Determination of benzo[a]pyrene sulfate conjugates from benzo[a]pyrene-treated cells by continuous-flow fast atom bombardment mass spectrometry

Abstract

The level of certain water-soluble hydrocarbon conjugates, such as benzo[a]pyrene sulfates (BP-SO4), is a direct measure of carcinogenic polycyclic aromatic hydrocarbon metabolism and an indication of exposure. A new method, based on continuous-flow high-resolution fast atom bombardment mass spectrometry, has been developed for the analysis of BP-SO4 in the medium of cell cultures treated with benzo[a]pyrene. An organic solvent extract of medium from cultures of the human hepatoma cell line (HepG2) was fractionated by reversed-phase SEP-PAK chromatography and microbore high-performance liquid chromatography (HPLC). The HPLC fraction containing BP-SO4 was collected, dried, and injected into a stream of acetonitrile/water/glycerol that was continuously flowing to the tip of the sample probe which was being bombarded continuously by a beam of high-energy xenon atoms. Molecular anions of BP-SO4 (m/z 347) desorbed from the liquid were analyzed by a high-resolution (m/delta m 5000) mass spectrometer and recorded as a function of time. As little as 1.5 pg of BP-SO4 could be detected with a S/N ratio of 8. The mass spectrometer response was linear with respect to the quantity of BP-SO4 injected over the range from 15 to 625 pg. The results obtained with this method show that the HepG2 cultures metabolized 3% of the benzo[a]pyrene into the BP-SO4 conjugate in 24 h. This procedure, which was used to detect and quantify directly BP-SO4 in culture medium without the use of a radiolabeled precursor, should be generally applicable for analyses of sulfated conjugates resulting from the metabolism of different hydrocarbons.