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Oral Presentations
Poster Presentations
Oral Presentations
Author: Richard A. Henry – Sunday, March 8 – 1:20 pm, Room S404bc
Co-Authors: Craig Aurand, Jennifer Claus, Jay Jones, Dave Bell
Title of abstract: Retention Mechanisms in Chiral Chromatography: LC-MS Analysis Using Macrocyclic Glycopeptide and Cyclodextrin Chiral Stationary Phases
Abstract Content:
Enantiomer separations on chiral stationary phases (CSP) are accomplished via a complex set of interactions between the analyte and the CSP. This complexity often leads to a time-consuming and trial-and-error approach to method development that can be unproductive. A systematic understanding of the underlying retention mechanisms would help analysts to make informed choices during chiral method development and optimization.
Macrocyclic glycopeptide and cyclodextrin-based CSPs have distinct and valuable benefits over cellulosic and amylosic CSPs. Because they operate in polar organic and aqueous-organic mobile phases, they are amenable to polar and ionizable analytes and mass spectrometric (MS) detection. LC-MS techniques may be utilized to run selectivity studies for a large variety of compounds in a short period of time. In this study, the LC-MS approach was utilized to study the impact of variables such as ion concentration, acid/base ratios, organic modifier type and pH on enantiomeric selectivity for a wide range of pharmaceutically-relevant compounds. The results of the study provide an improved understanding of the impact these variables have on chiral selectivity and thus offer a means to facilitate method development.
Author: Richard A. Henry – Sunday, March 8 – 2:00 pm, Room S503a
Co-Authors: Greg Baney, William Campbell
Title of abstract: Separation Mechanisms on 2.7 µm Superficially Porous Particle Phases for UHPLC
Abstract Content:
Recent advances in HPLC have focused on new instrumentation and particle technology to dramatically enhance column efficiency. However, even in UHPLC, knowledge of selectivity and control of analyte-phase interaction retains primary importance.
We have investigated C8, C18, RP-Amide and Phenyl phase chemistries on new 2.7 µm superficially porous silica particles to better understand the selectivity offered by these UHPLC columns. For small analytes, retention and selectivity can be treated as a competition for the analyte between stationary and mobile phases. Variation in selectivity can be attributed strictly to stationary phase differences if the mobile phase is held constant. Studies were carried out with designed sets of analytes such as toluenes, phenols, benzoic acids and anilines with para-substituents. Experiments included van’t Hoff plots to determine thermodynamic parameters for the analyte-phase interaction. Transition state enthalpies could be significantly related to selectivity for certain analytes when Amide, Phenyl, C8 and C18 phases were compared. A high degree of transition state order, or entropy, was also noted for Amide and Phenyl columns. Data suggests that both orientation and stability enhancements can accompany the use of stationary phases other than C18. The van’t Hoff relationships show that selectivity can be reversed in some cases by use of the temperature variable. Data correlate very well with widely accepted selectivity mechanisms for Amide and Phenyl phases and suggest that plots of retention versus temperature could be very useful for establishing how target analytes interact with a particular stationary phase.
Author: Craig Aurand – Tuesday, March 10 – 8:20 am, Room S503a
Co-Author: Dave Bell
Title of abstract: New Insights into Retention and Selectivity in Aqueous-Normal Phase/HILIC Separations
Abstract Content:
A significant interest in normal-phase chromatography using aqueous-organic mobile phases (aqueous normal-phase, ANP or also referred to as HILIC) has been observed in recent years [1,2]. In ANP, analyte retention increases with an increase in the organic component of the mobile phase. In previous studies, significant contribution of stationary phase chemistry toward the manipulation of retention and selectivity in ANP has been demonstrated [3]. The aim of this study was to enhance our knowledge of stationary phase chemistry contributions and, in turn, further our understanding of the governing retention mechanisms in this interesting mode of chromatography.
Through a combination of mass spectrometric selectivity and modern software tools, retention and selectivity data for a wide variety of analytes were obtained on bare silica and several polar, bonded phases in ANP/HILIC mode. Some of the columns included in the study were novel phases built on superficially-porous particles. Mobile phase variables thought to be important in this mode, such as buffer type and concentration have been contrasted and compared on each of the phases. Further evidence of bonded phase participation in the overall retention mechanism in ANP is shown. Insights into method development strategies as well as parameter considerations often neglected in reversed-phase processes are highlighted.
