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.