DATE Thursday, May 20, 2004

TIME 6:00 pm Informal Dinner

7:00 pm Presentation

LOCATION Advancis Pharmaceutical Corporation, Germantown, MD

SPECIAL NOTE: Due to security concerns, reservations are required for this

meeting. Reservations may be made by contacting Kate Rimmer at crimmer@nist.gov or (301)975-3651.

FEATURING DAVID S. BELL^1,2

Solute Attributes and Molecular Interactions Contributing to “U-Shape” Retention on A Fluorinated Stationary Phase

¹Department of Chemistry, The Pennsylvania State University

University Park, PA 16802

²Supelco Division of Sigma-Aldrich, 595 North Harrison Road, Bellefonte, PA 16823

ABSTRACT

The combination of high performance liquid chromatography (HPLC or LC) and mass spectrometry (MS) has become the dominant analytical tool in analysis of pharmaceuticals and for metabolite analysis. LC/MS, however, suffers from serious limitations in analysis of polar, low molecular mass (< 500 Da) metabolites, which are often poorly retained on common HPLC stationary phases. Inadequate chromatography can result in significant matrix effects and poor quantitation. In traditional chromatographic analysis, ion-pair reagents are often added to mobile phases to provide retention for polar, ionizable analytes. These reagents, however, are generally non-volatile and suppress ionization in LC/MS experiments. Other separation techniques such as capillary electrophoresis are also suitably employed for retention and separation of such analytes, but the predominantly aqueous solvents and high ionic strength buffers employed are less amenable to MS interfacing than many liquid chromatographic systems. It is therefore desirable to design stationary phases capable of retaining polar analytes using mobile phase constituents suitable for mass spectral analysis.

Fluorinated, silica-based stationary phases have shown unique retention for small, polar analytes. In particular, pentafluorophenylpropyl (PFPP) phases exhibit both reversed-phase and normal-phase retention for polar analytes, which has shown to be dependent on mobile phase composition. At lower percentages of organic modifier, solute retention resembles that of classical reversed-phase systems. At higher percentages of organic, however, behavior more typical of normal-phase separations is observed with increasing proportions of organic modifier. The normal-phase behavior is observed using mobile phase components common to reversed-phase LC that are highly compatible with mass spectrometry. To date, the retention mechanisms responsible for the normal-phase behavior and the fundamental properties of analytes that exhibit this phenomenon remain unclear.

Herein we report the retention characteristics for several classes of pharmaceutically relevant compounds on both PFPP and traditional C18 bonded stationary phases. Retention characteristics are related to analyte structure and the impact of the unique retention on the PFPP phase for LC/MS experiments is highlighted. Furthermore, results from fundamental studies aimed at establishing the molecular interactions responsible for the observed retention are presented.

SPEAKER BIOGRAPHY

After receiving his B.S. degree from SUNY Plattsburgh in 1989, Dave gained employment within the pharmaceutical industry where he was involved in analytical method development using various forms of chromatography and electrophoresis for 8 years. For the past 7 years, working directly in the chromatography industry, Dave has focused his efforts on the design, development and application of HPLC stationary phases. Of special interest is the understanding of molecular interactions that contribute to retention and selectivity in chromatographic processes. He is currently completing his graduate work toward a Ph.D. in Analytical Chemistry at The Pennsylvania State University where the focus has been the development of mass spectrometry platforms for the analysis of small, polar molecules.