[1] W. Naidong, Journal of Chromatography B 796 (2003) 209.
[2] D.S. Bell, Jones, A. Daniel, Journal of Chromatography A 1073 (2005) 99.
[3] D.S. Bell, Brandes, Hillel K., in 30th International Symposium and Exhibit on High Performance Liquid Phase Separations and Related Techniques, San Francisco, California USA, 2006.
Author: Michael May (Isotec) – Sunday, March 8 – pm session
Co-Authors: Vladimir Katovic, John E. Kuo, Elizabeth Paul, Scott Purdin, CT Tan
Title of abstract: Electrochemical and Isotopic Investigation of Oxalic Acid
Abstract Content:
On platinum disk electrode, cyclic voltammetry of 1.3-mole% oxalic acid in water indicates that electrochemical processes are occurring near each potential limit when cycling between E = -0.2 volt and E = +0.8 volt (silver-silver chloride reference electrode/ 3-molar potassium chloride). Similar oxalic acid voltammograms were observed in deuterium oxide over the scan rate range from 0.02 to 0.10 volt/sec. Utilizing chronoamperometry, the above platinum disk area was estimated to be 0.011 cm2 by applying a potential step to 0.004-molar aqueous potassium ferricyanide. In order to facilitate the analytical-scale electrolysis of isotopic materials, an Integrated Vacuum Electrochemical Reactor (IVER) was previously developed that converts electroactive (liquid-phase) analyte to product gases. Structurally an IVER comprises a tubular Electrolysis Station in spatial linkage with a tubular Chemical Reaction Station, upper Valve Station that accommodates a metering valve, gastight Reactor Connector, and internal headspace for product gas storage. The IVER body is preferably constructed of borosilicate glass. For particular isotopic materials, mass spectrometric analysis of the electrolytic product gas from an IVER can provide quantitative isotopic enrichment. In the case of highly enriched oxalic acid-13C2, IVER-MS technology provides the atom% 13C-enrichment via electrolytic generation of carbon dioxide followed by mass spectrometry (single batch 99.5 +/- 0.02 atom%-13C/ n=10 samples). At sufficiently low electrolysis voltage the major gaseous products detected were dihydrogen and carbon dioxide, along with a minor amount of dioxygen (m/z 32 signal intensity <0.5% that of m/z 45).
Author: Hillel Brandes – Tuesday, March 10 – pm session
Co-Authors: Richard Henry, Paul Ross, Wayne Way
Title of abstract: Extended Performance of LC Instruments with Fused-Core™ Particle Columns
Abstract Content:
Recent advances in liquid chromatographic performance have focused on gains in peak efficiency afforded by sub-2µm particles, while requiring new instrument engineering to accommodate higher backpressures (> 9000 psi). Fused-Core particle columns, however, provide similar efficiencies to sub-2µm particle columns, while not incurring the magnitude of penalty in generated backpressure. Therefore, another level of performance can be realized from conventional HPLC instrumentation in combination with Fused-Core columns. One major advantage of very high efficiency columns is that they now afford the use of shorter, smaller columns, as longer lengths are no longer needed to achieve a given resolution. Smaller columns, however, mandate attention to details of system volume not previously required with conventional porous particle columns.
System dispersion is a measure of band broadening inherent in the instrument, and is related to system volume. While design of injectors and detectors can be optimized to minimize dispersion, this is not in the realm of end-user system optimizations. What an end-user can affect is the volume of the fluid path downstream from the point of sample injection.
This paper will discuss measurement of system dispersion and demonstrate the performance of Fused-Core particle columns that can be achieved on a popular conventional HPLC, as a function of system configuration. The only modifications to configuration considered are the fluid path downstream from the injector, including detector flow cell. The relative effect of various system modifications on measured peak efficiencies and system dispersion are documented.
