The Laboratory Notebook

Answering important questions about quality.

By Robert Green

The Laboratory Notebook begins a little differently this month with an editorial on the (Oxygen Radical Absorbance Capacity) ORAC method of analysis discussed in the September issue of this publication. This will be followed by questions on screening supplements for pharmaceutical compounds and the analytical method of mass spectroscopy.

ORAC

The September article on ORAC claimed, in essence, that this method represents the definitive measure of antioxidant activity, and even suggested that one day there may be an RDI for antioxidants in ORAC units. We have heard this story before. In our opinion, ORAC has become more a marketing angle than a scientific treatise. Let’s explain.

The story starts with free radicals, which are highly energetic species generated as byproducts of chemical reactions that go astray. Free radical generation is directly related to “oxidation” (not necessary involving an oxygen atom) in foods and biological systems, and if uncontrolled can lead to some rapid decomposition/degradation of biological tissue (a bad thing). As a result, an antioxidant effect reducing or eliminating the creation of free radicals is a good thing. So now there is a race to develop a method(s) that can predict the total antioxidant capacity of a bioactive supplement, and marketers seek ways to claim their product has higher antioxidant activity than that of the competition.

Unfortunately, one cannot directly measure the power of an antioxidant in situ - in vivo (in place in a life system) due to the uncertainty of the free radicals generation and annihilation by interacting with the antioxidative material, so an indirect method must suffice. Experimentally, several methods have been described for this purpose. It is important to note there is not just one free radical, but many, and the methodologies available are generally targeted to only one or two types. The main methodologies involve superoxide radical scavenging (O•2); hydrogen peroxide scavenging (H2O2); hypochlorous acid scavenging (HOCl); hydroxyl radical scavenging (HO•) and peroxyl radical scavenging (ROO•). Among these are the methods that use azo-compounds to generate peroxyl radicals, such as the “TRAP” method (Total Radical-Trapping Antioxidant Parameter) and ORAC; the scavenging of radical cation by 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) such as the ABTS or “TEAC” method (Trolox Equivalent Antioxidant Capacity); the scavenging of stable radical by 2,2-diphenyl-1-picrylhydrazyl known as the DPPH method and the scavenging of radical cation by N,N-dimethyl p-phenylenediamine for the DMPD method.

Close examination of the published ORAC papers indicate that it is directed to the quenching of the peroxyl or hydroxyl radicals once they have been created. This makes it different from most of the other assays that involve pro-oxidants, which are species that could become oxidative (a bad thing) given the right set of circumstances. As a result, ORAC was designed to measure the ability of a compound to neutralize a free radical once created, and not the power of an antioxidant to prevent the creation of a free radical in the first place. So those claiming a high ORAC value shows their product has a “high” antioxidant value are not quite correct; at best a high ORAC value means they have a “high” ability to quench a free radical once created. An antioxidant which prevents the creation of free radicals could get a very low ORAC score. The perfect example is vitamin C, long known to be a powerful antioxidant preventing the creation of free radicals but scoring poorly in an ORAC assay because it has a limited ability to neutralize a free radical once created. Prevention is always better than the cure of an existing condition, so basing a decision solely on ORAC may lead you astray.

In our opinion the ORAC method also has serious technical flaws. The traditional ORAC assay is reported in Trolox equivalents. Trolox is a commercially available compound used to measure a radical cation. However, the ORAC method is designed to measure the quenching of a peroxyl radical. This is comparing apples to oranges. It’s not hard to beat the quenching power of Trolox on a peroxyl radical since Trolox was not designed to do that.

The ORAC assay also employs the use of a fluorescent molecule as the “visualization probe” of the end point. Basically, the time it takes for the fluorescence to diminish is considered to be proportional to the “power” of the antioxidant. But other factors can contribute to that rate of diminishment. Many foodstuffs possess some very similar fluorescence properties as the end point marker probe. This makes the fluorescence diminish slower (re­sulting in a higher ORAC value) but for reasons unrelated to their antioxidative power.

The fact of the matter is that measuring antioxidant capacity is very complicated and difficult, so no single measurement of antioxidant status is sufficient. To properly determine antioxidant potential in biological systems a battery of measurements is required. And that’s our opinion.

