Waters h class

For the enzyme assays, 2 μg samples of the enriched protein fraction were mixed with assay buffer (100 mM Tris, 200 μM MnCl₂, 1 mM DTT; pH 8.0), and the IAA conjugates, IAA-Ala, IAA-Leu, IAA-Asp, IAA-Gly and IAA-Trp, were added individually at a concentration of 1 mM, as substrates. The reactions were incubated at 37 °C for 1 h and terminated by the addition of 20 μl 1 N HCl. The aqueous phases were then extracted with 250 μl of ethyl acetate, evaporated, and the reaction products were dissolved in 100 μl of methanol for ultra performance liquid chromatography (UPLC) analysis. Aliquots (0.2 μl) of the methanol extracts were then injected into a UPLC instrument (Waters H-class, Waters, Millford, MA, USA) coupled to an autosampler (Jasco AS-1550), and equipped with a 2.1 × 100-mm Cortecs C18 2.7 μm, reversed phase column. 1% aqueous acetic acid and 100% methanol were used as the solvents. The solvent program was 15% methanol for 3 min, followed by a gradient from 15 to 90% methanol for 4 min, then a decreasing gradient from 90 to 15% for 3 min with 1% acetic acid throughout. Compounds were detected using ACQUITY FLR fluorescence (excitation 278 nm, emission 350 nm) and quantitated by the peak area.

The ultralow temperature refrigerator BCD-215DK was produced by Haier Company, Qingdao. The ultrahigh liquid chromatograph used was a Waters H-Class from Waters Corporation, USA. The time-of-flight mass spectrometer used was an ACQUITY UPLC Synapt G2-S HDMS produced by Waters Corporation, USA. The column used was an ACQUITY UPLC@HSS T3, 2.5 μm, 100 × 2.1 mm, from Waters Corporation, USA.
Belamcanda chinensis extract was prepared by the Liaoning Institute of Traditional Chinese Treatment (batch number: 20200601). Dexamethasone acetate tablets were obtained from Anhui Jintaiyang Biochemical Pharmaceutical Co. Ltd. An ELISA kit (96T) for guinea pig IL-4, IgE, IFN-γ, and TNF-α was obtained from Nanjing Jiancheng Bioengineering Institute. Methanol was purchased from Fisher Company, USA. Formic acid was also obtained from Fisher Company, USA. Ovalbumin was purchased from Sigma, Germany. Aluminum hydroxide was obtained from Tianjin Damao Chemical Reagent Factory.

Samples of YQJD formula were analyzed using an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q TOF/MS) system (Waters H Class, Waters Technology Co., Ltd., and Sciex TriPle TOF 4600 LC/MS, Shanghai Aibo Caisi Analyt. Instruments Trading Co., Ltd.). Both positive and negative ion modes were applied for parallel reaction monitoring, and mobile phases consisting of 0.1% formic acid in water (A) and acetonitrile (B) were used to separate analytes on a Waters ACQUITY UPLC ® HSS T3 column (2.1 × 100 mm, 1.8 µm). Gradient elution was set as follows: 0–5 min, 0% B; 5–15 min, 0%–15% B; 15–30 min, 15%–60% B; 30–35 min, 60%–95% B; 35–40 min, 95%–95% B; and 40 min, 95%–95% B. The flow rate was 0.3 ml/min, the injection volume was 3 μL, and the column temperature was 30°C. Thirty-four compounds were identified by comparing the multilevel mass spectra of the samples with available information from the Natural Products High Resolution Mass Spectrometry database (Natural Products HR-MS/MS SPectral Library 1.0.1 database) and related literature (Lee et al., 2017 (link)) (Supplementary Material).