The Laboratory Notebook

Answering important questions about quality.

By Robert Green

This month we tackle interesting questions on chelated minerals, the analysis of a drink product (which lends itself nicely to a discussion on lyophilization) and currently popular citrus bioflavonoids.

Q. I recently increased testing of my multi-mineral product that contains three chelated metals (calcium, chromium and magnesium) and have found the amount of these metals varies wildly from lot to lot. My custom manufacturer swears that he puts the same amount of each product in each lot. Do you have any idea what the problem is?

A. There may be a simple explanation for the problem,assuming that we can take your manufacturer’s word for it, and it lies in the inherent nature of chelated minerals. First let’s explore the unique nature of a chelate.

A chelate is an organic chemistry term used to describe a class of complex compounds consisting of a central metal atom attached to a large molecule in a cyclic or ring structure. The term “chelate” is said to have been first used in a paper published in 1920 in the Journal of The Chemical Society. Chela is the claw of the lobster, and a chelate was described as a caliper-like group with two associating units (the two claws of the lobster) that fasten to an atom in between. Chelates are actually associations of the metal atom and the large molecule and not fixed structures. What this means is that the ratio between the metal atom and the other molecule changes from batch to batch.

In our industry chelation is used with metals. Metals can be difficult to absorb into the body. To counter this, some postulate that by associating a metal with something that is readily absorbed, such as an amino acid, the metal will be dragged with the amino acid, aiding absorption. This is where chelated metals come from.

Using chelates requires extra care. With a true inorganic species such as magnesium oxide the amount of magnesium does not vary from lot to lot. So once you calculate the over-adjustment for the non-metal component, in this case the oxide, you can continue with that adjustment going forward. But because chelates are associations of metal ions with a large molecule, in this case an amino acid, the percentage of the desired metal content is not fixed, but rather, varies from lot to lot. The only correct way to use this material is to determine the metal content in each and every lot and make the appropriate adjustment in the formulation. In your situation this step was probably not taken. The manufacturer may ­indeed have added the same amount of chelated material in each lot, but the metal content in the formulation expectantly varied from lot to lot.

One final point on chelates. We can routinely determine the content of both the metal and amino acid in your product, but we cannot confirm that a chelate was actually formed. The fact that we find both could mean the presence of a chelate, or simply, that both are present without the necessary chelation reaction having occurred.

Q. I manufactured a liquid drink product containing several herbs and minerals. I sent a bottle of the finished product to a testing lab and they found no trace of some of those ingredients. Since I formulated the product myself I know they are in there. Do you have any idea what the problem is?

A. We are going to guess your problem is in the analysis and not the product. In order to conduct an HPLC ­analysis we must prepare a liquid sample for testing. Generally that means we put the appropriate amount of the contents of capsules, tablets or raw material in the appropriate type and amount of solution and then inject it into the HPLC apparatus. So one might think if the analyte to be tested is already in solution, in this case a drink, why not just pour off some of the liquid and inject it? The problem is the analytes are probably too dilute in the drink to be detected by the HPLC apparatus.

An HPLC analysis requires that the analyte of interest be in the appropriate concentration. If it is too dilute, the analyte will be below the instrument’s limit of detection, meaning that it will not be observed even if present.

So, you may ask, what is the appropriate procedure to analyze components in a drink. The answer is to concentrate the components first by removing the solution. But you have to be very careful doing that, for if done improperly you could harm the analyte of interest, which would also render a false analysis.

While there are several ways to concentrate an analyte in solution, probably the best is through a process called lyophilization, also more commonly known as freeze-drying. Lyophilization is the process of removing water from a product by sublimation. Sublimation is a process whereby a substance changes from a solid to a vapor without first becoming a liquid. This process is the key to freeze-drying.

This is how lyophilization works. First you freeze the drink to change its water into ice. Then you dry it (get it—freeze-dry). To dry it the lyophilization equipment then creates a vacuum, thereby lowering the pressure inside. Adding heat to this low-pressure environment allows the ice to sublimate into vapor. After drawing off the water vapor, the process is complete. You are left with the solids in the drink, which are unharmed and unaltered. Now you put it in solution in the correct concentration and conduct the HPLC analysis.

So ask your analytical lab if they concentrated the analyte of interest first before subjecting it to analysis, and if they did not, find someone who knows what they are doing. And yes, this is the same freeze-dry process used for coffee and astronaut food.

Q. I will be carrying a new product primarily containing a citrus bioflavonoid complex. The manufacturer certifies that the complex contains 50% bioflavonoids. Can you suggest what I should be looking for when analyzing the raw material?

A. A “bioflavonoid” is a natural phytochemical. Many are responsible for the brightly colored pigments in fruits and vegetables. Bioflavonoids include isoflavones, anthocyanidins, flavans, flavonols, flavones, and flavanones. Some of these have received recent media attention and we have seen increased interest in them.

Citrus bioflavonoids, quite logically, refer to bioflavonoids found in citrus fruits, such as oranges, grapefruits and lemons. Citrus bioflavonoids of interest include hesperidin, naringenin, rutin, quercetin and naringin.

Rather than bet on any one citrus bioflavonoid, supplement companies are selling a “citrus bioflavonoid complex,” which, as the name implies, purports to be a combination of citrus bioflavonoids. A proper analysis would confirm that a complex does indeed exist, and not just one citrus bioflavonoid.

We have seen purported analyses of citrus bioflavonoids that report the value of hesperidin only. As noted above, hesperidin, which is found in great abundance in grapefruit rinds, is just one of several citrus bioflavonoids. As a result, a finding of hesperidin is no evidence that a complex exists. What’s more, hesperidin can be purchased relatively inexpensively as a single ingredient, so the opportunity for spiking (adding hesperidin to some other material) is very real. The best way to confirm the nature of the complex is to get the purported formulation of the complex itemizing the amount of each citrus bioflavonoid claimed to be in the product and then confirm that, making sure that the relative amounts of each bioflavonoid are realistic for the species of fruit from which they originate.NW