Agilent 5977b gc ms

Bone marrow plasma and peripheral blood plasma samples were spiked in 15 of internal solution containing [¹³C₅]-labeled analytes for 2-HG, glutamate, and α-ketoglutarate. The proteins were removed by adding 260 μl of chilled methanol and acetonitrile solution to the sample mixture. After drying the supernatant in the speed vacuum, the sample was derivatized with a solution of O-ethylhydroxylamine in pyridine (20 mg/mL) for 1 h at 35 °C, followed by sylation with MtBSTFA + 1% tBDMCS for 1 h at 70 °C before it was analyzed on an Agilent 5977B GC/MS under electron impact and single ion monitoring conditions. Concentrations of α-ketoglutarate (m/z 360.2), 2-HG (m/z 433.2), and glutamate (m/z 432.2) were measured against a 12-point calibration curves that underwent the same derivatization. Finally, validation of the concentrations of glutamate and aspartate in each of the bone marrow plasma samples obtained by the GC-MS methodology was verified by LC-MS.

Feces samples were obtained 1 day before euthanization. They were weighed, suspended in 1 mL of water with 0.5% phosphoric acid per 0.1 g of sample, and stored in −30 °C immediately after collection until homogenization. Feces were homogenized for 2 min and centrifuged for 10 min at 14.8 RPM. The supernatant was isolated for ethyl acetate (300 μL) extraction, and the organic phase was collected for Agilent 5977B GC-MS (Agilent Technologies; Palo Alto, CA, USA) analysis. The GC instrument was fitted with a Nukol™ Capillary GC Column (30 m × 0.25 mm id, 0.25 μm df) and helium was used as the gas carrier, injected at 1 mL/min. The column temperature was 90 °C initially and then increased to 150 °C at 15 °C/min, 170 °C at 5 °C/min, and finally to 250 °C at 20 °C/min; this temperature was maintained for 2 min (total time of 14 min). Solvent delay was 3.5 min. The detector was operated in the electron impact ionization mode (electron energy of 70 eV), with scanning conducted in the 30–250 m/z range. The temperatures of the ion source, quadrupole, and interface were 230 °C, 150 °C, and 280 °C, respectively. Short-chain fatty acids (SCFAs) were identified based on the retention time of standard compounds and with the assistance of the NIST 08 and Wiley7N libraries.

GC–MS was used to identify compounds in the methanol extract of B. cernua (stem). Sample derivatization was conducted using the trimethyl silyl derivatization (TMS) method. Briefly, 20 µL of extract was dried using an oven (80 °C, 20 min). Immediately after, the sample was solubilized using 80 µL of methoxyamine hydrochloride solution in pyridine (2 mg/100 mL), mixed by vortexing for 1 min, and incubated for 90 min at 30 °C. Next, the solubilized sample was combined with 80 µL of MSTFA and incubated for 30 min at 37 °C prior to GC–MS analysis [21 (link)]. GC–MS analyses of B. cernua (stem) extracts were carried out using an Agilent 5977B GC/MS (made in USA). Pure helium gas was employed as the carrier gas (1 mL/min), and the injector temperature was set at 270 °C. The oven temperature program was set as follows: gradual increase from 70 °C to 200 °C (10 °C/min), then to 310 °C (10 °C/min), before holding at 310 °C for 5 min. The mass spectrometer was set to electron ionization mode at 70 eV with an electron multiplier voltage of 1859 V. The retention index of compounds was recorded using Mass Hunter GC/MS Acquisition 10.0.368 software. The phytochemical content determination was conducted by comparing the results to authentic compound spectral databases stored in the National Institute of Standards and Technology (NIST) library.