Dietary Supplements — How Safe are they?

Maybe you take a multi-vitamin pill every morning, or perhaps use a whey protein supplement to increase performance at the gym. But did you know that these consumer products are not subject to the same scrutiny as pharmaceutical drug formulations? A manufacturer does not need to demonstrate efficacy before sale to the general public. Indeed, it is only after the product has been demonstrated to be “adulterated or misbranded” by the FDA can action be taken to halt production, distribution, and sale. Recently, this has in fact happened with some dietary supplements containing compounds like aegeline or 1,3-dimethylamylamine (DMAA). These compounds are not APIs in any currently used pharmaceutical formulation and scientific study of their safety for human consumption is nebulous at best. On the contrary, they have been implicated in deleterious health issues up to and including death when taken in dietary supplement products.
                We investigated protein sport mix dietary supplements in our laboratory and were able to separate and detect both aegeline and DMAA in these samples. Use of LC-MS allowed for further specificity with EICs of the analytes. We used both reversed phase and ANP methods with the Cogent Bidentate C18 2.o™ and Diamond Hydride™ columns depending on the analytes. I think these method approaches will become more valuable in the years to come; as the FDA begins to crack down more heavily on dietary supplement manufacturers with tighter regulation, there will be an exigent need for reliable analytical methods.
                Not only did we discover aegeline or DMAA in the samples, we also found other ingredients not listed on the label. Here we observed creatine and caffeine present in one formulation. Although these compounds may not be as dangerous as aegeline or DMAA, this finding may be important for some people who need to reduce or eliminate their intake of creatine or caffeine yet are unaware of their presence in the product. The bottom line is that consumers should have a clear understanding of what the product contains and how it may impact their health to make an informed purchasing decision.  
                Click here to read the full study.

Say goodbye to complex sample prep!

Today’s laboratory is becoming increasingly automated and streamlined. Indeed, one of the most time-consuming tasks in your lab is likely to be sample prep which often requires several manual steps. Ideally, we would all like to use a simple “dilute-and-shoot” approach for every analysis but because of interferences from the sample matrix, this is not always possible. 

                I investigated three simple strategies which you can use to avoid sample cleanup steps like SPE. The first uses the Cogent Diamond Hydride™ column to retain the analyte by a different mechanism. When I tried to analyze folic acid in cereal by reversed phase, I found interfering peaks co-eluting with the analyte peak. In aqueous normal phase (ANP) with the Diamond Hydride™, most of the matrix peaks eluted at the solvent front while folic acid was retained.

                The next approach can be used where matrix contaminants build up on the column. You can elute these in a wash step incorporated into the injection sequence. How often you need to do the wash will depend on the complexity of the samples but I found every six injections for orange juice samples was enough.

                The third approach uses LCMS. More sophisticated detection methods can provide you with additional specificity. When analyzing histamine in red wine, I found vast differences between the complex total ion chromatogram (TIC) and the clean extracted ion chromatogram (EIC). Hence, peaks that can co-elute with histamine may be resolved using MS specificity.

                You may be able to avoid sample cleanup steps using one or all of these strategies. Read more in our Extended Application Note.

Cogent™ Columns —Discovering the Active Ingredients in Traditional Medicines

We received a tree bark sample (Brownea grandiceps) from the jungles of Brazil. They say that a tea brewed from the bark has medicinal properties, and we hoped to find out more about which compounds were responsible. If you can determine which one has the medicinal effects, you could make a pharmaceutical formulation of that compound or even improve upon its properties using a superior derivative. A good example is salicylic acid, a natural compound found in some types of tree bark. It has good analgesic properties but also a number of side effects. This has led to the development of acetylsalicylic acid, also known as aspirin. Our research here could lead to a similar useful drug if the active ingredients are determined.

                The first thing we did was prepare the tea the same way the Amazonian natives do. We took two pieces of bark and put them in boiling water for five minutes. After filtering, the samples were ready for the LC-MS. Next we used both the Diamond Hydride™ and the Bidentate C18™ columns to cover the whole range of polarity in the sample. In the LC-MS data, we identified various compounds present in the bark extract. These compounds included rutin, quercetin, isoquercetin, 6‐beta‐O‐2',3'‐dihydrocinamonyl‐12‐hydroxy‐(13)‐15‐en‐16,12‐olide‐18‐cassaneoic acid, camptothecin, and 9-methyoxy CTP. With further study, one of these compounds could be identified as being responsible for the medicinal properties of the Brownea grandiceps bark extract. Camptothecin for instance has been shown to have anti-cancer properties.

                To read more about the application, Click Here.

Bisphenol A Alternatives – Are they really safer?

