Restricted Access Media Column Research Articles

Characterization of benzodiazepine "combinatorial" chemical libraries by on-line immunoaffinity extraction, coupled column HPLC-ion spray mass spectrometry-tandem mass spectrometry.

To characterize combinatorial chemical libraries of small drug compounds, an automated column switching system incorporating an immunoaffinity extraction (IAE) column and two reversed-phase HPLC columns was coupled to a triple-quadrupole mass spectrometer. A Protein G column and antibodies to benzodiazepines were used to screen library components. A pH change in the mobile phase eluted the benzodiazepine-antibody complexes onto a C-18 restricted access media (RAM) column, thereby separating the selected benzodiazepines from the antibody. In a final step, backflushing the RAM column eluted the benzodiazepines onto a C-8 analytical reversed-phase column for separation before detection and preliminary structural characterization using ion spray mass spectrometry (MS) and tandem mass spectrometry (MS/ MS). A known 19-component library and an unknown 20-component library were analyzed. Full-scan IAE/LC/ LC/MS and IAE/LC/LC/MS/MS chromatograms suggested the feasibility of this combination of techniques, although the antibodies used were not highly specific. Inspection of MS/MS spectra of components in the unknown library compared to the MS/MS spectrum of a known standard (chlordiazepoxide) identified a subclass of benzodiazepines. Productions of the known standard and an unknown benzodiazepine were successively captured and fragmented (MSn experiments) using an iontrap mass spectrometer off-line, which confirmed that the unknown was an analogue of chlordiazepoxide.