Agilent masshunter qualitative analysis b 05

LC-MS/MS analysis was performed using Agilent QTOF 6550 at the LC-MS/MS laboratory, Monash University Malaysia, Selangor, Malaysia. The digested fraction was loaded into an Agilent Large Capacity Chip (Agilent, USA) comprising a 160 mL enrichment column and a 75 μM × 150 mm analytical column, which was packed with 5 μM of Zorbax 300SB-C18. The solvent systems used to elute the peptides were Solvent A (0.1% formic acid in MilliQ water), and Solvent B (9 : 1 ratio of 0.1% formic acid in acetonitrile : MilliQ water). The gradient used was programmed as: 3–50% of solvent B for 30 min, 50–95% of solvent B for 2 min, 95% of solvent B for 7 min, and 95–3% of solvent B for 47 min. The quadrupole time-of-flight (Q-TOF) polarity was set at positive, the capillary and fragmentor voltages were set at 2050 V and 300 V, respectively, and the gas flow was set at 5 L min⁻¹ and 325 °C. The peptide spectra were acquired using the Agilent MassHunter Workstation Data Acquisition software (Agilent Technologies, USA) in an auto MS/MS mode ranging from 110 to 3000 m/z for the MS scan, and from 50 to 3000 m/z for the MS/MS scan. The chromatograms were analyzed using the Agilent MassHunter Qualitative Analysis B.05.00 software (Agilent Technologies, USA).

Gummy texture of M. porteri's ethanolic extract (10 mg) was obtained from the drying process using a rotary evaporator. 1 mL of methanol was used to dilute the gummy extract to a final concentration of 10 mg/mL. Then, the extract was diluted again with methanol to a concentration of 1 mg/mL. The extract was filtered with a 0.22 μm pore size syringe filter before performing the analysis using LC-MS/QTOF (model 6520 Agilent Technologies, SA, USA). 2 μl of the filtered extract was injected into Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HT column (2.1 mm × 100 mm × 1.8 μm, Agilent Technologies, SA, USA) at the temperature of 40 °C. The flow rate used was 0.25 mL/min with solvent A (0.1% formic acid in distilled water) and solvent B (0.1% formic acid in acetonitrile). The gradient elution program was 0.00–18.00 min for 5–95% of mobile phase (B), 18–23 min for 95% of mobile phase (B) and 23.01 min for 5% of mobile phase (B). The total run time for analysis of the extract was 30 min. The mass spectrometer was set to positive electrospray ionisation (ESI) mode with an optimal gas temperature of 325 °C, gas flow of 11 L/min, and nebulizer pressure of 35 psi. The Agilent Mass Hunter Qualitative Analysis B.05.00 software (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the MS data and METLIN database was used to annotate the predicted chemical compounds [25 (link)].