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.

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.

Cogent Diol™ column is great for biological samples!

Many of you are analyzing blood samples for applications like clinical trials of pharmaceuticals, forensics testing, and metabolomics research. We have had a number of successful separations of these types of samples with the Diamond Hydride™ in ANP mode. You may be interested in seeing how the latest addition to our TYPE-C Silica™ line of HPLC columns, the Cogent Diol 2.o™, would work in these cases. We selected three test solutes that would be pertinent to blood sample testing: warfarin, hydroxybupropion, and codeine. Let’s take a look at each:

1) Warfarin: An anti-coagulant used for treatment of thrombosis. It does not have an amine group like the other two, which makes its retention lower. Warfarin does exhibit keto–enol tautomerism and its keto form is ionizable. In this form, it has a pKa of 5.0 due to an acidic hydrogen located between two electron-withdrawing carbonyl groups. Its retention was observed to be the lowest of the three.

2) Hydroxybupropion: The active metabolite of bupropion, an antidepressant and smoking cessation drug. Its main structural feature that influences retention in the ANP mode is a secondary amine. It eluted next, with baseline separation from codeine.

3) Codeine: An opiate used in many pharmaceutical formulations for its analgesic or antitussive properties. It has a tertiary amine which makes it amenable to ANP retention. Therefore it eluted the latest of the three compounds. The peak shape was highly symmetrical, which can sometimes be difficult to obtain for amines.

               We spiked the analytes in a real blood sample and separated them by LC-MS. Using extracted ion chromatograms, interferents from the complex sample matrix could be eliminated. What you will have left are three sharp, well-separated peaks corresponding to the analytes.

               The data you can obtain shows how ANP chromatography is not limited to the Diamond Hydride™ column. Any TYPE-C™ column can be used in the ANP mode. Another interesting feature we discovered is that acetone can be used instead of acetonitrile in the mobile phase. Acetone has the advantages to your laboratory of lower cost as well as lower toxicity.

              Click here for more information on this application.

 

Using Near UHPLC to separate an API from a Prodrug

I found that the Cogent Bidentate C8 2.o™ column is great for separations of mometasone furoate from its active form. The official USP assay method calls for an L7 stationary phase, and this column is ideal for such analyses. The method I used here has some useful features.
               First off, the Bidentate C8 2.o™ column has a small average particle size of 2.2µm, leading to high efficiency and rapid analysis; I was able to separate the prodrug, the API, and excipients in the cream matrix in under four minutes. This is a near-UHPLC phase but doesn’t require the specialized instrumentation of smaller particles. Hence you can take advantage of the benefits smaller particle size columns have to offer but without the associated drawbacks of UHPLC.
               Another convenient feature is that the method conditions are very easy to set up. No gradients or complicated buffers are required. The mobile phase consisted of only isocratic 50/50 acetonitrile/DI water. You can even find this available premixed from some solvent suppliers (just be sure it’s HPLC grade). The flow rate was 0.3 mL/min so solvent consumption was low.
               For sample prep, I weighed 5.0 g of the 0.1% mometasone furoate topical cream in an Erlenmeyer flask with a stirbar. After pipetting 50 mL of methanol, I capped the flask and let it stir for an hour. After filtering with a nylon membrane syringe filter, I had a stock solution that I would use for 1:5 dilutions. I prepared two diluted solutions but used different diluents for each. The first used methanol, and in this case the mometasone furoate prodrug should be present without degradation. This prodrug contains an ester bond which can be hydrolyzed to mometasone, the API. Here I used an acid diluent of 90/10 methanol/1N HCl to catalyze the hydrolysis. You need the methanol component for solubility reasons. Then I heated it in a dry bath at 80 °C. Under these conditions, the active form should be present in the solution.
               In the extract using the methanol diluent, I saw a small peak early in the run corresponding to one or more excipients. Aside from that, there was only one other peak in the chromatogram. In a methanol diluent, there will be no conversion to the active form so this peak should be mometasone furoate. However, with the extract using the 90/10 methanol/1N HCl diluent, I saw an extra peak eluting just before the prodrug. There was also a slight decrease in peak height of the prodrug, which indicates some of the mometasone furoate had been converted to the active form.
               As for the chromatography, the elution order made sense. An ester is a relatively hydrophobic functional group and therefore we would expect the prodrug to elute later than the active form in reversed phase conditions. Indeed, this is what is observed in the data.
               Hence, this column can distinguish between the prodrug and the active form with good resolution and a low run time. This may be useful for various analyses, such as studies investigating the rate at which the prodrug is converted.   
              Click here to read about the full study and see the chromatograms.

How Acetone Can Save Your Lab Time & Money

In most reversed phase HPLC separations today, acetonitrile is the organic solvent of choice. However, I found that use of acetone instead can have many advantages. In preparative chromatography, acetone is more volatile and therefore it is easier to obtain product from a collected fraction. In addition, its lower cost compared to acetonitrile translates to significant savings at the preparative scale. In analytical applications, it can be used as a means to change elution order in the case of some analytes. Sometimes the peak shape or efficiency may even be superior.

               I investigated the separation of a variety of test solutes using either acetonitrile or acetone. Due to the high UV cutoff of acetone, I selected analytes with strong absorption in the high UV/visible range. These analytes typically have a high degree of conjugation, such as dyes. If you’re not using a UV-based detection method though, acetone won’t be a problem for analysis of an analyte with little or no UV absorption. An example of this is our study of amino acids using acetone with LC-MS.

               In 2008–2009, there was a worldwide shortage of acetonitrile that prompted studies of alternative solvents. Often, methanol was found to be the next best choice, but it has its own drawbacks as well. In terms of safety, its toxicity is higher than acetone. Chemically, acetone is more similar to acetonitrile than methanol since both are polar aprotic solvents. For this reason, you could predict that in general retention using acetone would be comparable to acetonitrile in many instances. I found this to be the case for four of the five test solutes I tested, which showed very similar retention (Fig. 1).

               You can read about the full investigation here.

Fig. 1