Acquity uplc 1 class vion ims qtof

The total flavonoid content was extracted according to Liu et al. (2019) (link), with a minor modification. Sample powder (0.05 g FW) was sonicated in 1 mL of 100% methanol for 30 min at room temperature. After centrifugation at 12,000 rpm for 10 min, the sample supernatant was filtered through a 0.22 μm syringe filter (Sartorius, Gottingen, Germany) into an UPLC vial. Target flavonoids were detected using an Acquity UPLC I-class/VION IMS QTOF (Waters Corp., Milford, MA, United States) instrument, equipped with a photodiode array detector (PDA). The separation of the target flavonoids was achieved on a C₁₈ BEH (1.7 μm, 2.1 × 100 mm) column equipped with a C₁₈ BEH (1.7 μm) precolumn (Waters) at 45°C, and the mobile phases consisted of eluent A (0.1% formic acid in Milli-Q water) and eluent B (0.1% formic acid in MeCN, Dingguo Corp., Beijing, China) with a flow rate of 0.4 mL min^–1. The procedures were as follows: 5–20% B (0–3 min), 20–100% B (3–10 min), 100% B (10–12 min), 100–95% B (12–15 min), 95% B (15–19 min). Finally, 1 μL of the sample was injected into the liquid chromatograph-mass spectrometer (LC-MS) system and the UV wavelength was set at 254 and 335 nm for the detection of genistein and scutellarin. The mass spectrometric analysis and construction of the standard curve were performed according to Xia et al. (2014) (link).

The linking arm and carboxyl group were introduced at the α-cyano group in the Fen structure as a hapten; the synthetic procedure of Fen haptan is shown in Figure S1, according to the previous study [28 (link)]. Then, the Fen hapten was identified by the liquid chromatography–mass spectrometry (LC–MS) analysis according to the method reported by Martínez et al. (2006) [29 (link)]. Briefly, the LC–MS conditions were performed with the Acquity UPLC I-class/VION IMS QTOF (waters, Milford, MA, USA) according to Martínez, with a slight modification: solvent A was set as acetonitrile and solvent B was set as ammonium formate 50 mM and 5% acetonitrile, acidified at pH 3.5 by adding formic acid to perform the program (30% B in 0–3 min, 30–20% B in 4–8 min, 20–0% B in 8–10 min, 0% B in 10–13 min). The haptens were identified according to the liquid chromatographic peak and mass spectral molecular weight.