Author Archives: Vera

The cytochrome P450 (CYP) family is composed of a large group of monooxygenases that mediate the metabolism of xenobiotics and endogenous compounds. CYP2C9, one of the major isoforms of the CYP family, is responsible for the phase I metabolism of a variety of drugs. The aim of the present investigation is to use rational design together with MetaSite, a metabolism site prediction program, to synthesize compounds that retain their pharmacological effects but that are metabolically more stable in the presence of CYP2C9.

The model compound for the study is the nonsteroidal anti-inflammatory drug celecoxib, a COX-2 selective inhibitor and known CYP2C9 substrate. Thirteen analogs of celecoxib were designed, synthesized, and evaluated with regard to their metabolic properties and pharmacologic effects. The docking solutions and the predictions from MetaSite gave useful information leading to the design of new compounds with improved metabolic properties.

Chemical modifications of drugs induced by phase I biotransformations significantly affect their pharmacokinetic properties. Because the metabolites produced can themselves have a pharmacological effect and an intrinsic toxicity, medicinal chemists need to accurately predict the sites of metabolism (SoM) of drugs as early as possible.

However, site of metabolism prediction is rarely accompanied by a prediction of the relative abundance of the various metabolites. Such a prediction would be a great help in the study of drug–drug interactions and in the process of reducing the toxicity of potential drug candidates. The aim of this paper is to present recent developments in the prediction of xenobiotic metabolism and to use concrete examples to explain the computational mechanism employed.

Microalgae have recently become a popular functional food due to their health benefits. Sulfolipids, a class of substances abundant in this matrix, have been reported to have interesting bioactivities, such as anti-carcinogenic activity. However, despite the potential interest in sulfolipids, a dedicated analytical method for their characterization is currently lacking but would significantly increase the coverage of sulfolipids with respect to the direct lipidomic analysis.

To achieve this goal, in this work a procedure, based on graphitized carbon black solid phase extraction, was developed for clean-up and enrichment of sulfolipids (sulfoquinovosyldiacylglycerols and sulfoquinovosylmonoacylglycerols) and it was applied to spirulina (Arthrospira platensis) microalgae. A careful study of the solid phase extraction conditions was performed, first to maximize the recovery of reference standards, then to increase the total number of identified sulfolipids from the spirulina lipid extract. All samples were analysed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry and lipids were tentatively identified by Lipostar, for a reliable lipid structure assignment. The developed method was compared to the direct lipidomic analysis without enrichment, to establish the enrichment efficiency in terms of number of identifications.

From the comparison, the enrichment procedure proved better and allowed the tentative identification of 199 sulfolipids, which is the largest number reported so far for the Arthrospira platensis species. The described method was validated in terms of precision, accuracy, recovery, limit of quantitation and detection for two sulfolipids. Finally, a relative lipid quantitation based on peak area was carried out on the microalgae sample, which indicated nine abundant sulfolipids as representing ca. 60% of sulfolipids in spirulina microalgae. 

The determination of phospholipids in olive oil is challenging due to their low concentration. For this reason, a comparison of two solid phase extraction procedures, namely weak anionic exchange (WAX) and graphitized carbon black (GCB), is presented for the enrichment of phospholipids. Analyses were performed by liquid chromatography-high resolution mass spectrometry (LC25 HRMS) and lipids were identified by Lipostar software. Compared to the WAX solid phase extraction, GCB demonstrated the best performance and provided 82 identified phospholipids vs only 32.

The final method was validated for some representative phospholipids, showing good repeatability and recovery (63-101%). High sensitivity was reached, with detection limits in the range 9-36 ng g-1, never reported before for phospholipids in olive oil. A semi-quantitative analysis indicated phosphatidic acids and phosphatidylglycerols as the most abundant species, both in number and concentrations. The GCB-LC-HRMS-Lipostar platform can be successfully applied for a comprehensive polar lipidomic characterization of olive oils.

The chemical composition of Cannabis sativa L. has been extensively investigated for several years; nevertheless, a detailed lipidome characterization is completely lacking in the literature. To achieve this goal, an extraction and enrichment procedure was developed for the characterization of phospholipids and sulfolipids. Firstly, a study on the solid-liquid extraction was performed, to maximize the recovery of the considered lipids; the best procedure consisted of a simple extraction with a mixture of methanol and chloroform (1:1, v/v).

The hemp extracts were analyzed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry and lipids were tentatively identified by Lipostar. To improve the number of identifications, an enrichment method, based on graphitized carbon black solid phase extraction, was evaluated to fractionate phospholipids and sulfolipids into separate eluates. Recovery and matrix effects of the procedure were determined on a mixture of standard lipids, containing representative compounds for each considered lipid class. The optimized method allowed the tentative identification of 189 lipids, including 51 phospholipids and 80 sulfolipids, in the first and second fractions, respectively. The detection of only 6 sulfolipids in the first fraction and 9 phospholipids in the second fraction proved the efficacy of the fractionation method, which also allowed the number of lipid identifications to be increased compared to the same procedure without enrichment, which scored 100 lipids.

Finally, a semi-quantitative analysis permitted the hemp polar lipidome to be characterized. The results of this study allow knowledge of the hemp chemical composition to be improved with a detailed description of its phospho- and sulfolipid profiles. Graphical abstract.