HPLC of Promethazine Tablets — Overcoming Analytical Challenges

If you’ve done method development for a pharmaceutical impurities analysis, you know it can be a tricky endeavor. I find there are two main obstacles to deal with. The first issue is the sensitivity. Impurities are typically present in low levels in the formulation, yet must be adequately quantitated to ensure safety to the consumer. In order to obtain the required sensitivity, it may likely be necessary to overload the main peak. If a gradient is used, interference from the baseline can also be a problem as well in detecting impurity peaks. The second issue pertains to the chromatographic resolution. Impurities tend to share many structural similarities with the API, as they are often side-products from the synthesis, degradants, etc., and therefore exhibit similar chromatographic behavior. As such, a traditional C8 or C18 column may not be enough to obtain separation.

 I used a Cogent UDC-Cholesterol™ column in this case, which I found was necessary to separate promethazine from the impurity isopromethazine. Shape selectivity from the UDC moiety can help separate isomers in many instances. As for the problem of sensitivity, I found it hard to observe the phenothiazine peak, which was the last to elute, due to the sloping baseline from the gradient. Here, I chose to switch the wavelength to a local UV max of 320 nm, in which interference from the baseline is negligible.

I used this method, as well as a separate isocratic assay, for quantitation of the API and the specified degradant promethazine sulfoxide. Further assessment of the methods was conducted by comparing the data to established system suitability criteria. I found the methods to be reliable for routine analyses of promethazine formulations in a QC environment.

                Click here to read the full study.

Solvent evaporation in your sample vial —How to avoid it

Suppose you have a set of samples to run but the instrument is being used by another analyst. You take a look at their injection sequence and find that it won’t be finished until the next morning. You can leave your samples in the autosampler tray overnight and add them to the end of the sequence, but will they be okay sitting out for hours?

                Left in ambient conditions over a significant period of time, the solvent in your sample vials can evaporate even when capped properly. For this reason, it’s important to know what factors affect solvent evaporation. I studied these effects by altering different variables and calculating the percent loss of solvents in capped autosampler vials. In doing so, I aimed to discover which factors were most significant in contributing to evaporative loss. Does a snap cap or a screw cap provide a better seal? Will a methanol diluent lead to greater loss than an acetonitrile/water diluent? Do pre-slit septa have a disadvantage in terms of allowing solvent to escape the vial more readily?

                The graphs I obtained helped answer these questions. You can compare the slopes of each set of experimental conditions to determine which parameters had the greatest effect on evaporation. The traces with the highest slopes had the fastest rate of evaporation. So if two traces differ by only the solvent used in the experimental conditions, you can conclude that the one with the faster rate of loss was likely due to the solvent.

               I averaged multiple replicates from the same set of experimental conditions in each case in order to obtain more reliable trends in the data. That is, I wanted to ensure that any differences in the evaporation were in fact due to the variable under study, rather than tolerance differences in how a particular vial and cap sealed together.

This information can aid in the selection of suitable caps, solvents, etc. for your particular application. For example, if you’re deciding between methanol and acetonitrile/water as a diluent for your method, the results may provide you with information to make a more informed choice.

                Click here to read the full study.

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.