OEX024422
|
Salvia_Root_Neg |
Metabolomic |
Sample collection: Each plant was separated into root and leaf samples, with 3 biological replicates. The 2 parts of each plant were crushed into homogeneous powders respectively and sieved by 40-mesh sieve.
Extraction: All samples were ground into a fine powder, and 10 mg prepared powder was extracted with 1 mL of 70% methanol (v/v) containing warfarin (5 μg/mL) as the internal reference for 1 h in an ultrasonic bath (53 kHz, 350 W) at 4 °C. After centrifuging at 12,000 x g at 4 °C for 30 min, the supernatant was used for UHPLC-QTOF-MS analysis.
Chromatography: An Acquity UPLC system (Waters) coupled with a Xevo G2-XS QTOF mass spectrometer (Waters) was used for the metabolic analysis. The samples were first separated using an Acquity UPLC T3 column (2.1 mm x 100 mm, 1.8 μm). The column temperature was kept constant at 40 °C, and the flow rate was 0.40 mL/min with an injection volume of 1.0 µL. The mobile phases for gradient elution consisted of 0.1% (v/v) formic acid/water (solvent A) and 0.1% (v/v) formic acid/acetonitrile (solvent B). The elution gradients were 98-80% A over 0-7 min, 80-78% A over 7-11 min, 78-40% A over 11-20 min, 40-35% A over 20-25 min, 25-28 min at 35% A, 35-5% A over 28-30 min, 30-33 min at 5% A and final re-equilibration at 98% A for 5 min.
Mass spectrometry: A Xevo G2-XS with an electrospray ionization source was used to collect MS data. MS was performed in both positive ion and negative ion modes under 30 V cone voltage, with a capillary voltage of 3.0 kV (positive ion mode) or 2.5 kV (negative ion mode). The desolvation temperature was set at 450 °C with the desolvation gas flow rate of 600 L/h, and the source temperature was set at 150 °C with a cone gas flow rate of 50 L/h. All data were collected in the MSE mode, with the following parameters: MSE range, 50-1200 m/z; MSE low energy, 6 eV; and MSE high energy, 15-30 eV. To calibrate the instrument, a sodium formate solution (0.5 mM) was used. Continuous acquisition of leucine enkephalin was used as an external standard for mass correction. All data were viewed in MassLynx v4.2 (Waters).
Data transformation: The raw data were first converted to mzML format (Ms convert Gui) and the analysis base file (ABF Converter; https://www.reifycs.com/AbfConverter/) format before being imported into the MS-DIAL v4.60 software. The parameter settings were as follows: the tolerances for MS1 and MS2 were 0.01 Da and 0.02 Da, respectively. The mass range of MS1 and MS/MS was set between 100 and 1000, with the MS/MS amplitude cutoff at 800. Retention time range was set between 1.0 and 29.5 min, with a retention time tolerance of 0.15 min. The width of the mass slice was 0.1 Da. Adduct types such as [M-H]-, [M+HCOO]-, [M+Na-2H]-, [M+K-2H]-, [2M-H]- and [2M+FA-H]- were selected for the negative ion mode. [M+H]+, [M+Na]+, [M+K]+, [M+NH4]+ and [2M+H]+ were selected for the positive ion mode. Each of the obtained feature tables was split into two peak tables based on the retention time ranges, which were according to the accumulation level of phenolic acids (1.0-18.5 min for both root and leaf samples) and ATDs (16.5-29.5 min for root samples; 14.0-29.5 min for leaf samples).
Metabolite identification: Reference standards were applied to identify the known abietane-type diterpenoids. Diagnostic neutral losses were used to identify the chemical modifications of rings and methyl groups at C-20 from abietane-type diterpenoids. |
2023-07-14 |