Petra Kotnik

Bioactivation of bisphenol A and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes

Bisphenol A analogs are a class of chemicals known as diphenylmethanes, which contain two benzene rings separated by one central carbon atom, usually with a para-hydroxy group on both benzene rings. Bisphenol A (BPA) can induce an uterotrophic response in immature CD-1 mice and elicits estrogenic responses in many other experimental systems. Besides highlighting endocrine effects, a number of metabolic studies provide strong support for the idea that reactive species of BPA are formed in vitro and in vivo that can form covalent adducts with nucleophilic macromolecules and/or produce oxidative stress. We used a liquid chromatography with a triple quadrupole tandem mass spectrometry (LC-MS/MS) for the detection of metabolites and glutathione conjugates of BPA and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes (HLM) or with recombinant CYP isozymes in the presence of NADPH and GSH as a trapping agent. We have confirmed that BPA and its structural analogs form hydroxylated metabolites and electrophilic species during bioactivation in HLM and CYP isozymes. These results provided important mechanistic insight into the metabolic fate of BPA structural analogs in vitro.

Phase equilibria in systems containing α-tocopherol and dense gas

Abstract Solubility of α-tocopherol in CO 2 and propane was determined at temperatures 303, 313, 333 and 353 K and over a pressure range from 79 to 286 bar for CO 2 and 16 to 112 bar for propane. A static-analytic method was applied. The solubility of α-tocopherol in dense CO 2 under the conditions investigated was in the range of 0.2–17.0 mg/g CO 2 . The maximal solubilities of α-tocopherol in propane were approximately up to ten times higher than in CO 2 and were in the range from 38.9 to 171.9 mg/g propane. Phase equilibrium data for Milk Thistle seed oil in supercritical CO 2 were determined at temperatures 313, 333 and 353 K and pressures ranging from 100 to 300 bar. The oil was previously additionally vitaminized and contained 1.9 wt.% of α-tocopherol and 1.7 wt.% of free fatty acids (FFA). The solubility of oil in CO 2 was in the range from 1.3 to 17.9 mg/g CO 2 . The distribution coefficients of α-tocopherol and FFA between light and heavy phase were determined on a solvent free basis. The obtained separation factors increased with temperature and pressure to approximately 160–200 bar and decreased with a further increase of pressure.

HPLC–MS/MS method optimisation for matrix metalloproteinase 3 and matrix metalloproteinase 9 determination in human blood serum using target analysis

A quantitative analysis of zinc endopeptidases matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 3 (MMP3) from human blood serum are presented. Both matrix metalloproteinases (MMP) are present in human blood serum and can be used as biomarkers for different diseases. The analysis was performed using LC-MS/MS with a triple quadrupole mass spectrometer, based on two specific peptides of each MMP in comparison with an enzyme-linked immunosorbent assay (ELISA). While the conditions for the LC-MS/MS analysis of MMP9 peptides were previously reported for bronchoalveolar lavage fluid, the analysis of MMP3 peptides was newly quantified for human blood serum herein for the first time. For MMP3, the linear behaviour was determined in the concentration range from 1.0-200.0ng/mL (R2=0.997) with an LLOD of 0.5ng/mL. For MMP9, linearity was determined in the concentration range from 6.5-65.0ng/mL (R2=0.995) with an LLOD of 2.0ng/mL.