Nature-Inspired Chemistry of Complex Alkaloids: Combining Targeted Molecular Networking Approach and Semisynthetic Strategy to Access Rare Communesins in a Marine-Derived Penicillium expansum.

Communesins are rare alkaloids isolated from fungi of the genus Penicillium. In this work, the extract of a marine-derived Penicillium expansum strain was studied using targeted molecular networking approach allowing to detect 65 communesins including 55 new ones. A fragmentation pattern for dimethylvinyl communesins was established and a script was implemented allowing to predict the structure and map all communesins in a global molecular network. A semisynthetic strategy was carried out to obtain some minor congeners from the two isolated communesins A and B. Nine communesins were then synthetised: two of them were already described as produced by the studied strain; four are new natural products which occurrence in the extracts was confirmed; three are new semi-synthetic analogues never described so far. These communesins were evaluated for their cytotoxicity on two human cancer cell lines KB and MCF-7 leading to a preliminary study of their structure-activity relationships.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

A rapid and sensitive method for characterization and quantification of polyglycerol esters by supercritical fluid chromatography coupled to high-resolution mass spectrometry (SFC-HRMS).

Polyglycerol esters of fatty acids (PGEs), a very complex mixture of various isomers, are widely used as green surfactants in different industrial fields such as in cosmetic, pharmaceutic and food industries. However, no study related to the purification and the absolute quantification of these compounds has been described yet. In this study, we developed a rapid and efficient method for characterization and quantification of PGEs using Supercritical Fluid CO2 coupled to High-Resolution Mass Spectrometry (SFC-HRMS). The SFC conditions were first considered including the stationary phase, the nature of mobile phase, the column temperature, the back-pressure regulator. The MS parameters (drying-gas temperature, capillary voltage, nozzle voltage, fragmentor voltage) were then investigated to get the best sensitivity for the PGE analysis. The MS/MS-based structural characterization of targeted PGE, triglycerol mono-oleate (PG3+1C18:1), was established and is helpful to study complex mixtures of PGEs with numerous isobaric PGEs. PG3+1C18:1 was then purified at lab scale and used as standard for quantification. This enabled to develop a rapid quantification method for PG3+1C18:1 within 12 min with good linearity (R2 = 0.9997) as well as sensitivity (picogram level). The validated method was then successfully applied to quantify PG3+1C18:1 in commercial products in order to evaluate their composition.

Comparison of internal energy distributions generated by supercritical fluid chromatography versus liquid chromatography hyphenated with electrospray high resolution mass spectrometry.

The internal energy distributions of thermometer ions dissolved in supercritical and liquid solvents and produced in the gas phase by electrospray (ESI) were measured and compared by the survival yield method. The influence of different chromatographic conditions such as the nature of solvents, the composition of the mobile phase, the pressure of the back pressure regulator was studied for supercritical fluid chromatography (SFC) whereas the influence of the composition and of the flow rate of the mobile phase was investigated for liquid chromatography (LC). The MS instrumental parameters were studied in parallel for SFC and LC showing that the drying gas temperature and the fragmentor voltage affected the internal energy distribution, whereas the capillary voltage did not modify the internal energy distribution. A comparison of the internal energy distributions generated by SFC and LC was carried out leading to conclude that SFC led to higher internal energy, i.e. more in-source fragment ions, than LC.

Annotation and quantification of N-acyl homoserine lactones implied in bacterial quorum sensing by supercritical-fluid chromatography coupled with high-resolution mass spectrometry.

In recent years, use of supercritical-fluid chromatography (SFC) with CO2 as the mobile phase has been expanding in the research laboratory and industry since it is considered to be a green analytical method. This technique offers numerous advantages, such as good separation and sensitive detection, short analysis times, and stability of analytes. In this study, a method for quantification of N-acyl homoserine lactones (AHLs), signaling molecules responsible for cell-to-cell communication initially discovered in bacteria, by SFC coupled with high-resolution mass spectrometry (HRMS) was developed. The SFC conditions and MS ionization settings were optimized to obtain the best separation and greatest sensitivity. The optimal analysis conditions allowed quantification of up to 30 AHLs in a single run within 16 min with excellent linearity (R2 > 0.998) and sensitivity (picogram level). This method was then applied to study AHL production by one Gram-negative endophytic bacterium, Paraburkholderia sp. BSNB-0670. Nineteen known AHLs were detected, and nine abundant HSLs were quantified. To further investigate the production of uncommon AHLs, a molecular networking approach was applied on the basis of the SFC-HRMS/MS data. This led to additional identification of four unknown AHLs annotated as N-3-hydroxydodecanoylol homoserine lactone, N-3-hydroxydodecadienoyl homoserine lactone, and N-3-oxododecenoyl homoserine lactones (two isomers).

Metabolomics-Driven Discovery of Meroterpenoids from a Mussel-Derived Penicillium ubiquetum.

Penicillium ubiquetum MMS330 isolated from the blue mussel Mytilus edulis collected on the Loire estuary in France was here investigated. As very few secondary metabolites have been documented for this species, its metabolome was studied following the OSMAC approach to enhance as many biosynthetic pathways as possible. Interestingly, HPLC-HRMS based hierarchical clustering analysis together with MS/MS molecular networking highlighted the selective overproduction of some structurally related compounds when the culture was performed on seawater CYA (Czapek Yeast extract Agar) medium. Mass-guided purification from large scale cultivation on this medium led to the isolation of nine meroterpenoids including two new analogues, 22-deoxyminiolutelide A (1) and 4-hydroxy-22-deoxyminiolutelide B (2), together with seven known compounds (3-9). The structures of 1 and 2 were elucidated on the basis of HR-ESIMS and NMR spectroscopic data analysis. Furthermore, NMR signals of 22-deoxyminiolutelide B (3) were reassigned.