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. Product ions of the known standard and an unknown benzodiazepine were successively captured and fragmented (MS(n) experiments) using an ion-trap mass spectrometer off-line, which confirmed that the unknown was an analogue of chlordiazepoxide.