The Laboratory Notebook
Answering important questions about quality.
By Robert Green
We had a great time demonstrating the latest Waters HPLC instrumentation at SupplySide West. While most are familiar with the term HPLC analysis, this was the first time many have actually seen one conducted. Because we had so many questions about the HPLC and its operation, we thought we would briefly review HPLC principles. This is followed by questions on dissecting a multi-component finished product, fatty acids and ginkgolic acid.
High Pressure Liquid Chromatography (HPLC) is a high-tech version of the age-old scientific method of chromatography, which is simply a way to separate complex mixtures by passing them through a media that selectively absorbs materials. With HPLC, the material being tested is a liquid (a solvent is added to all samples before they are injected into the test equipment) under pressure that increases the ability to separate complex mixtures and expedites the process. These systems are computer-controlled using sophisticated software.
While we may refer to an HPLC as one instrument, in fact it is very versatile and can be configured in many different formats, depending upon its intended use. Once configured properly, the instrument is instructed to run a specific protocol for the sample under analysis. The actual protocols involve countless permutations depending upon the sample, including the correct combination of solvents, absorption media used and detector to measure the material. HPLC instrumentation is truly remarkable, but it is only effective when placed in the hands of a skilled analytical chemist with the required knowledge and resources.
Q. We have an encapsulated multivitamin/mineral formula and we would like to confirm the capsules contain both the stated items and respective quantities as listed on the label. The capsules contain 22 items all mixed together. How can you identify and measure each one of them?
A. The answer lies in technology. As an analytical laboratory we test raw materials and finished products every day. With finished products, one important skill is to separate and recover each item of interest, being certain (a) to collect all of the item(s) of interest present and (b) not to damage the material in the process.
As we have discussed before, there is no machine that can identify and quantify a mixed component product. Rather, different instrumentation is used for different types of material. Even when one instrument is used for several items, the instrument may not be able to identify and measure several items simultaneously. For example, while many items of interest are analyzed using HPLC, each may require a unique configuration, so they must be analyzed independently. Therefore, these items must first be separated and recovered before the analysis can begin.
We generally achieve this through a process known as “Selective Solvent Extraction.” Quite simply, we apply a particular solvent to the product that we know will extract the item(s) of interest without harming them. For example, to isolate water-soluble vitamins (such as the B vitamins) we would apply a highly aqueous solvent. For fat-soluble vitamins (like D and K) we would use hexane or isopropyl alcohol. In each of these instances the group of similar analytes is separated from the whole, and is now ready for analysis by HPLC.
Other items of interest require different treatment. For certain components in salt form, such as pantothenic acid, we must neutralize the salt before the extraction process can begin. For others that are too small to be detected, such as biotin and folic acid, we actually incorporate a salt before extraction.
Minerals and metals are an entirely different story. We conduct this analysis by Energy Dispersive X-Ray Fluorescence. This is a non-destructive technique that quantifies the metal content by measuring the fluorescence spectra of metal ions when irradiated by high-energy x-rays. In one analysis this state-of-the-art technique detects every element in the Periodic Table of Elements. One beauty of this technique is that neither extraction nor other manipulation is required; the product is placed in a slide and the instrument does the rest. Another technique for metals and minerals is ICP-MS, which requires a nitric acid digestion before analysis.
So you see, sophisticated techniques are available to take apart a finished product and analyze the individual parts. Great care must be taken not only in selecting the appropriate methods, but in executing them, for if the extraction process goes awry the analysis will be incorrect.
Q. I have a mixed fatty acid product encapsulated in a softgel. I have had two laboratories test capsules from the same bottle; one lab said the capsules meet label claims and the other said there was insufficient material present. Do you have any idea what the problem is?
A. There can be any number of reasons why the lab results are contradictory, but here is something to look into. Usually when we test a finished capsule we remove the material from the capsule and work solely with the contents. That’s not true with many softgel products, especially those containing fatty acids. Here the softgels have a tendency to absorb the fatty acid material. Now there is nothing wrong with that, since the consumer digests both the capsule and its contents and is therefore still obtaining the full dosage, but this effect has great consequences when it comes to analysis. If the lab follows the usual practice of discarding the softgel, it will also be discarding some of the contents. The resulting analysis will in all likelihood show the product’s contents is under label claims. Here the proper procedure is to include the softgel in the extraction process.
Q. I am about to purchase a supply of ginkgo biloba extract and heard some material contains high levels of ginkgolic acid, which is undesirable. Can you explain the issue and determine if a sample has an undesirable level.
A. Ginkgolic acid, which is a natural component of ginkgo biloba, is structurally similar to the urushiols found in poison ivy that cause allergic reactions. As a result, persons sensitive to poison ivy will often cross-react to ginkgo products. To protect consumers it is generally understood that the ginkgolic acid level should be less than 5 ppm (parts per million).
Since ginkgolic acid is well known, quantifying its level in a sample is routine. We conduct the analysis by HPLC, through which the ginkgolic acid level can easily be determined.NW