TYPE-C™ Columns and Synergistic Approaches to Scientific Investigation

In my opinion, significant advances in science are often achieved by the amalgamation of techniques from various fields of study. The challenges facing scientists today may be too complex for a cadre of specialists from a single discipline. Consider for instance the research of Dr. Kyu Rhee from Weill Cornell Medical College and co-workers. Dr. Rhee has performed metabolomics studies which elucidate the mechanism of action and discovery of new treatments for bacterial pathogens such as Mycobacterium tuberculosis. Using the Diamond Hydride™ column, profiling of polar metabolites in these investigations was made possible. Hence, technologies from the fields of both medicine and chromatography were applied together to produce innovative results.

In another related example, correlation of zeta potential measurements and analyte retention for TYPE-C Silica™ materials has shed light on the nature of the Aqueous Normal Phase (ANP) mechanism. The data have demonstrated the contribution that adsorbed hydroxide ions on the stationary phase surface has on ANP retention. In this case, theory from both zeta potential techniques as well as chromatography was used to understand the nature of complex solute–sorbent interactions.

The successful scientist will be able to make use of any and all methodologies at his or her disposal to solve a given problem. Demarcations between physics, chemistry, biology, and so on may be helpful to the student, who learns more easily by the organization of this information into discreet subjects, but it is the mark of a real scientist when one can synthesize these disparate concepts into a cohesive strategy of experimental design.

Refractive Index – Detection of Non-UV Absorbing Compounds

You probably use UV absorption detection for most of your routine HPLC methods. Indeed, it is simple to use and maintain, and sensitivity is often suitable for many typical analyses. Not every compound can be detected by UV, however. If a compound lacks chromophores, detection can’t be achieved at any wavelength. In these cases, you will need to use an alternative method.

                LC-MS has become more prevalent in recent years, as advances in technology have allowed for greater ease of use, reliability, and detection limits. Even so, it is relatively sophisticated and expensive instrumentation, and many QC laboratories might find it more than is required for routine assays where great sensitivity is not required. A good example of this is the food and beverage industry, where refractive index might be more suitable for typical analysis goals. In these applications, levels of ingredients are often relatively high, and the high sensitivity of LC-MS may not be needed. Although refractive index generally has much lower sensitivity, it is often suitable for these food and beverage applications. Unlike the complex apparatus used in LC-MS, all that is required for refractive index is to flush the reference cell with the mobile phase and to use a thermostat to avoid baseline drift.

                I investigated the use of refractive index detection for a taurine application. I was able to observe a nice peak of the standard and obtain good retention. This latter point is important because taurine is very polar and hence difficult to retain by reversed phase methods. With the Cogent Diamond Hydride column, the compound could be readily retained by an ANP mechanism. I demonstrated the ANP behavior by comparing retention times at 70 and 80% acetonitrile; retention increased at higher organic content.

                So I used two strategies to address the analysis of taurine. The first was the use of a “universal detector,” refractive index, suitable for detection of any type of compound, whether UV-absorbing or not. The second strategy dealt with the retention. Here, I used ANP chromatography to readily retain a compound that might be poorly retained by traditional retention modes.

                Click the link to see the application note.

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.