Agilent 6400

For quantitative analysis of cholesterol, cells were homogenized in PBS and lipids were extracted by modified Bligh and Dyer extraction method^{21 (link),22 (link)}. Briefly, 100 μl of chloroform plus methanol (2:1) was added to 100 μl of PBS including homogenized cells after counting the cell number. After centrifugation, the chloroform layer was collected and dried with a centrifugal dryer. The dry matter was lysed in 100 μl of ethanol to measure free cholesterol using a Cholesterol Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Fluorescence signals were read with an Envision Multilabel Reader (PerkinElmer). Total lipids including sterols in spinal fluids from patients were extracted by the same method, with the extracts being subjected to LC–MS/MS-based quantification (Agilent 6400). Briefly, 100 μl of chloroform plus methanol (2:1) was added to 10 μl of spinal fluids mixed with 90 μl of PBS. After centrifugation, the chloroform layer was collected and dried with a centrifugal dryer. The dry matter was then lysed in 100 μl of ethanol, filtered with a 0.2-μm centrifugal filter tube, and measured for cholesterol by the use of Agilent 6400.

The phytochemicals constituents in methanolic extract were identified using GC-MS (Perkin Elmer Clarus 500, Shelton, CT, USA) and LC-MS. The GS-MS was equipped with a flame ionization detector and capillary column (30 m length × 0.25 mm ID coated with 5% phenyl 95% dimethylpolysiloxane) with a film thickness of 0.25 μm. Helium gas used as mobile/carrier gas was operated at flow rate of 1 mL/min. During operation, the temperature of the injection port was kept at 280 °C, and analysis was carried out with 1 μL injection volume. The temperature of stationary phase was at 60–300 °C. The mass spectrum was prepared with full scan mode considering the range of 40–450 Daltons [49 (link)]. LC-MS profile was studied with Agilent 6400 (Santa Clara, CA, USA) Series Triple Quadrupole System equipped with an electrospray ionization (ESI) source. The separation was done with Phenomenex Luna PFP analytical column at 40 °C at with injection volume of 10µL. For LC analysis the column temperature was 40 °C and a combination of 0.1% aqueous solution of formic acid and methanol (eluent A and B ratio was 80:20) was used as mobile phase at a flow rate of 0.40 mL/min under isocratic elution conditions. The obtained peak was identified with the standard libraries from NIST (The National Institute of Standards and Technology) library and Wiley.

Determination of the fatty acid profile in the various yeast strains was performed as follows: the yeast powder (0.05 g) of each strain was mixed with 3 ml of KOH–methanol in the 10-ml tubes and incubated at 70°C for 3–5 h. Then, 2 ml of boron trifluoride–methanol solution was added to the mixture, followed by incubation at 70°C for 1.5 h (after adjusting the pH values to 2.0) to achieve methylation. The fatty acid methyl esters (FAMEs) were recovered by mixing with 3-ml n-hexane. The upper liquid layer was isolated for gas chromatography (GC) analysis (Agilent 6400, Redwood City, CA, United States). For determination of the fatty acid profile in the half-seed, the cotyledon sample was crushed using a glass rod and added to a 500-μl mixture of ether: petroleum ether (3:2) at room temperature for 4 h. Then, 250 μl of KOH-methanol was added and incubated at room temperature for 2 h. Tubes were shaken for 1 min after 500 μl of H₂O was added. Subsequently, 100 μl of the supernatant was pipetted out for GC analysis. Fatty acids were identified by comparing their retention times against the FAME standards (Sigma Chemicals Co., St. Louis, MO, United States) separated on the same GC machine. The seeds collected from parental lines and each line of the populations were used for oil content analysis by near-infrared spectroscopy.