Preparing HPLC Mobile Phase Solutions Consistently

Have you ever read what seemed like simple method conditions and found yourself pondering how exactly something was done? For example, what does “0.1% ammonium acetate” mean? Is the 0.1% in terms of w/v? What about w/w or v/v percent? I always try to avoid ambiguities such as these when describing some method conditions. What may be obvious to assume for one person may not be so with another. This becomes especially crucial when you want a method that’s reproducible across different laboratories and analysts. Ideally, anyone should be able to read the method conditions and arrive at the same end result as the person who wrote it.

What about premixed mobile phases with an aqueous buffer and an organic solvent? I often have some of the same questions about these. If a 90 : 10 acetonitrile : aqueous buffer is said to be “10 mM ammonium acetate,” is that just for the buffer component or for the total solution? It would actually make a big difference in the ammonium acetate concentration depending on how it was interpreted (a factor of ten in this case).

For this reason, we thought it would be helpful to show you a simple step-by-step walkthrough on how to make a premixed 90 : 10 acetonitrile : 10 mM ammonium acetate buffer. This mobile phase solution is used quite frequently in ANP methods and LC-MS, so it would be good to have a handy resource to refer to when you need it.

See the link below for our tutorial:

http://kb.mtc-usa.com/article/AA-03145

New Location for Manufacturing/Applications Laboratory

I am pleased to announce that MicroSolv’s laboratory division, previously located in upstate New York, has now relocated to Wilmington, NC. Situated in the CREST Research Park of UNCW, this new facility will allow for collaboration between MicroSolv and those at the university. In addition, the new lab is very well-equipped and up-to-date, which will allow our operations to be very streamlined and productive.

                I have spent seven years at the previous laboratory and watched it develop over the years, ever since its very inception. It has been very gratifying to have seen the laboratory acquire new instruments and capabilities while at that location, and I think our new facility here in Wilmington will allow for even more of this kind of growth in the future.

                I look forward to seeing how the new location will enhance our processes in both manufacturing and research applications. Stay tuned for more updates on our progress with the new laboratory!

Eastern Analytical Symposium 2017

Recently I had the privilege of giving a lecture at the annual Eastern Analytical Symposium in Plainsboro Township, NJ. This year, the symposium was held in an elegant new venue, the Crowne Plaza Princeton-Conference Center. I have been attending EAS since 2011 and I think this has been my favorite one so far.  

                My presentation was at 11:00am on Wednesday the 15^th. The subject of my lecture was about a method my colleagues and I had been working on for chromatographic assay and organic impurities detection of chlorpheniramine maleate formulations. The current USP method calls for a time-consuming multistep liquid–liquid extraction procedure for the assay, and no method is provided for impurities analysis. Consequently, there was a lot of room for improvement in bringing these methods up-to-date.

As a basic compound, chlorpheniramine may be prone to prone to chromatographic tailing via undesirable electrostatic interaction with residual silanols present on the surface of some traditional silica-based stationary phases. Hence, the moderately hydrophobic Si–H-based surface of Cogent™ columns was postulated to be advantageous in terms of minimizing tailing contributions from this phenomenon.  

                We encountered a few obstacles in method development. The first involved peak splitting for the first two peaks. I had previously selected a diluent with 50% acetonitrile in an effort to ensure solubility of the more hydrophobic analytes, but this choice had the undesired effect of peak splitting. Reducing the organic content in the diluent to 10% resolved this issue. Another encountered problem dealt with trying to use a high enough extract concentration such that we could detect impurity peaks at the 0.1% level while being careful not to overload the column so much that we lost resolution of the critical peak pair. In this case, adjusting the gradient and using a lower mobile phase concentration of trifluoracetic acid (0.05% instead of 0.1%) helped to improve selectivity enough that resolution was sufficient at the higher extract concentration.

                Despite these setbacks, we produced a nice method for subsequent validation studies and I think the audience was quite pleased with the end result. This was my second year as a speaker at EAS and look forward to the opportunity to possibly present again at future meetings! Hope to see you there in 2018!