Author: Craig Aurand – Tuesday, March 10 – pm session
Co-Authors: Hillel Brandes, David Bell, An Trinh, Paul Ross
Title of abstract: Phospholipid Depletion in Bioanalysis using HybridSPE Technology
Abstract Content:
Analysis of biological samples is often hindered due to interferences carried through the sample preparation technique. Protein precipitation is a widely accepted sample preparation method for biological plasma samples due to simplicity and gross level removal of proteins. Though widely used, protein precipitation methods often result in chromatographic irregularities due to co-extracted endogenous species such as phospholipids that negatively affect chromatographic analysis. A more thorough sample cleanup can be achieved using solid phase extraction (SPE), but at a cost of time and method complexity. This presentation details a new platform developed to process various plasma samples using a simplified two-step procedure which produce biological samples depleted of phospholipids prior to LC-MS/MS analysis.
Author: David Bell – Tuesday, March 10 – pm session
Co-Authors: Craig Aurand, Jennifer Claus, Dan Shollenberger, Jay Jones
Title of abstract: Chiral LC-MS Analysis of Drug Substances (Beta-Blockers) from Plasma Using Macrocyclic Glycopeptide Chiral Stationary Phases
Abstract Content:
The analysis of enantiomers in a clinical setting requires rapid and sensitive methodology. The tool of choice is often liquid chromatography coupled with mass spectrometry (LC-MS). The separation of chiral drug compounds within biological samples is often problematic using traditional amylosic and cellulosic chiral stationary phases (CSPs) as the mobile phases utilized to provide separation are often not amenable to LC-MS. Macrocyclic glycopeptide and cyclodextrin-based CSPs often provide enantiomeric selectivity using polar organic solvents or aqueous-organic mixtures as mobile phases. These mobile phases are readily amenable to LC-MS sources and, when coupled to the mass spectrometer, can provide the speed and sensitivity required for clinical analyses.
In this study, enantiomeric separation for several beta-blockers was investigated using MS-compatible mobile phases on CSPs based on different macrocyclic glycopeptide molecules. Applicability of the methodology toward clinical analyses is demonstrated using the analysis of selected beta-blockers from rat plasma.
Author: John Kuo – Tuesday, March 10 – pm session
Co-Authors: Michael Gray, Steve Klekar, Michael May, Lisa Roth, David Schory, CT Tan
Title of abstract: Distribution of 1H and 2H in Deuterated NMR Solvents and 2H atom % Determination using Binominal Model
Abstract Content:
With the fast advance of NMR technology, various demands on Deuterated NMR solvents have been steadily increasing. These demands include not only a wider diversity of NMR solvents, but also a higher Deuterium enrichment and ultra high solvent purity.
While to increase the Deuterium enrichment for deuterated NMR solvents has been specified as one of major goals in Stable Isotope industry, one should realize that there is a hydrogen isotope distribution for any deuterated solvent. This distribution is attributed to protium/deuterium exchange operations adapted by today's industry. For example, even highly deuterated acetonitrile-d3 contains a minor quantity of acetonitrile-d2, trace amounts of acetonitrile-d1, and perhaps even acetonitrile-d0. The 1H and 2H distribution can be observed using proton NMR and deuteron NMR, and under certain circumstances, the observed distribution can be used to quantitate the overall solvent deuterium enrichment using a binomial distribution model.
In this poster presentation, the hydrogen isotope distribution of three NMR solvents, namely acetonitrile-d3, DMSO-d6, and acetone-d6, are depicted. Their chemical shifts, peak patterns, 1H-2H nuclear coupling, and peak area measurements are also displayed. Numerical examples of overall enrichment for 2H atom% determination are presented, and conditions under which a binomial distribution can or cannot be applied are proposed.
Author: Michael Ye – Wednesday, March 11 – pm session
Co-Authors: Craig Aurand, Charles Mi, An Trinh
Title of abstract: High Throughput Removal of Both Phospholipids and Proteins in Bioanalytical Sample Preparation
Abstract Content:
Sample preparation is a must in bioanalysis due to complex sample matrices such as serum and plasma, and delicacy of subsequent analytical instruments, typically liquid chromatography-mass spectrometry (LC-MS). Currently, the commonly used technology is 96-well protein precipitation for its fast speed and simplicity. However among the critical endogenous interferences in the matrices, it removes only proteins. Other critical interferences, such as phospholipids, still remain in the sample. It is well documented that the presence of phospholipids is one of the main causes for ion-suppression in mass spectrometry analysis, leading to low recovery and high variation of analytical results. Phospholipids could also accumulate in the analytical HPLC column, generating high column backpressure and unwanted chromatography peaks.
