The Laboratory Notebook

Answering important questions about quality.

By Robert Green

It’s been another interesting couple of weeks in the world of supplement testing and our mailbag is full of great questions. This month we’ll discuss issues on formulation, different test methodologies and product adulteration.

Q. My formulator encapsulated a three-component capsule. We tested all of the components and took into account your previous warning on the difference between percentage purity and percentage assay and we made all the necessary calculations and adjustments. The formulator then mixed the components for hours and, seeing no clumping or other problem, commenced the encapsulation. When that was complete we tested capsules from the beginning, middle and end of the run and found wide variations; the capsules at the beginning of the run had too much of one component and not enough of another and the capsules at the end of the run were almost the exact opposite. What went wrong?

A. It appears that the problem was in the formulation and suspect the cause was unequal particle size. When you look at two plastic bags of the same weight with different dry powdered material you might say they are both powders and therefore the same size. However, each bag is made up of zillions of individual particles, that is, the smallest component in the bag. If you look carefully through a microscope you will see the individual particles and may notice that within the bag they are of different sizes. Most likely the particle size between two ingredients will be different.

Particle size is a crucial consideration in many areas. For example, pharmaceutical companies go to great lengths to determine which particle size is required for optimum bioavailability of their drugs. For our purposes, particle size is critical for proper formulation.

The laws of physics prevent a completely homogeneous mixture of ingredients with different particle sizes. You can mix the product until the cows come home, but the “fines” will separate from the coarser particles. What’s worse, as the mixture sits the fines will fall to the bottom. The result is that there will be more fines at the beginning of the run (and less of the coarser material) and the opposite will occur at the run’s end. Quality formulators will insure that all particles are within an acceptable range before encapsulation.

Q. I had a bilberry sample tested for anthocyanosides by a laboratory that used a spectrophotometric method. The same material was tested using an HPLC method and the level of anthocyanosides was lower. Can you explain this?

A. While there can be any number of reasons why laboratory results conflict, let’s focus on the different methodologies, which could easily be the cause.

Spectrophotometric analysis em­ploys the use of a spectrophotometer (there is a logic to these things). Simply, a spectrophotometer consists of two instruments, namely a spectro­meter for producing light of any selected color (wavelength) and a photometer for measuring the intensity of light. The instruments are ar­ranged so that a sample is placed between the spectrometer beam and the photometer. The amount of light absorbed by the sample is measured by the photometer and then recorded. The sample must be in liquid state and it is prepared very simply by placing the sample in a solvent. This means that you may be measuring the ab­sorbance of more than one component in the sample and not just the one of interest like, in the case of bilberry, anthocyanosides.

HPLC also measures the amount of light absorbed by a sample with one dramatic difference—before the light absorbance is measured the HPLC separates the components to be tested from the balance of the sample. As a result, the measurement taken is only of the components at issue. This eliminates components in the sample that may absorb similarly to the components in question, resulting in a higher number recorded spectrophotometrically. It is now apparent that HPLC is the more exacting methodology because it is component specific. HPLC also makes it harder for someone to spike a sample with a compound that he/she knows will absorb similarly to the component at issue but which is less expensive, thereby achieving higher results at a decreased cost.

Q. I sell a glutamine powder product and received a customer complaint that a bottle purchased was tainted. The customer insists that the safety seal was intact. I obtained the bottle and it does indeed look different. What do I do now?

A. Product adulteration has become a recurring problem. Fortunately though, it can be combated with the help of science and a skilled laboratory.

The first thing to do (which you already did) is to get the product in question. If the customer insists on saving material “as evidence,” ask for the container and a large amount of product and let the customer retain a sample.

Once you have the adulterated product, launch a two-pronged investigation. One prong is your examination of the production of the lot in question (the lot number should be on the container). Review your manufacturing records and procedures for the lot and examine a large number of containers from that lot. This should indicate or rule out the possibility that the adulteration (intentional or accidental) occurred on your end.

While you are running your investigation, a skilled laboratory should be asked to conduct a technical analysis of the product sent by the customer. The key here is for the lab to keep it simple. If this is a case of customer adulteration the odds are the foreign substance is a generally available household product, with detergent, baking soda, salt, sugar, fertilizer and flour being the usual culprits. There is no need to launch a high-powered and expensive investigation looking for all sorts of exotic chemicals that are generally not available to the typical consumer.

Now science becomes our ally. Every chemical substance has a series of characteristics. Many characteristics are shared in common with many substances, but each substance has one or more that makes it different. The lab’s job here is to narrow down the identity of the substance so you can rule out the possibility of adulteration during manufacturing and reasonably conclude that it was consumer-initiated. To do this you may not need to know the exact identity of the foreign substance; its general classification may be sufficient.

Generally speaking, the lab’s first step is to separate the foreign substance. This may be accomplished simply by running the suspect product and an untainted sample (which is the control) through a series of sieves. Different components have different particle sizes (even though they all must be within a reasonable range for proper formulation as discussed above) and the sieves separate these components by that size. This particle size analysis should have different results for the two samples on account of the foreign substance. Simply collect the fraction with the greatest weight difference since that is where the majority of the contamination is.

Now we are zeroing in on the foreign substance. Next is the examination of this additional material under a microscope to further separate the foreign matter from other material it will still be mixed with. This examination could also yield clues as to the nature of this material.

By now a good sample of the foreign material has been collected. To further narrow its nature a simple melting point analysis will determine if it is organic or inorganic. As its name implies, melting point analysis will determine the temperature at which a compound melts; if there is a melting point the foreign substance is probably organic, whereas no melting point (meaning the substance never melts but instead turns to ash when the temperature gets hot enough) indicates that the substance is probably inorganic (such as a salt) or polymeric (like flour).

There are additional relatively simple tests to employ, including determining if the substance dissolves in water, alcohol or acid. The result of each of these tests helps classify the foreign substance and the combination of these results leads to the foreign substance’s identification, at least in a general sense.

If you need to identify the exact substance you can do so with high technology. The most likely analysis would be by mass spectrometry, which is a powerful tool used to determine the molecular weight of a substance. With this in hand the identity of the foreign substance could be definitive.

And of course, many times experienced lab personnel can look at, touch and smell a foreign substance and then name it (“hey, that’s baking soda”), shortening the entire process.

The above is just an example of one way to pursue a product-adulteration situation. Each situation has its own set of circumstances that determines how to proceed. The point to make is that science is a friend that can help in many difficult situations so long as it is put in the hands of qualified and experienced personnel, and it doesn’t have to cost a fortune to employ it.NW