Analysis of benzo(a)pyrene metabolites by mass spectrometry
Abstract
A continuous-flow fast atom bombardment mass spectrometry (FABMS) method was developed for analysis of benzo(a)pyrene (BP) conjugates. Continuous-flow FAB mass spectra of BP-sulfates and BP-glucuronides acquired in the negative ion mode gave characteristic molecular anions and fragment ions. About fifty times more sample was required to produce spectra with similar signal-to-noise ratio by standard FABMS. A method based on high resolution continuous-flow FABMS was developed for the analysis of BP-sulfates isolated from medium of BP-treated human hepatoma cell cultures (HepG2). A detection limit of less than 1 pg was obtained using high resolution (m/$\Delta$m 5000) single ion monitoring at the molecular anion (m/z 347.0378) of BP-sulfate. HPLC Fractions from BP-treated cells corresponding to the retention time of authentic BP-sulfates gave a mass spectrometric response and fractions from medium of untreated HepG2 cells had UV-absorbing material but gave no mass spectrometric response. The amount of BP-sulfates in the medium was calculated based on a calibration curve and recovery rates obtained by using authentic standards. This continuous-flow FABMS method with some modification was applied to analyses of BP-glucuronides isolated from medium of BP-treated hamster embryo cells, the mouse hepatoma cell line Hepa-1c1c7 and HepG2 cell cultures. A detection limit of 1 pg was obtained by using high resolution (m/$\Delta$m 10,000) single ion monitoring at the molecular anion of BP-glucuronide (m/z 443.1129). An on-line LCMS method, based on high resolution continuous-flow FABMS, was developed for the rapid detection of BP-conjugates from cell cultures. Analysis of medium from BP-treated HepG2, hamster embryo, and Hepa-1c1c7 cell cultures by a reversed phase microbore HPLC system that separates BP-sulfates from BP-glucuronides and also resolves the isomeric 3-BP-glucuronide and 1-BP-glucuronide and showed that the majority of the BP-conjugates in HepG2 cell culture were BP-sulfates and both Hepa-1c1c7 and hamster embryo cell cultures produce at least two isomeric BP-glucuronides. These highly sensitive continuous-flow FABMS methods allow the analysis of polycyclic hydrocarbon metabolites at levels formed in living cells without requiring the use of radioisotope labelled hydrocarbons and should be generally applicable for the direct analysis of hydrocarbon glucuronides and sulfates formed in biological systems.
Degree
Ph.D.
Advisors
Baird, Purdue University.
Subject Area
Analytical chemistry
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