# An Integrated Metabolomics Study of Glucosinolate Metabolism in Different Brassicaceae Genera
## 4 Materials and Methods
### Targeted Analysis
#### 4.4.2 Targeted Analysis of Glucosinolates  
Sample Preparation  
Fresh vegetables were washed, snap-frozen in liquid nitrogen, and then freeze-dried. The freeze-dried vegetables were ground into fine powder and then immediately stored at -80 \({}^{\circ}\)C. one hundred mg of each freeze-dried sample was incubated in 1 mL of 70% methanol containing 150 ng/mL of internal standard (Glucosinalbin, Santa Ana, CA, USA) at 70 \({}^{\circ}\)C for 10 min. After chilling in an ice bath, the mixture was centrifuged at 15,000\(\times\)\(g\) for 15 min, with the supernatant transferred to a new tube. The extraction procedure was repeated twice, and the three supernatants obtained were combined, evaporated to dryness at 40 \({}^{\circ}\)C, reconstituted in 3 mL of 70% acetonitrile (ACN), and filtered through a 0.22 \(\upmu\)m nylon filter for analysis.  
Quantification of Glucosinolates  
Quantitative analyses of glucosinolates were performed on an Agilent 1290 UHPLC system (Waldbronn, Germany) coupled to a 6490 QQQ mass detector (Agilent, Santa Clara, CA, USA)equipped with iFunnel Technology and an electrospray ionization source. The elution of glucosinolates was conducted on a Waters ACQUITY UPLC(r) BEH HILIC column (2.1 \(\times\) 100 mm, 1.7 \(\upmu\)m) at a column temperature of 35 \({}^{\circ}\)C using mobile phases C and D at a flow rate of 0.4 mL/min with the following gradient: 0-1 min, 100-100% D; 1-5 min, 100-95% D; 5-8 min, 95-80% D; 8-10 min, 80-15% D. The autosampler was cooled at 4 \({}^{\circ}\)C, and 5 \(\upmu\)L of the extract was injected. The mass spectrometer was maintained in negative ion mode with the following parameters: Drying gas (N\({}_{2}\)) temperature, 200 \({}^{\circ}\)C; drying gas flow, 14 L/min; nebulizer, 30 psi; sheath gas temperature, 400 \({}^{\circ}\)C; sheath gas flow, 11 L/min; capillary voltage, 3000 V; nozzle voltage, 800 V. Glucosinolates were quantified (with available standards) or semi-quantified (without available standards) in MRM mode with time segments. Data acquisition and processing were conducted with MassHunter software version B.05.00 (Agilent Technologies, Santa Clara, CA, USA).  
The quantitative method was validated for limit of detection (LOD), linearity, accuracy, precision, and recovery. Briefly, each standard was dissolved in 70% acetonitrile and diluted to give a series of standard solutions with gradient concentration to make the calibration curves. All the solutions were stored at -20 \({}^{\circ}\)C. Method precision was determined by injecting the same mixed sample solution six times consecutively, both in one day for intraday variation and in three successive days for inter-day variation. The reproducibility of the method was determined by analyzing six independently prepared samples from the same mixed vegetable powder. The recoveries were evaluated by spiking glucosinolate standards in three different amounts (approximately equivalent to 0.8, 1.0, and 1.2 times of the concentration in the matrix) into the mixed vegetable sample in triplicate, and were extracted and quantified as described before [48]. The method validation details are presented in Supplementary Materials Table S4.