Miniaturized method for the quantification of persistent organic pollutants and their metabolites in HepG2 cells: assessment of their biotransformation.

Biotransformation can greatly influence the accumulation and, subsequently, toxicity of substances in living beings. Although traditionally these studies to quantify metabolization of a compound have been carried out with in vivo species, currently, in vitro test methods with very different cell lines are being developed for their evaluation. However, this is still a very limited field due to multiple variables of a very diverse nature. So, an increasing number of analytical chemists are working with cells or other similar biological samples of very small size. This makes it necessary to address the development of analytical methods that allow determining their concentration both inside the cells and in their exposure medium. The aim of this study is to develop a set of analytical methodologies for the quantification of polycyclic aromatic hydrocarbons, PAHs (phenanthrene, PHE), and polybrominated diphenyl ethers, PBDEs (2,2',4,4'-tetrabromodiphenyl ether, BDE-47), and their major metabolites in cells and their exposure medium. Analytical methodologies, based on miniaturized ultrasound probe-assisted extraction, gas chromatography-mass spectrometry-microelectron capture detector (GC-MS-µECD), and liquid chromatography-fluorescence detector (LC-FL) determination techniques, have been optimized and then applied to a biotransformation study in HepG2 at 48 h of exposure. Significant concentrations of the major metabolites of PHE (1-OH, 2-OH, 3-OH, 4-OH-, and 9-OH-PHE) and BDE-47 (5-MeO-, 5-OH-, and 3-OH-BDE-47) were detected and quantified inside the cells and in the exposure medium. These results provide a new method for determination and improve information on the metabolization ratios for a better knowledge of the metabolic pathways and their toxicity.