Robert Green04.01.02
The Laboratory Notebook
Answering important questions about quality.
By Robert Green
Once again our mailbag is full of great questions. This month’s topics are GMO testing, gravimetric analysis and determining the standardization rate of a raw material after it’s encapsulated with other ingredients.
Q. I have been selling a soy protein product for years and lately I am getting questions about whether it contains genetically modified organisms (GMOs). Can you explain this issue?
A. GMOs are plants that have had their DNA modified by scientists in the laboratory. “RoundUp Ready” soybeans, for example, are resistant to the RoundUp herbicide allowing farmers to spray while the plants are growing, and StarLink corn produces insecticidal proteins that kill the devastating corn borer. GMO food crops in production include corn (maize), soybeans, rapeseed (canola), potato, squash, papaya, chicory, melon, sugar beet, rice and tomato. The U.S. is by far the largest grower of GMO crops.
While everyone acknowledges this technology is remarkable, many fear it may be harmful to human health and the environment. Recent events tend to support some of these fears. You may recall that StarLink corn was found in taco shells and corn chips even though it is not approved for human consumption, resulting in widespread recalls. What’s worse, only yellow corn has been genetically modified yet StarLink was found in white corn, meaning that the genetic modification unintentionally passed to another species.
In response to the GMO controversy many countries require labeling of products containing GMOs. While there is no such labeling requirement in the U. S., consumer awareness is growing and increasingly food companies are anticipating this trend by instituting GMO testing.
So how do we detect GMOs? DNA engineered into a crop consists of a number of elements, which govern its functioning. These are typically a promoter sequence, engineered structural gene and terminator sequence. GMO identification is made by detecting at least one of these elements. There are a limitless number of engineered structural genes and most are kept confidential by the inventors, so the information required to identify them is not available. Fortunately, virtually all known genetic modifications use the same promoter and terminator sequences. By screening for both the promoter and terminator we are able to simultaneously detect multiple GMO varieties, including unknown ones.
While there are several methods to detect GMOs, the most sophisticated is a biotechnology technique called polymerase chain reaction (PCR). Very simply, the process starts with the extraction and purification of DNA material in the sample. The DNA is then “amplified” or replicated millions of times using PCR technology, enabling the detection of extremely low levels of the promoter and terminator sequences.
We are seeing growing interest in the GMO issue and expect it to accelerate in the future.
Q. A manufacturer provided me with a certificate of analysis stating that the saponin content of its Tribulus terrestris was 60% as determined by gravimetric analysis. My lab reported a saponin content of 45% as determined by HPLC. Can you explain the different results?
A. Gravimetric analysis is a method of quantitative analysis in which the constituent sought (in your case saponins) is selectively dissolved into a solvent that can be separated from the sample. The remaining solids are then dried and weighed. The amount of the particular constituent in the sample is determined from the difference between the original weight and the observed weight of the remaining solid.
As you can imagine, this method only works when you are certain only the desired constituent will be solvated. If not, you will be measuring the desired constituent plus other material and your result will be inflated. This is exactly why gravimetric analysis generally fails for botanicals.
Saponins are of sugar origin. In a gravimetric analysis it is impossible to separate the saponins from other sugar or sugar-containing species that are present in tribulus. So while someone may say they determined the amount of total saponins by gravimetric analysis, in fact they measured saponins and other sugar type compounds together, thereby inflating the saponin content.
HPLC methodology, on the other hand, first separates the saponins from all other present material (including all other sugar type compounds) and then measures the saponins of choice alone, thereby providing an accurate measurement of the desired material.
We have seen this same issue with boswellin (a gravimetric analysis will include the desired boswellic acids plus other present organic acids) and shilajit (where fulvic acid and other organic acids will be aggregated).
With the advent of HPLC technology and its application to botanical analysis, we do not believe gravimetric analysis is suitable for most botanicals and we would not assess material based on one.
Q. I have a mixed botanical finished product and would like to confirm the specification of one of the standardized botanicals. Specifically, the label claims the product contains 200 milligrams of citrus aurantium standardized at 6% synephrine, and I would now like to confirm that the citrus aurantium is indeed so standardized.
A. We have had a similar question from a reader who wanted to determine the specification of an ingredient in a mixed finished product and the answer then still applies—so sorry, too late.
To explain the answer it is helpful to describe how we confirm the quantity of a standardized herb in a finished product. Quite simple, first we determine the amount of the standardized component (also referred to as the “marker”) in the product. Let’s say we find 12 mg of synephrine. Knowing that the Citrus aurantium is standardized to 6% synephrine, a mathematical calculation determines that the total quantity of Citrus aurantium is 200 mg(6%/12mgs=100%/Xmgs).
The reader would like to confirm the 6% number. Well we now know there are 12 mg of synephrine in the product, but how can we use that data to confirm the standardization rate? Well we can’t. There is no objective formula we can use to translate a marker quantity into a standardization rate.
But wait. Some creative soul might say you can work the above formula in reverse. With a label claim of 200 mg of Citrus aurantium and a finding of 12 mg of synephrine, why not calculate back to get the standardization percentage? The answer is that it’s not scientifically legitimate to do so.
While analytical chemistry is in fact a science, much of what we do involves assumptions. Obviously, one can assume anything, but legitimate analytical chemistry demands that there be a reasonable basis for each assumption made. It is perfectly reasonable for us to assume your Citrus aurantium is 6% synephrine since that is the industry standard. If you asked us to make calculations based on Citrus aurantium standardized to 12% we could not legitimately make that assumption because there is no reasonable basis for it. (Of course, in this scenario if you could first demonstrate your Citrus aurantium raw material was 12% we could then use it.)
The reverse calculation argument would require us to assume the finished product contains exactly 200 milligrams of Citrus aurantium. We have no reasonable basis to make that assumption; maybe it is 200 milligrams, but maybe it’s 220 or 180.
All of the above results in the generally accepted analytical practice that you should not determine the percentage of a marker compound of a raw material by analyzing a mixed product sample. First, such an analysis requires an assumption (the amount of the material in the mixed product) for which there generally is no reasonable basis to make. Second, the introduction of additional materials in the finished product presents additional complications. The proper method to measure the marker compound is with an analysis of the independent raw material. NW