Tripletof 5600 q tof

A mixer mill (MM400, Retsch) was used to ground the samples into fine powders (1.5 min) at 30 Hz. Lyophilized powder (100 mg) was used to extract metabolites at 4°C using 0.8 ml of 70% aqueous methanol (methanol: H₂O₂, 70:30, v/v). After adding pure methanol, centrifugation was conducted for 10 min (10000 g). We gathered, homogenized, and sieved the supernatants accordingly (SCAA-104, 0.22 mm pore size; ANPEL Shanghai, China, www.anpel.com.cn/). Samples were mixed into tissue-specific samples, namely root tissue and rhizosphere soil, to assess the inter-tissue variations in the different compounds using LC-MS/MS analyses. Biomarker biocloud platform (www.biocloud.net) was used to carry out instrument stability after combining the samples. After that, UHPLC system (1290, Agilent Technologies) containing of a TripleTOF 5600 (Q-TOF, AB Sciex) and UPLC BEH Amide column (1.7 μm 2.1*100 mm, Waters) was used to carry out LC-MS/MS analyses. More details regarding LC-MS/MS analyses, metabolites processing, and annotation were documented in previous studies (Fallah et al., 2022 (link); Yuan et al., 2022 (link))

The LC-MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies) with a UPLC BEH Amide column (1.7 μm 2.1 mm × 100 mm, Waters) coupled to Triple TOF 5600 (Q-TOF, AB Sciex). The mobile phase consisted of 25 mmol/L NH₄OAc and 25 mmol/L NH₄OH in water (pH = 9.75) (A) and acetonitrile (B) and was used with an elution gradient as follows: 0 min, 95% B; 7 min, 65% B; 9 min, 40% B; 9.1 min, 95% B; and 12 min, 95% B, which was delivered at 0.5 mL min⁻¹. The injection volume was 2 μL. The Triple TOF mass spectrometer was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the full-scan survey MS data as it collects and triggers the acquisition of the MS/MS spectra depending on preselected criteria. In each cycle, 12 precursor ions with an intensity greater than 100 were chosen for fragmentation at a collision energy (CE) of 30 V (15 MS/MS events with a product ion accumulation time of 50 ms each). The ESI source conditions were set as follows: ion source gas 1 at 60 Psi, ion source gas 2 at 60 Psi, curtain gas at 35 Psi, source temperature at 650°C, and ion spray voltage floating (ISVF) at 5000 V or −4000 V in positive or negative modes, respectively.

The UHPLC system (1290, Agilent Technologies) with a UPLC BEH Amide column (1.7 μm 2.1 × 100 mm, Waters) coupled with Triple TOF 5600 (Q‐TOF, AB Sciex) was used for LC‐MS/MS analyses. 25 mM NH₄OAc and 25 mM NH₄OH in water (pH = 9.75) (A) and acetonitrile (B) were used as the mobile phase. The elution gradient was as follows: 0 min, 95% B; 7 min, 65% B; 9 min, 40% B; 9.1 min, 95% B; and 12 min, 95% B. The flow rate of the mobile phase was 0.5 ml/min. The injection volume of analytical solution was 3 μl. The Triple‐TOF‐MS was used for its ability to acquire MS/MS spectra on an information‐dependent basis (IDA) during an LC/MS experiment. In this mode, the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria were surveyed by the acquisition software (Analyst TF 1.7, AB Sciex; Fraga, Clowers, Moore, & Zink, 2010). In each cycle, 12 precursor ions with intensity greater than 100 were chosen for fragmentation at collision energy (CE) of 30 V (15 MS/MS events with product ion accumulation time of 50 msec each). ESI source conditions were set as following: ion source gas 1 as 60 Psi, ion source gas 2 as 60 Psi, Curtain gas as 35 Psi, source temperature 650°C, ion spray voltage floating (ISVF) 5,000 or −4,000 V in positive or negative modes, respectively.