Method Validation Tip: Do Robustness Studies First

After method development, I find that a lot of chemists will delve into method validation studies like accuracy, precision, etc. but leave robustness for the end. However, I think it is better to do robustness first. The reason for this is that the results can help you further refine the method in ways that you may not have considered during the method development stage.

                Let’s say for example that you do your method development have a perfectly satisfactory separation where your critical peak pair is baseline-resolved. You move on to validation and go through all the necessary studies (e.g. accuracy, linearity, repeatability, intermediate precision, LOD, LOQ, etc.). Then in the robustness studies you find that alteration of one of the method variables actually leads to a superior separation. This was what I found when I varied the TFA concentration. I chose a 0.1% concentration originally during the method development stage but hadn’t given much thought to it at the time. Then in robustness studies, I found that the critical peak pair, which had been just baseline-resolved with 0.1% TFA, became vastly better separated upon a decrease to 0.05%. Before proceeding with the rest of the validation, I could use the 0.05% concentration in my method.

                I hope this information helps you as much as it helped me. I am always learning new tips and tricks to make chromatographic analytical techniques more streamlined and efficient.

UV Trace Drift in Gradients

Gradient elution mode is an invaluable tool for us as analytical chemists, but the slope of the UV trace that results can present issues. You can understand why this happens by considering how each of the solvent systems has its own UV absorption profile. Some of the drift is due to differences in the refractive indices between the two solvents as well. Because of these differences, the UV readout will change continuously over the course of the gradient, producing the slope that we observe. If it is too steep, it can obscure eluting peaks, reducing sensitivity. It would be ideal to have the slope as shallow as possible if we can do so.

                The other day, I was working on a method development project for the USP that called for low pH. We observed tailing for some of our peaks and speculated that the lower pH was needed for this reason. To this end, we tried an additive of 0.5% formic acid in the mobile phase solvents but found a rise in the UV trace of 200 mAU over the course of our 30 min gradient. In an impurity method, where low detection levels are of critical importance, this degree of noise would clearly not be practical. By using TFA instead, however, we were able to reduce the additive concentration by a factor of five and still obtain the good peak shapes that we sought. With the lower additive concentration, the trace slope had risen by about 20 mAU, a mere tenth of the slope obtained previously! This clearly represented a much more viable option for a prospective USP method.

                You just have to remember a few things with TFA, though. It can be prone to oxidation from the atmosphere, so extraneous peaks or a steep UV trace can result if it degrades. In that case, you would have the same problem as before! Your best bet is to try single-use ampules of TFA, since these will have minimal contact time with air. If you use a resealable bulk bottle, you can try adding a blanket of argon after every time it’s opened to ensure it is kept free of air. This oxidation is a slow process, so it only becomes an issue after storage of the opened bottle. Then of course, another thing to keep in mind about TFA is its MS-incompatibility, as it contributes significantly to ion suppression. So if you develop a nice method for UV-based analyses with TFA, just be aware that its applicability will be more limited than with something like formic acid. Even so, TFA can do wonders for some tailing peaks of basic compounds, so it is a good tool for the analytical chemist to keep at the ready.

                I hope these are some helpful tips for you to try in your own method development process. I was always a fan of TFA for those situations where symmetrical peak shapes may be difficult to obtain otherwise. The effect of the trace slope can be one of the more tricky aspects of a UV-based gradient method. And if you can, maybe also consider using a more gradual solvent gradient. This will have a direct effect on how steep the resulting UV trace is.

HPLC Troubleshooting Tutorial: Problems with the UV Baseline

I was using a Cogent Bidentate C8 2.o™ column in my HPLC method recently when I observed a strange phenomenon. Instead of a normal, flat baseline in the UV monitoring readout, there was a highly erratic pattern; the signal would descend and jump back to its starting point in a recurring “sawtooth” like waveform. Meanwhile, the pressure was stable and all other instrument parameters indicated normal operation. My isocratic mobile phase was fairly simple (50% DI water/ 50% acetonitrile/ 0.1% formic acid) and other columns I tried did not have the issue. No amount of equilibrating seemed to remedy the situation.

