The Laboratory Notebook

Answeringimportant questions about quality.

By Robert Green

This month we tackle interesting questions on gas chromatography (GC), what to do if a third party fails your product and the Folin-Ciocalteu analytical method.

Q. I usually see analyses of nutritional supplements conducted by HPLC, but recently I saw a product analysis for fatty acids determined by GC, which I am not familiar with. Can you explain what GC is, and which is better?
A. GC stands for gas chromatography, a well established technique that can be used to separate volatile organic compounds. Quite simply, GC involves a sample being vaporized with the resulting gas analyzed chromatographically (hence the term “gas chromatography”). Chromatography is simply the process by which the component(s) of interest are separated first and then analyzed.

A gas chromatograph consists of an injection port, a flowing mobile phase, a separation column containing the stationary phase and a detector. The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.

First the sample to be analyzed (dissolved in an appropriate solvent if it is not initially in a liquid state) is drawn into a syringe needle and injected into the injection port of the GC. The injection port is maintained at a higher temperature than the boiling point of the least volatile component of the sample mixture. The volatized sample is then transported by the mobile phase, which is an inert gas such as helium, argon or nitrogen.Since the partitioning behavior is dependent on temperature, the separation column is usually contained in a thermostat-controlled oven. Separating components with a wide range of boiling points is accomplished by starting at a low oven temperature and increasing the temperature over time to elute the high-boiling point components.

Once separated, the analyte of interest is identified and measured by a detector. There are many detectors, which can be used in gas chromatography, each with a different type of selectivity. A non-selective detector re­sponds to all compounds except the carrier gas, a selective detector re­sponds to a range of compounds with a common physical or chemical property, and a specific detector responds to a single chemical compound.

Gas chromatographic detectors include: flame ionization detector (FID) (the most common), thermal conductivity detector (TCD), electron capture detector (ECD), photo ionization detector (PID) and flame photometric detector (FPD). All of these produce an electrical signal that varies with the amount of analyte exiting the chromatographic column. Another GC detector that is also very powerful (and very expensive) is a mass spectrometer.

As stated previously, GC can only be used for the analysis of volatile organic compounds, since the analyte must be vaporized into a gas for the method to work. In the supplement industry a GC analysis is used for phytosterols (such as those in nettle root), some singular compounds (such as MSM) and for fatty acids following suitable derivitization to increase their volatility. Compounds that cannot be vaporized, such as metals/minerals and most botanical analytes, cannot be analyzed by this method.

Liquid chromatography, as in HPLC, entails the separation of components, while they are in a liquid state. Since most components of interest in the supplement industry can be put into a liquid state but cannot be vaporized into a gas, HPLC is the dominant method of analysis with GC used for a distinct group of products.

Getting back to your specific question, fatty acids have traditionally been analyzed by GC following derivitization. However, recently HPLC techniques have been developed which are also extremely sensitive and accurate. Either method is ac­ceptable and would provide comparable results for the identical material, with the determining factor being the qualifications and quality of the lab conducting the analysis.

Q. One of my finished products was tested by an “independent third party” and the results were not good. I believe the product met all specifications. What should I do now?
A. In the last few years we have been asked numerous times to review these situations, sometimes with surprising results. While these testers (which include industry organizations, the news media and private enterprises) have appointed themselves as watch dogs over the supplement industry, there are no requirements or oversight on their operations so there is no assurance their findings are accurate. Unfortunately, some lack the qualifications for the role they are playing. Many times the analytical work is sub-contracted to the lowest bidder.

The first thing to do is confirm the same lot of your product that was tested does indeed meet specifications. You do not want to create a fuss if you are wrong, since that will only make matters worse. (And if your product is below grade then shame on you; take your medicine and don’t let it happen again.) Also confirm your product was tested before its expiration date; if not (which we have seen happen) you are home free.

The next step will require someone with expertise. Have them obtain and review the method of analysis AND the supporting data generated during the testing. This should also include a review of the equipment and standards used. Your goal is to uncover any facts, which cast doubt on the accuracy of the analysis.

In America we subscribe to the proposition that you are innocent until proven guilty, and this principle should apply to the supplement industry as well. You should insist that all relevant material be disclosed before publication of the negative finding. In one instance where the lab refused to disclose the analytical method used (shame, shame) the tester voluntarily withdrew the analysis. While you may need to assert your rights, so long as your products meet label claims, you should be able to vindicate yourself.

Q. I see that some labels of grape seed extract products are posting levels of total phenolics using the Folin-Ciocalteu method. Can you explain what that method is?
A. Folin-Ciocalteu is a colorimetric method, that is, upon the completion of the method a color appears and the intensity of the color provides the analytical result. More specifically, this method uses gallic acid, one polyphenol, as a reference standard. Gallic acid contains two hydroxyl groups, which react with a specific reagent (not surprisingly called the Folin-Ciocalteu reagent) by turning blue. The intensity of the blue color can be measured by a color meter. The final step is to compare the blue intensity from the sample under analysis to a colorimetric index of a known quantity of gallic acid and viola—you have the amount of total polyphenols in the product….or do you?

This method assumes that all polyphenols contain two hydroxyl groups. The problem is that this assumption is not true. Many polyphenolic constituents contain varying numbers of hydroxyl groups, but only those with two groups will be counted. Furthermore, colorimetric methods are non-specific, meaning they count anything that reacts with the reagent by turning the desired color (in this case blue), and not just the desired constituents. For example, proteins, nucleic acids and ascorbic acid turn the same blue as does polyphenols, so with this method they would be included in the calculation of total polyphenols. We have found that this method generally overstates the actual polyphenol content in the product, which is why many manufacturers may like to use it.

Remember from the earlier discussion of GC, modern chromatographic methods (GC, HPLC) first entail the separation of the component(s) of interest and then its independent measurement. Colorimetric methods are non-specific, meaning the component(s) of interest are not separated first and then analyzed. This creates the opportunity for other components present in the product to similarly react with the item of interest, thereby either increasing or decreasing the analytical result.

We are routinely asked to use the Folin-Ciocalteu method, which we gladly do since, despite its shortcomings, it is a legitimate analytical method and we always note its use on the product analysis. However, modern technology has given us the ability to first separate and then measure the exact components we desire, so our preference is to use these more refined methods.NW