination and seed dispersal. Hundreds of anthocyanins have been identified.

The actual color created by an anthocyanin is dependant upon the pH environment. A typical anthocyanin is red in acid, violet in neutral and blue in basic environments. Therefore, a single type of anthocyanin can be responsible for different colors in different plants, all dependant on the pH.

Anthocyanidins, on the other hand, lack the attached sugar moieties. There are six common anthocyanidins: cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin.

Studies in humans indicate that anthocyanidins are the species that are actually active in the body at the site of action. However, they are insoluble in water and therefore cannot be easily absorbed by the body. Some therefore postulate that anthocyanins should be ingested and allow the body to convert these to anthocyanidins at the site of use.

Let’s go one step further. When many anthocyanidins are linked, the polymer is called a proanthocyanidin, or more technically, oligomeric proanthocyanidins. This is where the abbreviation “OPC” comes from.

Q. I received a certificate of analysis for grape seed material and the sole reported specification was “total phenols by Folin-Ciocalteau.” Please explain what this means and its sufficiency.

A. Folin-Ciocalteau is an assay used to measure total phenols (also called phenolics). It has been around for a long time; the first paper describing it was published in 1927 authored, not surprisingly, by Drs. Folin and Ciocalteau.

This is a colorimetric oxidation/reduction assay. Colorimetric means that the measurement is based on color and oxidation/reduction refers to the addition and removal of oxygen. As with most assays a standard must be used for comparison, and here it is gallic acid. Gallic acid is used because it is stable and available in pure form, and the response to it is generally equivalent to most other phenolics. The result of this assay is typically expressed as gallic acid equivalents (GAE).

In technical terms, the Folin-Ciocalteau reagent is added to the prepared liquid sample. The reagent oxidizes the phenolates and reduces the heteropoly acids, turning the sample blue. Using a spectrophotometer reading at 740 nm, the blue of the sample is compared to the blue of the standard, and the determination of GAE is made.

While the Folin-Ciocalteau method has its place, we believe it should not be relied on as the sole method of analysis in today’s supplement industry. In days of old there was a close connection between the manufacture and sale of materials. As such, there was little concern of product identity and adulteration. You knew the dried blueberry in your possession was in fact dried blueberry because you knew and trusted the provenance of the material. In today’s commercial world most of us have no clue as to the pedigree of the material at hand, so we must be ever-diligent.

The Folin-Ciocalteau method quantitates all phenolic groups (e.g., anthocyanins, quercetin, catechins, etc.) present without distinction. (Yes, that’s right, our friend anthocyanin is a phenol sub-group). You cannot be sure the “correct” phenols are present and the “incorrect” phenols are not. Thus, confirmation of identity is not possible and adulteration cannot be detected. In addition, using this assay you cannot differentiate the types of phenol present (is it a monomer, dimer or trimer). Interferences are also problematic. The presence of sugars, proteins, nucleic acids and ascorbic acid can alter the response factor.

Our preference is always to use the most specific method available. For phenols that’s HPLC. If we are assaying grape seed, let’s zero in on the anthocyanins, or catechins for green tea. A total phenol number by Folin-Ciocalteau may also be useful, but only in conjunction with an identity confirmation.NW