6. LC/MS Diskussionstreffen, 29. Nov. 2005, Wuppertal ABSTRACTS

Abstracts: 6. LC/MS Diskussionstreffen, 29. Nov. 2005, Wuppertal 5
Turbulent Flow Chromatography as an Effective Tool for Reducing Matrix
Effects in ESI-MS-Based Enzymatic Bioassays
André Liesener and Uwe Karst
University of Twente, Chemical Analysis Group and
MESA + Institute for Nanotechnology, P.O. Box 217,
7500 AE Enschede, The Netherlands
In the analysis of samples originating from enzymatic bioassays by means of electrospray
ionization mass spectrometry (ESI-MS), one of the major issues regarding the reliability of
the method is the possible influence of matrix constituents. ESI-MS is known to be very
susceptible to both, ionization suppression and ionization enhancement of low molecular
weight analytes in the presence of large biomolecules such as enzymes. In order to minimize
these disturbing effects, it is desirable to remove matrix components prior to the ESI-MS
analysis. Due to the typically large number of samples, which have to be analyzed during a
complete assay, sample preparation has to be fast and automated. A sample preparation
method fulfilling these criteria is the so-called turbulent flow chromatography (TFC). In this
approach, the samples are directly injected into a high flow rate aqueous mobile phase
stream and transferred onto a microbore column containing a large particle size stationary
phase, where the low molecular weight analytes are retained, while the high molecular
weight compounds and salts are rapidly washed off.
The effectiveness of TFC as means to reduce matrix effects in samples originating from
simultaneous multianalyte mass spectrometric bioassays is explored. In this study, the
effects of enzymes present in the sample on the signal response of five analytes were
simultaneously investigated over a protein content range from 0 �g/mL to 38 �g/mL by
means of direct flow injection mass spectrometry. As model enzymes, trypsin, thrombin and
chymotrypsin were selected. Without TFC, the matrix effects caused both signal suppression
and signal enhancement depending on the nature of the analyte and the amount of matrix in
the sample and were found to be generally intolerably large. The deviation from the mean
signal response as a measure of distortion was found to be between 14% up to 112%. The
addition of an excess of methanol as means of sample clean-up was investigated and found
not to be sufficient. By employing TFC for on-line sample preparation, it was possible to
reduce the matrix effects to a minimum for all model systems investigated. In case of trypsin,
the distortion could be lowered from 41.9% to 2.6%, thus demonstrating clearly the efficiency
of the TFC-based on-line sample clean-up.