1290 infinity 2

Approximately 200 mg of fresh samples of broccoli seedlings were weighed, frozen immediately in liquid nitrogen, and stored at −80 °C. Glucosinolates were extracted with 5 mL of pre-cooled 80% methanol, and the extract was passed through DEAE Sephadex columns, followed by treatment with sulfatase (Sigma-Aldrich, Saint Louis, MO, USA). Extract solutions (5 μL) were then analyzed using ultra-high-performance liquid chromatography (UPLC) (1290 Infinity II, Agilent Technologies) according to the procedure previously described [26 (link)], and sinigrin (Sigma, USA) was used as an internal standard. The mobile phases were water (A) and methanol (B), and the gradient program was set as follows: 0 min, 0% solvent B; 7 min, 25% solvent B; 8.6 min, 60% solvent B; 9.2 min, 100% solvent B; 9.2–9.8 min, 100% solvent B; 10.6 min, 0% solvent B; and 10.6–13.0 min, 0% solvent B. The flow rate was 0.4 mL min⁻¹, and desulfoglucosinolates were identified using a UV detector at 229 nm.

The separation and detection of the target analytes were carried out by a UHPLC system (Agilent 1290 Infinity II) coupled to a triple quadrupole (QqQ) mass analyser (Agilent Technologies 6430 Triple Quad), equipped with a binary pump, a degasifying system, an automatic injector and an electrospray ionization (ESI) interface. The separation of the analytes was performed using an ACE UltraCore 2.5 SuperC18 (2.1 mm × 100 mm, 2.5 µm, Avantor, Symta, Madrid, Spain) chromatographic column that has a working pH range of 1.5–11, equipped with an UltraCore Super C18 UHPLC guard precolumn placed in an ACE UHPLC guard holder (both purchased from Avantor, Symta). The column temperature was maintained at 35 °C and 7 µL was selected as injection volume.
For the mobile phases, UHPLC water (A line) and MeOH (B line) were used at pH 2.5 (0.1% HCOOH) and 10.5 (0.05% NH₄OH) to ensure ionization of all compounds. The flow rate was set to 0.3 mL min⁻¹, and it was continuously in-line filtered through an ACE UltraCore 5 SuperC18 (2.1 mm × 30 mm, 5 µm, Avantor, Symta) column placed before the injector in order to reduce interfering compounds coming from the LC equipment. Further details on LC-QqQ analysis are included in Sect. 2.2.2 in SI.

A preparative HPLC-MS chromatography machine (Agilent 1290 Infinity II) was used coupled to a UV-VIS detector with a mass LC/MSD detector (Agilent InfinityLab) and an Agilent Prep-C18 column (5 μm, 21.2 mm × 50 mm, Waters, Milford, MA, United States) to obtain the final products. Analyzed samples were dissolved in 50% MeOH before injection. The mobile phase was methanol (A):H₂O (B) with a flow rate of 20 mL min^–1 and linear gradients (0 min, 10% B; 0–12 min; 90% B) were used.

Secondary metabolites were analyzed by reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) [51 (link),52 (link)]. The plant sample after evaporation (prepared as above) was dissolved in 10% methanol containing 0.01% formic acid and centrifuged at 12,000 g for 10 min and filtered through PES syringe filter 0.22 µm (VWR, Radnor, PA, USA). The resulting solution was analyzed on a LC system 1290 Infinity II (Agilent, Santa Clara, CA, USA) interfaced to Q-TOF maXis II (Bruker, Billerica, MA, USA). A Zorbax C18 reverse-phase silica-based column was used for separation (150 × 2.1 mm, 3.5 μm; Agilent, Santa Clara, CA, USA). The compound quantities were compared with their respective standard curves. The standards were purchased from Sigma-Aldrich (USA) and Carl Roth (Germany) in ≥98.0% purity (HPLC).

Following pretreatment, plasma metabolic profiling was performed on a UHPLC system (1290 Infinity II; Agilent Technologies, Santa Clara, CA, USA) coupled to a time-of-flight MS (Agilent 6530; Agilent Technologies, USA) platform. The sample pretreatment, UHPLC-TOF/MS analysis conditions, and equipment parameters used for metabolic profiling are described in detail in the Supplementary Material, section Methods. The feature peak extraction, peak alignment, retention time (RT) correction, and data filtering to extract peak areas were performed using the XCMS package based on the R platform.

The lipid profiling of samples was performed with an LC-QqQ(LIT)-MS/MS system composed of a LC-20A Prominence series HPLC (Shimadzu, Kyoto, Japan), and a 3200 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer with Turbo V™ ion source (Sciex, Concord, ON, Canada), as previously described [3 (link)]. Enhanced mass scan (EMS) was used for the lipid signal survey with the positive electrospray ionization (ESI+) mode. The tandem MS (MS²) analysis of PQ lipid molecular species was carried out under the mode of EMS with independent data acquisition and enhanced product ion scans (EMS-IDA-EPI). Meanwhile, for high-resolution mass measurement, the LC-Q/TOF MS analysis was performed with a 1290 infinity II high-performance liquid chromatograph and a 6530 Accurate-Mass Q-TOF mass spectrometer equipped with a JetStream source (both, Agilent Technologies, Santa Clara, CA) in ESI+ mode [3 (link)]. A lock mass solution including purine (m/z 121.0509) and HP-921 (hexakis (1H,1H,3H-tetrafluoro-pentoxy) phosphazene) (m/z 922.0098) was utilized for the real-time lock mass correction. For chromatographic separation of total lipids, 2 µL of sample was injected into a CERI L-column2 ODS (100 × 2.1 mm, 3 µm, Tokyo, Japan), held at 40 °C. Mobile phase solvents and gradient conditions were as previously described [3 (link)].