In this presentation, we will present a new technology that retains the speed and simplicity of protein precipitation plate while removing both proteins and phospholipids in biological samples. The technology utilizes zirconia-coated particles that exhibit selective binding towards phospholipids while remaining non-binding towards a range of basic, neutral and acidic compounds under the experimental conditions. Packing that material into a 96-well format becomes a highly desired tool for bioanalytical sample preparation. We will discuss the technology, demonstrate with experiments the removal of phospholipids and reduction of ion-suppression in mass spectrometry, and show the analytical results of a wide range of pharmaceutical compounds by applying this technology.
Author: William Betz – Wednesday, March 11 – pm session
Co-Authors: Mike Keeler, Craig Aurand, Katherine Stenerson, Len Sidisky
Title of abstract: New 300Å Zirconia and Titania Micropipette Tips for the Concentrations and Analyses of Phosphopeptides in Biological Matrices
Abstract Content:
Micropipette Tips have emerged as an effective analytical tool for the sample preparation of trace levels of biological samples. Micropipette tips are micro-scale forms of solid phase extraction (SPE) cartridges, and function to enrich the concentrations of specific biological molecules in micro-volume samples. Relative to SPE, the micropipette sample volumes are approximately 1000 times less than SPE sample volumes.
In this micropipette technique a sorbent is packed and/or attached to the inner walls of the pipette tip with an adhesive at the working end of the tips. The same analytical principles used in SPE apply for the sample preparation with micropipette tips (i.e., enrichment followed by elution). Micropipette tips are used to handle samples volumes as low as 1.0 µL. The samples are desalted or concentrated using these tips and analyzed by ESI-MS, MALDI-TOF-MS, LC-MS, HPLC and mass spectrometry/chromatography techniques.
The use of zirconia and/or titania adsorbents for the enrichment/binding of phosphopeptides in acidic environments followed by elution in basic environments is well documented. Two new patented composites have been prepared with 300Å mesopores for the analyses of phosphopeptides. One composite is a zirconia-silica composite and the second is a titania-silica composite. These 50um spherical composites are subsequently adhered to the inside of the working tip area using a proprietary, patented adhesive (US patent # 5,599,445).
A nitrogen porosimeter was used to study the surface areas, pore size distributions and total pore volumes of the composites. A helium pycnometer has been used to determine the helium densities. Adsorbent capacities and reversible adsorption characteristics were determined using the micropipette sample preparation processes for phosphopeptides.
Author: Robert E. Shirey – Wednesday, March 11 – pm session
Co-Authors: Craig Aurand, Katherine Stenerson, Yong Chen, Len Sidisky
Title of abstract: Newly Developed SPME Fibers Specifically for HPLC Use
Since SPME was introduced, fiber development has been primarily focused for extraction of analytes followed by thermal desorption into a GC inlet. The emphasis was on the development of coatings for extraction of small, thermally stable molecules < 300 AMU. These coatings have good thermal stability and properties that retain analytes either by adsorption or absorption mechanisms.
When SPME is used for the extraction of analytes requiring HPLC for analysis, solvent desorption is required. Even though the current SPME coatings are stable in many solvents, swelling of the phase can occur. If swelling of an exposed fiber occurs, the swollen coating may be stripped off the core by the outer SPME needle when the fiber is retracted. This makes using SPME for extraction of analytes requiring HPLC difficult.
A new line of SPME fibers has been developed that is designed primarily for HPLC use. These fibers contain bonded silica embedded in a non-swelling binder. This highly stable binder is very inert and allows molecules <700 AMU to pass through and interact with the coating bonded to the silica. Also, the binder is biocompatible in that it repels large macromolecules such as proteins.
This presentation will discuss some of the properties of the coatings. A variety of applications in clinical, environmental and pharmaceutical areas will be presented. The differences between the coating types will be shown. Examples will be given that show the ability of the fibers to extract analytes directly out of biological fluids without pretreatment of the fluids.