Bisphenol A (BPA) is a widely used epoxy resin that has come under scrutiny in recent years. It is so ubiquitous that it was detected in 93% of surveyed test subjects over the age of 6. You probably handle BPA-containing products already on a regular basis, such as receipts from the grocery store, plastic bottles, and many other sources. In today’s world, exposure to BPA is virtually unavoidable.
                As a compound that mimics the action of estradiol, BPA is an endocrine disruptor. In addition, a wide variety of studies have reported a number of other possible health effects. Some companies that have used BPA in their consumer materials have since replaced it with other bisphenol compounds and labeled the new materials as “BPA-free.”  This may lead consumers to believe that these products  are now safe, but the fact of the matter is that some of these BPA-substitutes are just as toxic, if not more so.
                Two examples of these BPA alternatives are bisphenol F (BPF) and bisphenol S (BPS). They are structurally similar to BPA and hence exhibit similar physical properties, making them ostensibly good BPA substitutes. In terms of health effects however, some reports claim they are actually 100 times worse than BPA [1].
                Similarity in structure also means that the compounds may be difficult to distinguish chromatographically. Nevertheless, it will be important to have HPLC methods capable of separating these three bisphenol compounds from each other for analyses of “BPA-free” products. Therefore I investigated such a separation using the Cogent Bidentate C18 2.o™ column. I used reference standards of the three bisphenol compounds and found excellent selectivity amongst them using the column.
                I was able to obtain a separation in under five minutes using a simple, premixed isocratic mobile phase. The method uses reversed phase conditions and a formic acid additive, which would be amenable to transfer to LC-MS. In more complex analyses such as plasma testing for bisphenol compounds, LC-MS may be the preferred choice. In any case, the Bidentate C18 2.o™ would be suitable for bisphenol separations in various types of samples. Analyses such as these may become more important once the health effects of the BPA substitutes are studied more in the future.
                To read more about the application, Click Here.

 Reference:

[1] “100 Times the Damage: Avoid at all Costs – BPA, BPF, BPS ,” Mass Report,  http://massreport.com/100-times-the-damage-avoid-at-all-cost-bpa-bpf-bps/, 2014-12-31. Retrieved 2015-03-25.

Before you blame the column…

…Rule out other possibilities first! Problems you can encounter with peak shape, retention, and so on can often be traced to a component of the HPLC system. For example, consider the following real-world situation which we encountered in our laboratory.

              We were running an HPLC method for ethylbenzene (0.1mg/mL concentration) with a Cogent UDA™ column and observed some problems with the chromatograms (see Figure A). We noticed that our analyte peak was retained longer and longer after each injection and that the peak became broader. Was this changing chromatographic behavior due to column damage? We know the columns are very stable so we investigated other more likely possibilities first.

               We were using a 0.5mL/min isocratic mobile phase from two solvent reservoirs (70% solvent A, 30% solvent B). Thinking there may be a problem with one of the solvent lines, we prepared a premixed mobile phase instead and only used one mobile phase reservoir (solvent A). Under these conditions, we obtained the results in shown in Figure B. Notice how the retention time precision is now very robust and the efficiency is high. This demonstrates that the column performance is fine and the problem was with the solvent B line. Perhaps the issue was at the mixing stage, in which the solvent proportioning valve was not mixing the correct amount of solvent from the B line.

               Also apparent from Figure A was a second, smaller peak that sometimes would show up in the chromatograms and sometimes would not. As before, we investigated system issues as the possible cause. It turned out that it was a problem with the injector. We were using an injection volume of 1 µL, and when we increased it to 5 µL, the peak was consistently present in every run (Figure B).

               So before you blame the column, be sure to inspect everything else thoroughly:

• Check your mobile phase. Are the A and B lines connected to the correct inlet ports? Has the flow rate been calibrated for each line? Is the solvent mixer proportioning valve functioning accurately? When was the mobile phase prepared and does it need to be replaced?

• Check your method. Are you using an appropriate mobile phase/gradient for your compounds? Use an ANP gradient for polar compounds and a reversed phase one for less polar analytes. Are you operating at a proper detection wavelength for your compounds’ UV absorption properties? Before running a sequence, look through your method settings and make sure someone else who used the instrument before you didn't accidentally change anything.

• Check your sample prep. Are the analyte concentrations suitable? Ensure that overload of the column or the detector will not occur at these concentrations and dilute if necessary. Have your samples been properly filtered to remove particulate matter from entering the system and creating blockages? Have you performed percent recovery studies to demonstrate that the analytes are completely present in the final sample for analysis?

• Check your injector. Is the needle going down far enough to reach the liquid sample? Is the syringe aspirating the correct volume of sample?

This is of course not a comprehensive troubleshooting list but it can give you some idea of the type of things that can malfunction. An HPLC analysis can be complex and involves many factors so do not start with blaming the column.