Msd enhanced chemstation

Information regarding the suspected sample's composition was almost always provided by the drug user. Then, MALDI data were analyzed using Tracefinder (Thermo Scientific) and a database created for this specific drug checking application. Identification was based on the mass over charge ratio, the isotopic pattern, and the library search based on MS/MS spectra comparison. Regarding the samples containing a substance that was not indexed in the library, Xcalibur was used combined with different external library search such as Metlin or m/z cloud. The data were analyzed using MSD Enhanced ChemStation (Agilent Technologies) and compound characterization was performed using mass spectra computerized databases, such as NIST Version 2014 (National Institute of Standards and Technology), Wiley Edition 10, MPW Version 2011 (Maurer, Pfleger, Weber, Drugs, Poisons, Pesticides, Pollutants, and Metabolites), DD Version 2014 (Drug Design and Discovery) and custom databases from the University Institutes of Legal Medicine of the Faculty of Medicine of Geneva (CURML). Quantitative results were treated using Analyst software version 1.6.2.

Samples from four independent liquid cultures were analysed. Each sample of 20 mg fresh weight (freeze-dried) was used for replicate analyses and results from WT and the ΔZtCBR1-1 mutant strain were compared using Student’s t-tests. Non-saponifiable lipids were extracted as reported previously (Kelly et al., 1995 (link)). Samples were dried in a vacuum centrifuge (Heto) and derivatized by addition of 100 μl of 90% bis(trimethylsilyl)-trifluoroacetamide (BSTFA) – 10% trimethylsilyl (TMS) (Sigma–Aldrich) and 200 μl anhydrous pyridine (Sigma–Aldrich) and heating for 2 h at 80 °C. Gas chromatography–mass spectrometry was performed using a VG12-250 mass spectrometer (VG Biotech) with splitless injection. Individual sterols were identified by reference to relative retention times, mass ions, and fragmentation patterns. Data were analysed using MSD Enhanced ChemStation (Agilent Technologies). Statistical analyses were carried out using the GenStat (2014, 17th edition, 297 © VSN international Ltd, Hemel Hempstead, UK) statistics package.

Single colonies of Candida albicans strains were grown for 18 hr in YEPD at 37°, 200 rpm; ∼15 ml of culture was harvested and cells were washed twice with ddH₂O. Cells were then resuspended in 1 ml ddH₂O and split equally into two tubes, one sample for the determination of the dry weight of cells, the other for sterol extraction.
Nonsaponifiable lipids were prepared and extracted as reported previously (Kelly et al. 1995 (link)). An internal standard of 10 µg of cholesterol was added prior to extraction with hexane. Samples were dried in a vacuum centrifuge (Heto), and were derivatized by the addition of 100 ml 90% N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/10% trimethylsilyl (TMS) (Sigma) and 200 ml anhydrous pyridine (Sigma), and heating for 2 hr at 80°. TMS-derivatized sterols were analyzed and identified using GC/MS (Agilent 5975C Inert XL GC/MSD) with reference to retention times and fragmentation spectra for known standards. GC/MS data files were analyzed using Agilent software (MSD Enhanced ChemStation, Agilent Technologies, Stockport, UK) to determine integrated peak areas, and enable calculation of the percentage of total sterols and the amount of sterol/dry weight of cells.