                I really wasn’t sure if the column had somehow been damaged but had no way of running a standard QC test if I could not observe analyte peaks amongst the unstable UV baseline. Next I thought maybe there was some contaminant on the column and it needed to be washed out. Initial trials using solvent systems such as 1:1 methanol: DI water proved unsuccessful, and then I had the idea that perhaps there was some trace immiscible solvent in the column and use of an aqueous-based mobile phase would not be appropriate.

                So I switched to pure isopropanol and gave it a try. Miscible with both reversed phase and normal phase solvents, isopropanol can often be an effective choice in general column cleaning. Soon after introducing the new solvent to the column, the UV baseline resolved into a typical, flat signal. Now the real test would be to see how my original solvent system behaved. Upon reintroducing the original mobile phase of 50% DI water/ 50% acetonitrile/ 0.1% formic acid, I was pleased to observe that the baseline had returned to normal.

                How can we account for this behavior? The outcome of the experiment seemed to indicate to me that the column did indeed have some minor amounts of a nonpolar solvent in it, which would create immiscibility issues with the reversed phase solvents I was using. These could have been residual packing solvents and will pose no problems once completely removed.

This just shows you if you think a column may be defective, try to investigate all your possibilities before prematurely concluding that it has been damaged. You can save your laboratory money by getting the most out of each valuable HPLC column instead of replacing it. I find issues like this can crop up in one form or another quite often; a column is suspected to be defective when in reality, there is a simple solution to resolve the issue. So the next time your HPLC column exhibits some unexpected behavior, try to ask yourself what might be causing it and whether it can be fixed.

 

New Uses for Cogent™ Columns: Ketorolac and Sucralose

Here at MicroSolv, we are always investigating new and exciting applications for our Cogent line of HPLC column products to address the ever-expanding needs of today’s analytical chemists. I am particularly pleased with these two latest application notes from our laboratories. Check them out, as you may indeed find them interesting as well!

In the first one, we examined the selection of the extraction solvent in influencing the extraction efficiency of ketorolac pharmaceutical formulations. We observed that use of DI water led to superior efficiency compared to ethanol. If you have encountered similar problems of low recovery in your sample prep of pharmaceutical extracts, you may pick up a pointer from the application. As for the chromatography, the compound was well-retained in reversed phase using the Cogent Phenyl Hydride™ column:

http://kb.mtc-usa.com/article/AA-02936/0/

The second application is for sucralose, commonly known by its brand name of Splenda®. In addition to being significantly sweeter than ordinary table sugar, sucralose has the further advantage of not contributing to elevated blood sugar levels among diabetics. If you’ve analyzed sucralose in your laboratory, you may have had problems with obtaining adequate sensitivity. UV absorption is low in this compound but it can be detected at sufficiently high concentrations and low wavelengths. Alternatively, you can try a universal detection method such as refractive index (RI). We tried both approaches in our application:

http://kb.mtc-usa.com/article/AA-02934/0/

I am looking forward to seeing what unique applications will be devised with these columns next!

Cogent TYPE-C Silica™ columns and ANP methods keep your MS source cleaner than HILIC methods

Does this situation look familiar?

Corroded Ion Source for Agilent TOF when used with HILIC Columns

It’s a common problem for many of us working with LC-MS and with polar compounds in particular. Our method calls for a mobile phase with a high concentration of buffer, and bam, your MS source quickly becomes contaminated (60 mM concentration and 500 injections in this case). The frequent required cleaning of the ion source reduces our throughput and takes up valuable time. What can we do to avoid problems like this?

The solution is to avoid using these high salt concentrations. The Cogent TYPE-C Silica™ materials allow you to do this by way of a aqueous normal phase (ANP) retention. Due to differences in the stationary phase properties compared to HILIC columns, a high salt mobile phase is simply not required. Typically we use 5–10 mM buffer where a HILIC column might require 50 or 60 mM for the same compounds. What this means for us the chromatographers is that the MS ion source will not be contaminated with salt precipitate as readily due to the lower concentration.  More uptime!, Less costs!

The difference is due to the stationary phase being more polar in HILIC phases than Cogent columns. Therefore a higher amount of salt in the mobile phase may be needed in HILIC to get compounds that are retained on the stationary phase to elute in a timely manner. By the way, this is also a great feature when doing prep HPLC as well.

Save yourself from a few headaches and try one for your next LC-MS method! You will be pleasantly surprised with the results!

