Liquid chromatography/chemical reaction interface mass spectrometry as an alternative to radioisotopes for quantitative drug metabolism studies.
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.
Goldthwaite
C A
CA
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
Hsieh
F Y
FY
Womble
S W
SW
Nobes
B J
BJ
Blair
I A
IA
Klunk
L J
LJ
Mayol
R F
RF
D000818:Animals; D002065:Buspirone / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017366:Serotonin Receptor Agonists / Q000032:analysis / Q000378:metabolism / Q000493:pharmacokinetics; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet
D004364:Pharmaceutical Preparations; D017366:Serotonin Receptor Agonists; D012997:Solvents; D002065:Buspirone