6420 triple quadrupole lc ms

After identifying the HPLC fractions with the highest antioxidant activity, they were further purified using the same RP-HPLC conditions. The purified peptides were then subjected to analysis by the 6420 Triple Quadrupole LC/MS (Agilent Technologies, Inc., USA), coupled with an HPLC system using a nanoelectrospray ionization source, which recorded the mass spectral range of 50–1506 m/z. To determine the amino acid sequence, the de novo sequencing method was employed, and the MS/MS spectra were analyzed by PEAKS Studio Version 4.5 SP2 [Bioinformatics Solutions Inc., Waterloo, ON, Canada] for peptide identification. Three purified peptides were synthesized by Smart Science Co., Ltd. (Pathum Thani, Thailand), with a final purity of ≥98% verified by HPLC (Gilson L7420, Hitachi Limited, Tokyo, Japan). The synthesized peptides were used for further analysis.

Betaine and proline in intra/extracellular metabolites was analyzed by LC–MS/MS under multiple reaction monitoring (MRM) methods. Ten microliters of each prepared sample was analyzed by an Agilent 6420 Triple Quadrupole LC/MS (CA, USA) coupled to an Agilent 1260 Infinity Binary LC (CA, USA). Separations were performed using an Agilent Zorbax HILIC Plus column (4.6 × 100 mm, 3.5 μm) and a flow rate of 500 μL min⁻¹. Eluent A was water containing 0.1% v/v formic acid, and B was 100% acetonitrile. The gradient applied was as follows: 0–3 min, 80% eluent B; 3–10 min, linear decrease to 20% eluent B; 10–13 min, 20% eluent B; 13–14 min, linear increase to 80% eluent B; 14–23 min, 80% eluent B. The flow rate was at 0.5 mL min⁻¹. The capillary temperature was 300 °C, and an electrospray ionization spray voltage was used at 4 kV.