Cogent TYPE-C™ Silica Columns and Mesquite Flour Separations

A new research article using Cogent TYPE-C Silica columns has been published in LC-GC Magazine. Our study investigates separation and identification of components in mesquite flour. Using these unique HPLC columns, we were able to identify a wide variety of compounds in these natural products, some of which have been unreported in previous works. Some analytes were quite polar and could only be retained in the ANP mode, while others were more amenable to reversed phase. Use of both of these complementary retention modes allowed for a more complete characterization than might be possible using either mode alone. Further study on the analytes could help elucidate their role in some of the health benefits of mesquite flour consumption, such as their antioxidant properties.


I feel very privileged to have the opportunity be a part of such innovative research. It is wonderful to see how many novel applications these columns have in disparate industries such as foods & beverages, pharmaceuticals, and clinical studies. I look forward to seeing what our research in the future may yield in addressing some of the challenges facing the modern analytical chemist.


You can read the full article here:
 
http://www.chromatographyonline.com/lc-ms-characterization-mesquite-flour-constituents

Exciting updates for the Cogent™ HPLC column product line

I have been working for MicroSolv for over five years now and have had the privilege of seeing our Cogent brand of HPLC column products expand significantly during that time. For example, we have introduced near-UHPLC small particle phases to complement our standard 4 µm columns, allowing users to combine the benefits of high efficiency particles with the unique retention and selectivity of TYPE-C Silica™. Several new types of bonded phases have also been added to our catalog in recent years, such as the Cogent Diol™ and Cogent UDA™.

                The future of Cogent TYPE-C Silica is bright as we anticipate several upcoming additions to the product line. Firstly, we will be releasing the Cogent Amide™ phase, which I have found to have tremendous potential for retention of polar sugar compounds. This material avoids many of the common pitfalls associated with amine phases, often considered the workhorse column for sugar analysis, such as poor robustness and low column lifetime. The Cogent Amide offers the best of both worlds because the bonded ligand can help retain sugars while not sharing the undesirable chemical reactivity of an amine column.

Another upcoming product we look forward to introducing is useful for compounds in which stainless steel column hardware can create detection/recovery problems. Now for the first time, you can choose PEEK hardware for your Cogent TYPE-C Silica column instead of stainless steel. Our laboratory has performed comparisons of anionic compounds such as nucleotides using both hardware types and has noted greater LC-MS detection levels using PEEK hardware.

              In light of these additions to the Cogent catalog, we are excited to announce a new logo for our Cogent line of products! Sleek and modern, this new look reflects our focus on updating the Cogent brand to better serve our Community and the growing needs of today’s practicing scientists. For more information, we invite you to check out our webpage and keep up with the latest news on these upcoming Cogent products:

www.Cogent-HPLC.com

Red Wine and the French Paradox

France is renowned for its wine cultivation and ubiquitous incorporation of the beverage into its everyday culture. Interestingly, the French have been noted for their low incidence of coronary heart diseases, a phenomenon popularly termed the “French Paradox.” Some have speculated that this may be linked to the relatively high consumption of red wine, which is known to have a number of antioxidants present. Among these is a stilbenoid called resveratrol. You may have read about resveratrol in the news in recent years. Ever since an influential 1997 article published in the prestigious journal Science demonstrated its chemopreventative activity, there has been ongoing research to explore its therapeutic effects on human health, addressing such disparate conditions such as heart disease, neurological disorders, and senescence.  

                Although there remains much to be done in resveratrol research, particularly in translating efficacy results to in vivo human clinical trials, this intriguing compound seems to show promise as a therapeutic agent. For this reason, I thought it would make a pertinent area of focus for research using the Cogent TYPE-C Silica™ columns. In the study, I addressed two major problems with resveratrol quantitative analysis in red wine samples: low sensitivity and poor specificity.

