Validation of LC/MS/MS method for assessment of the "in vitro" activity of selected rat cytochrome P450 isoenzymes : application to early drug metabolism screening

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous determination of seven metabolites of CYP450 model substrates (acetaminophen, 4-hydroxytolbutamide, 4'-hydroxymephenytoin, 1-hydroxybufuralol, 6-hydroxychlorzoxazone. 1'-and 4-hydroxymidazolam) in rat liver microsomes was developed. The assay used Kinetex analytical column and a gradient mobile phase consistent of acetonitrile and water with addition of 0.1\% formic acid. The analysis was performed in selected reaction monitoring (SRM) mode both in positive and negative (for 6-hydroxychlorzoxazone) mode. The method was validated over the concentration ranges of 10-2000 ng/mL for 4-hydroxymephenytoin and 4-hydroxytolbutamide, 50-2000 ng/mL for 1-hydroxybufuralol and 25-2000 ng/mL for the rest of the analytes. The intra- and inter-day precision (2-12%) and accuracy (93-119%) were within the limits set by the FDA and EMA guidelines. The developed method was successfully applied to assess the activity of selected CYP450 isoenzymes in rat liver microsomes after addition of ketoconazole

Validation of LC/MS/MS method for assessment of the "in vitro" activity of the selected rat cytochrome P450 isoenzymes : application to early drug metabolism screening

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous determination of seven metabolites of CYP450 model substrates (acetaminophen, 4-hydroxytolbutamide, 4í-hydroxymephenytoin, 1-hydroxybufuralol, 6-hydroxychlorzoxazone, 1í- and 4í-hydroxymidazolam) in rat liver microsomes was developed. The assay used Kinetex analytical column and a gradient mobile phase consistent of acetonitrile and water with addition of 0.1% formic acid. The analysis was performed in selected reaction monitoring (SRM) mode both in positive and negative (for 6-hydroxychlorzoxazone) mode. The method was validated over the concentration ranges of 10-2000 ng/mL for 4í-hydroxymephenytoin and 4-hydroxytolbutamide, 50-2000 ng/mL for 1-hydroxybufuralol and 25-2000 ng/mL for the rest of the analytes. The intra- and inter-day precision (2-12%) and accuracy (93-119%) were within the limits set by the FDA and EMA guidelines. The developed method was successfully applied to assess the activity of selected CYP450 isoenzymes in rat liver microsomes after addition of ketoconazole

Equilibria of complexes in the aqueous cobalt(II)–N-(2-hydroxybenzyl)phenylalanine system and their biological activity compared to analogous Schiff base structures

Due to their excellent prospects in biological applications, Schiff bases and their complexes are a source of continuing interest. The present study examines the formation of four cobalt(II) complexes with the reduced Schiff base N-(2-hydroxybenzyl)phenylalanine (PhAlaSal) in alkaline aqueous solution by pH-metry. UV–Vis and ESI–MS studies confirmed the model of proposed species. Kinetic analysis indicated that the single- and bi-ligand cobalt(II) complexes transitioned from octahedral to tetrahedral structures. The Schiff base and its complexes detected under physiological pH were tested for antimicrobial abilities and compared with analogous structures of the Schiff base derivative, N-(2-hydroxybenzyl)alanine (AlaSal). The ability of these structures to influence cell growth was tested on L929 mouse fibroblasts and on cervix and gastric adenocarcinoma cancer cell lines. N-(2-hydroxybenzyl)phenylalanine demonstrates greater antimicrobial efficacy than N-(2-hydroxybenzyl)alanine but also higher cytotoxicity; however, it is nonetheless effective against cancer cells. In turn, AlaSal demonstrates low cytotoxicity for fibroblasts and high cytotoxicity for gastric adenocarcinoma epithelial cells at bacteriostatic concentration for Helicobacter pylori and Candida strains. The presence of these microorganisms in the gastric milieu supports the development of gastritis and gastric cancer; AlaSal therapy may be simultaneously effective against both. Due to their cytotoxicity, Schiff base complexes are not suitable for use against fungal and bacterial infections, but may effectively prevent cancer cell growth. Data availability: Data will be made available on request

Equilibria in Aqueous Cobalt(II)—Reduced Schiff Base N-(2-hydroxybenzyl)alanine System: Chemical Characterization, Kinetic Analysis, Antimicrobial and Cytotoxic Properties

The present study describes the coordination properties of a reduced Schiff base, N-(2-hydroxybenzyl)alanine, towards cobalt(II) using potentiometric as well as spectroscopic (UV-Vis and ESI-MS) methods. The results indicate the formation of six mononuclear complexes showing high stability in aqueous solution. Coordination occurs in the {O−phenolic,N,O−carboxyl} and {N,O−carboxyl} chelation modes, depending on the degree of ligand deprotonation. Examination of the complexation equilibria at pH ca 7, which is important from a biological point of view, allowed to identify two species: [CoL] and [CoL2H]−. The kinetic analysis showed a structural change of those cobalt(II) complexes from octahedral to tetrahedral in accordance with a first-order time relationship. The antimicrobial properties of N-(2-hydroxybenzyl)alanine, cobalt(II) nitrate and of the Co(II) – ligand complexes were determined against Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis), Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Helicobacter pylori) and a fungal strain (Candida). The results indicate that the complexes are more active for more strains than the ligand alone. Nevertheless, the complexes induce a higher decrease in the metabolic activity of cells but without damage to nuclei. Tetrahedral structures show stronger anti-cellular toxicity than octahedral complexes, which is most likely due to the higher accessibility of the cobalt(II) center