Oct 21, 2014 – MSDG Meeting

I. Kevin Schug, Ph.D.

Shimadzu Distinguished Professor of Analytical Chemistry, Department of Chemistry & Biochemistry, The University of Texas at Arlington

“On-Line Preparation, Separation, and Quantitative Determination of Small and Large Molecules in a Multipath Liquid Chromatography – Mass Spectrometry System”

II. Fred Regnier,

J.H.Law Distinguished Professor of Chemistry Emeritus, Purdue University and C.E.O of Novilytic. West Lafayette, Indiana

“Sample Preparation: The Achilles Heel of Rapid Mass Spectral Analysis”

Abstract for Talk I:

Many precious biological samples may contain multiple analytes of interest; these may span the range from small molecule metabolites to large proteins. Typically, sample preparation, analytical separation, and quantitative determination schemes for different classes of chemical compounds can vary substantially – so much so that they require completely separate analyses. It has been our goal to develop a multipath liquid chromatography – mass spectrometry system, which enables sample preparation and analysis of multiple chemical compound classes from a single injection. Through integrated restricted access media, multiple valves, and flow-paths, it is possible to segregate the components of a complex biological mixture and facilitate their separation on appropriate chromatographic phases, followed by ultra-trace detection on a triple quadrupole mass spectrometer. This could be desirable to streamline analysis of, for example, multiple biomarkers or small and large molecule conversion products of biotherapeutics, simultaneously in one analytical run. This talk will describe our progression of work aimed towards those ends, including development of methods for: Ultra-trace determination of small molecules using bulk derivatization and restricted access media; optimization of multiple reaction monitoring schemes for intact protein analysis; and integration of these components into a single multipath instrument for segregation and simultaneous separation and determination of small and large molecule analytes.

Abstract for Talk II:

The issue of how to rapidly identify and quantify multiple substances in biological samples is a subject of great interest today, particularly in drug metabolism and pharmacokinetics, biomarker discovery and validation, clinical diagnostics, and translational medicine. The potential of mass spectrometry (MS) in this arena is a function of how fast data can be acquired from highly complex biological samples. Although MS can identify and quantify hundreds of analytes within msec, sample preparation frequently takes 12-24 hours. Clearly sample preparation is a substantial problem in addition to being boring, labor intensive, and costly. This presentation will examine ways in which this problem can be circumvented.

The fact that biological samples contain cells along with greater than 10⁵ molecular species means that multiple types of prefractionation must occur before introduction into an MS. Cell removal, analyte extractions and enrichment, and subtraction of interfering substances are all part of the sample preparation process. Chemical modifications of analytes ranging from derivatization to digestion are also necessary in many cases. Finally there is the issue of sample origin. When samples are derived at a source other than the MS lab there is the issue is how to get them to the laboratory, what happens to them en route, and following their identity as they pass through multiple preparation steps.

Clearly these problems can be minimized by automation. But the problem is more complicated than that. Automating multiple individual steps still takes a lot of space and probably does not reduce sample preparation time. A focus of this presentation will be on how to automate multiple steps of sample preparation simultaneously in a single device.