In drug development, the combinatorial synthesis of drug libraries is common use, therefore efficient tools for the characterization of drug candidates and the extent of interaction between a drug and its target protein is a central question of analytical interest. While biological activity is tested today by enzyme assays, MS techniques attract more and more attention as an alternative for a rapid comparison of drug-target interactions. CE enables the separation of proteins and drug-enzyme complexes preserving their physiological activity in aqueous media. By hyphenating CE with ESI-MS/MS, the binding strength of enzyme inhibitors can be deduced from MS/MS experiments, which selectively release the inhibitor from the drug-target complex after CID. In this study, alpha-chymotrypsin (CT), a serine protease, was chosen as a model compound. Chymostatin is a naturally occurring peptide aldehyde binding to CT through a hemiacetal bond and electrostatic interaction. First, a CE separation was developed, which allows the analysis of alpha-CT and a chymotrypsin-chymostatin complex under MS-compatible conditions. The use of neutral-coated CE capillaries was mandatory to reduce analyte-wall interactions. ESI-quadrupole ion trap-MS was worked out to demonstrate the selective drug release after CID. Fragmentation of the drug-enzyme complex was monitored in dependence from the excitation energy in the ion trap, leading to the V(50) voltage that enables 50% complex fragmentation as a reference value for chymotrypsin-chymostatin complex. A stable CE-ESI-MS/MS setup was established, which preserves the drug-enzyme complexes during ionization-desolvation processes. With this optimized setup, different CT inhibitors could be investigated and compared.

Determination of trace constituents in biological and environmental samples usually requires a pre-concentration step. While solid-phase extraction (SPE) has been widely used, it is slow, labor intensive and adversely affected by analytical errors from handling. On-line SPE eliminates some of the flaws but often suffers from solvent compatibility problems with the subsequent chromatography separation. In this study, we are presenting a technical solution for overcoming some of these compatibility issues, by utilizing a fully automated, focused SPE sample transfer technique utilizing narrow-band solvent plugs, for seamless hyphenation with high-performance liquid chromatography (HPLC) or flow injection mass spectrometry (MS). A wide range of pharmaceutical compounds was studied in different sample matrices. Short plugs of high elution strength solvent were generated by means of an electrically actuated sample loop and enrichment and transfer steps monitored using on-line SPE-MS. The impact of the solvent plugs on chromatographic separation was studied using hyphenated SPE-LC-MS. By carefully examining elution profiles of solvent plugs of different compositions, optimum conditions for quantitative elution within well-defined volumes were found for all substances. In addition, the highly focused elution bands resulted in excellent retention time and peak area reproducibilities when injected on-line onto HPLC columns. Finally, to demonstrate proof-of-principle, the fully integrated on-line SPE-LC-MS system was applied to the analysis of spiked urine and river water samples.

This article was originally published in Electrophoresis 2010, 31, 1248–1255, DOI

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