Chemical reaction interface mass spectrometry (CRIMS) was coupled on-line with HPLC using a Vestec particle beam interface. A helium-assisted nebulizer provided added stability with no loss in accuracy or precision as compared to the thermospray nebulizer at flow rates of up to 1.0 mL/min using isocratic conditions. However, mass spectral response was found to be solvent-dependent for both the helium-assisted and thermospray nebulizers. Postcolumn solvent addition of methanol eliminated solvent-dependent decreases in mass spectral response. This allowed gradient HPLC elutions to be performed. Under these conditions, the flow of solvent into the particle beam interface was 2.5 mL/min, so a conventional thermospray nebulizer had to be used instead of the helium-assisted nebulizer. Experiments were conducted with the antianxiety agent buspirone in order to validate the methodology. Metabolites from in vitro incubations of [15N]/[14C]buspirone with rat liver slices were analyzed by gradient LC/CRIMS and by gradient LC/[14C] radioactivity counting. The response from LC/CRIMS analysis for individual metabolites was then compared with that obtained by LC/[14C] radioactivity counting. An excellent correlation was observed between the two methods for metabolites with quite different HPLC characteristics. Thus, gradient LC/CRIMS in combination with stable isotopes provides an alternative to using radioisotopes for carrying out drug metabolism studies.
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