                Some laboratories may use UV detection while others require LC-MS. Using either detection method, these obstacles can be overcome. With UV detection, I employed a sample cleanup/preconcentration technique using Cogent TYPE-C Silica™ phases packed in a microextraction bed. This helped remove interfering peaks in the resulting chromatograms while also providing a 2X concentration increase, affording higher sensitivity. For LC-MS, you can obtain excellent sensitivity due to the nature of the MS detector, which is far greater than UV for this type of analyte. Furthermore, use of extracted ion chromatograms (EICs) allows you to obtain a clean chromatogram corresponding to the m/z of the resveratrol [M + H]⁺ ion.

                You can use these method strategies in your investigations of resveratrol in not just wine, but a variety of other matrices such as plasma samples. In particular, LC-MS is a powerful tool that will undoubtedly be a part of these quantitative studies more and more in the future. Cogent TYPE-C Silica™ columns offer advantages for these types of analyses, which I describe in detail in a research article published in LC-GC North America magazine.

Click here for the article!

A great way to do phospholipid analysis

 In a recent article published in the Journal of Chromatography A, Cífková et al. have investigated the chromatographic behavior of various phospholipids such as (lyso)phosphatidic acids and (lyso)phosphatidylserines. If you have ever done these kinds of compounds then you know they present a challenge to say the least; they contain polar functional groups which make them less suitable for chromatographic methods used to retain more hydrophobic lipids.

Furthermore, identification of discreet lipid classes is greatly facilitated by the high specificity of LC-MS, but previous methods reported in the literature tend to use ion pair reagents, which are not amenable to MS.  This can be painful.

The authors present a comparison of columns for the LC-MS separation of these compounds in real world samples (porcine brain and kidney extracts). In terms of peak symmetry, the Cogent Diamond Hydride™ produced tailing factors in the range 1.0–1.6 for all the studied analytes. Two analytes in particular tailed so severely on HILIC columns that a tailing factor could not be calculated; in contrast, the same two compounds produced only moderate tailing (1.4 and 1.6) on the Diamond Hydride™.

If you struggle with phospholipid analysis, this paper is well worthwhile reading as it is full of new insights.

REFERENCE:

E. Cífková, R. Hájek, M. Lísa, M. Holčapek, Hydrophilic interaction liquid chromatography–mass spectrometry of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipid classes, J. Chromatogr. A (2016), in press.

http://dx.doi.org/10.1016/j.chroma.2016.01.064

How you can get better precision and faster runs using Cogent TYPE-C Silica™ columns

I have heard many chromatographers describe problems with HILIC methods they use. Common complaints include long equilibration times and inconsistent retention when using gradients. Given the nature of the HILIC retention mechanism, this is not surprising. In HILIC, hydrophilic silanol groups on the stationary phase surface cause a thick water layer to form, which allows for the partitioning responsible for analyte retention. The problem with this retention mode is that the water layer is not a static environment but rather is constantly changing as the mobile phase changes over the course of a gradient. Then, during re-equilibration, it takes a significant number of column volumes to fully regenerate the hydration shell to its original form. It is this variability in the water layer that gives rise to both long equilibration times and poor retention precision.

So what solution is there to this kind of problem? It would be better to rely on a retention mode that does not involve partitioning with a water layer. With Cogent TYPE-C Silica™ columns, hydrophilic silanols are replaced by silica hydride groups. Because the latter are moderately hydrophobic, they do not result in the formation of a thick water layer, as in conventional silica based phases. Polar compounds can still be retained using these columns in a mode called Aqueous Normal Phase (ANP). Mechanistic studies involving measurements of zeta potentials have demonstrated that the retention in ANP is primarily adsorptive in character rather than due to partitioning. Indeed, the greater precision and lower equilibration times observed using ANP methods compared to HILIC are consistent with these findings.

What this means to you is that you can obtain more reliable data with greater throughput. Solvent savings can also be realized due to the minimal equilibration. Furthermore, the same column can be used in some instances for both ANP and reversed phase chromatography, allowing for more streamlined analyses. With HILIC columns, only polar analytes may be suitable for retention while more hydrophobic species may elute at the solvent front. Hence, you can get more versatility out of one column with Cogent TYPE-C Silica™ phases.

Ever-increasing globalization in many industries that rely on HPLC has created more competition than ever before. In today’s business environment, laboratories need to maximize their resources in order to stay competitive. Using Cogent TYPE-C Silica™ columns is one way to help you achieve these goals.

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.

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