Q. I have read that the Japanese government has discovered that some nutritional supplements illegally contain pharmaceutical products. Can you determine if my product contains any?

A. Yes, with a caveat. As to the specific issue raised, we understand that the pharmaceutical Fenfluramine was detected in a Traditional Chinese Medicine weight loss product. During the last few months we screened many samples from both American companies hoping to export weight loss supplements into Japan, and Japanese companies hoping to import these products, for Fenfluramine and other pharmaceuticals.

One surmises the criminal theory is to increase a product’s effectiveness by adding one or more pharmaceuticals with a complementary effect. So for weight loss products the possible adulterants are sennosides (laxatives), triiodothyronine (T3) and thyroxine (T4) (metabolism enhancers), and N-Nitroso-fenfluramine and Fenfluramine (appetite suppressants), and we are screening diet aids for these substances.

While there are several methods available to conduct screening, we are fond of using our GC-MS (gas chromatograph-mass spectrometer). In September’s column we discussed GC, which is a separation technique isolating the components of interest. In the question below we discuss MS, which is the process we use to qualify the components. We have a very powerful MS that has an on-board library of virtually every known pharmaceutical, which greatly aids identification.

Now back to your general question and the caveat. You had asked if we could screen your product for “any pharmaceutical.” The answer is that we could indeed do that, but it would be very expensive. The problem is that there is no one instrument that can detect everything. Different compounds have different identifying characteristics, and one of the arts of analytical chemistry is to match the compound of interest with the appropriate method of analysis. So, in ef­fect, for general commercial analytical chemistry you need to know what you are looking for. For the potential diet adulterants noted above, we know all will be detected by our GC-MS methodology, so detection is commercially feasible. Now, if you want us to screen for “all pharmaceuticals,” we would have to analyze the product by several methods using various instruments, and that starts to get expensive. But it is perfectly reasonable to screen a diet aid for diet-related pharmaceuticals, just like we would screen a joint aid for arthritis-related pharmaceuticals.

Q. There is an adulterant in my product that I would like identified. It has been suggested that the adulterant be analyzed by “mass spec.” Can you explain what that is?

A. “Mass spec” is short for mass spectroscopy or the instrument used, a mass spectrometer (also referred to as MS), which is an extremely powerful instrument that can be used for identifying unknown compounds as well as confirming the presence of desired compounds. The instrument provides information about the molecular weight and chemical structure of the compound by measuring the masses of individual molecules that have been electrically charged (which are called “ions”). Since molecules are so small, typical units of measurement (like milligrams) don’t work so for the mass of individual molecules we use a unit known as the Dalton (Da for short).

First, the mass spectrometer creates charged particles (ions) from molecules. There are several ionization methods, the most common being Electron Impact (EI), Chemical Ionization (CI), Electrospray (ESI), Fast Atom Bombardment (FAB) and Matrix Assisted Laser Desorption (MALDI). Next stop is the analyzer of which there are also several to chose from, including Quadrupole, Sector (Magnetic and/or Electrostatic), Time-of-Flight (TOF) and Ion Cyclotron Resonance (ICR). To get the correct results, the analytical chemist must select the right combination of the ionization method and analyzer.

Reasonably pure compounds can be introduced directly into the instrument. However, because two compounds present could create an overlapping or mixed spectrum making unambiguous identification impossible, compounds in a mixture must first be separated. This can be accomplished by several methods, the most popular being gas chromatography (GC) and liquid chromatography (LC). Nowadays a GC or LC is coupled directly to the mass spectrometer (creating a GC-MS or LC-MS), allowing compounds to enter the mass spectrometer separated in time, so that the components of mixtures can be detected and analyzed sequentially. Today’s sophisticated instruments also contain on-board libraries that assist in the identification of the compounds present.

Mass spectrometry is a very powerful tool in the analytical arsenal. However, not every substance is suitable for such an analysis, and it can be expensive. Before you jump to an MS analysis, you should explore with your analytical lab simpler, less expensive methods to identify the substance and get some assurance that an MS analysis will be successful in your instance.NW