Author: Olga Shimelis – Thursday, March 12 – am session
Co-Authors: An Trinh1, Michael Ye1, Anna Karin Whilborg2, Brian Boyd2
1) Supelco, Bellefonte, PA, USA
2) MIP Technologies AB, Lund, Sweden
Title of abstract: Simplification of Sample Cleanup for Difficult Matrices Using Molecularly Imprinted Polymers
Abstract Content:
Extraction of trace residues in sample pre-treatment is often elaborate and time consuming. Solid phase extraction (SPE) phases based on molecularly imprinted polymers (MIP) eliminate the need for multiple extraction steps, simplifying the pretreatment procedure. MIP phases in most cases reduce the sample handling time, yield cleaner extracts, lower detection limits and improve MS compatibility. MIP phases contain pre-formed cavities that are complimentary in shape and chemical properties to the target analytes. It allows selective extraction of either single molecular species or ‘classes’ of molecules containing the same functional groups.
Analysis of antibiotics in food is important for monitoring food quality. In this presentation, we will report using MIP SPE to clean up complex food matrices to achieve lower detection limit of chloramphenicol and fluoroquinolones, two widely used antibiotic classes found in food products, such as milk and honey. The MIP phases have been developed specifically for the extraction of chloramphenicol and fluoroquinolones. The extracted samples were analyzed by LC/MS/MS without the need for matrix-matched standards. The presentation will present the method performance data and compare MIPs SPE to the previously used sample pre-treatment methods.
Author: JT Lee – Thursday, March 12 – am session session
Co-Authors: William Campbell, Jennifer Claus, Dave Bell
Title of abstract: Benefits of Preparative Chiral Separations with Macrocyclic Glycopeptide CSPs in Reversed-Phase and Polar Ionic/Polar Organic Modes
Abstract Content:
For the chiral purification of enantiomers using elution chromatography, aqueous-based reversed-phase systems and those employing polar organic solvents have distinct and valuable benefits over traditional normal phase systems. These benefits can be exploited to improve sample solubility, capacity, throughput and yields. Also, the predominantly aqueous mobile phase systems are less toxic and reduce the amount of organic solvents consumed and must be disposed of. However, few chiral stationary phases (CSPs) are able to accommodate the highly-polar aqueous reversed-phase environment.
The exception has been the CSPs based on macrocyclic glycopeptides bonded to high-purity silica particles. These CSPs (termed “CHIROBIOTIC”) operate in any solvent system, including very polar and halogenated solvents, allowing the user to optimize the preparative separation based on a balance between enantioselectivity and sample solubility. The macrocyclic glycopeptide CSPs also have desirable preparative attributes of predictable scale-up because the same chemistry is employed on all particle sizes. Stable chemistry for no-bleed operation and long-term stability is achieved through the multiple covalent linkages that attach the CSP to the silica surface.
This poster will present data demonstrating the preparative performance of the macrocyclic glycopeptide CSPs in reversed-phase and polar organic/polar ionic mobile phase systems. Additionally, procedures to isolate the purified enantiomers post-separation will also be presented.
Author: JT Lee – Thursday, March 12 – am session
Co-Author: William Campbell
Title of abstract: Chiral Purification Using Macrocyclic Glycopeptide CSPs with Simulated Moving Bed (SMB) Technology.
Abstract Content:
Chirality has long been regarded as one of the critical issues in drug design and the discovery processes in pharmaceutical and biotechnology industries. During the early development stages, the pharmacokinetic aspects of chiral drugs need to be addressed because each enantiomer can behave differently in terms of absorption, distribution, metabolism and excretion in clinical studies. Thus, small amounts of pure enantiomers are needed for these studies for any chiral molecules under development.
LC chiral stationary phases (CSPs) made by bonding macrocyclic glycopeptides to silica particles have demonstrated very wide chiral selectivity and excellent robustness since their introduction in 1995. Also, the advances in simulated moving bed (SMB) technology have been successfully applied to many large-scale binary separations of sugars, hydrocarbons and other small molecules.
This poster will explore the methodology of utilizing macrocyclic glycopeptide CSPs in SMB systems to purify chiral pharmaceutical compounds in various mobile phase systems. The advantages of this approach over traditional batch preparative chromatography will be discussed in terms of overall throughput, purity of the enantiomers and the reduction of solvent waste. The ease of method development and ruggedness of this system and the chiral stationary phases will also be demonstrated.
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