Liquid Chromatography/Chemical Reaction Interface Mass Spectrometry as an Alternative to Radioisotopes for Quantitative Drug Metabolism Studies

Abstract

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.

Flow injection with chemical reaction interface-isotope ratio mass spectrometry: an alternative to off-line combustion for detecting low levels of enriched 13C in mass balance studies

“…The urine results showed a ␦ 13 C value 2.8‰ above the control 13 C/ 12 C ratio using a 1% aliquot (8 -16 mL) of a 24 h human urine sample. This elevated ratio converts to detecting an 83 Ϯ 52% excretion of the administered 13 C aspirin dose into urine. Nakagawa et al [2] measured ␦ 13 C and ␦ 15 N in rat urine, feces, liver, and hair, again by combustion and measurement of the resulting CO 2 and N 2 by IRMS.…”

“…We can detect trace amounts of a 13 C-labeled drug (3Ј-azido-3Ј-deoxythymidine, AZT) in urine or feces. Our ability to quantify less than 100 ng/mL of excess 13 C (ϳ1 g/mL of 13 C-labeled AZT) from a sample equivalent to 10 L of urine is superior to previous detection limits for 13 C in urine that use off-line combustion methods. Parallel studies using 14 C-labeled AZT showed that our stable isotope method provides comparable percent excretion data for urine and feces.…”

“…They used the conventional method where a sample of urine was combusted and the resulting CO 2 was admitted into an IRMS. Their studies of variability in the baseline for the 13 C/ 12 C ratio from urine showed only small [SD ␦ 13 C PDB Ӎ 1‰ (this terminology is explained in the Methods section)] fluctuations over a 10 day period. This was reassuring because variations in diet will be reflected as variations in the baseline and could significantly limit the utility of stable isotope methods where differences below 1‰ can be measured.…”

“…This was reassuring because variations in diet will be reflected as variations in the baseline and could significantly limit the utility of stable isotope methods where differences below 1‰ can be measured. Knowing that the reproducibility of 13 C ratios of urine was good enough to permit this type of experiment, one subject was given a 0.32 mg dose of 13 C-labeled aspirin along with a 320 mg dose of unlabeled aspirin. The urine results showed a ␦ 13 C value 2.8‰ above the control 13 C/ 12 C ratio using a 1% aliquot (8 -16 mL) of a 24 h human urine sample.…”

“…Knowing that the reproducibility of 13 C ratios of urine was good enough to permit this type of experiment, one subject was given a 0.32 mg dose of 13 C-labeled aspirin along with a 320 mg dose of unlabeled aspirin. The urine results showed a ␦ 13 C value 2.8‰ above the control 13 C/ 12 C ratio using a 1% aliquot (8 -16 mL) of a 24 h human urine sample. This elevated ratio converts to detecting an 83 Ϯ 52% excretion of the administered 13 C aspirin dose into urine.…”