Xr ods c18 column

The Shimadzu LCMS-IT-TOF system (Tokyo, Japan) was used in positive ion mode with an electrospray ionization (ESI) source. A Shim-Pack XR-ODS (C18 column (2.0 mm i.d. ×75 mm, 1.6 μm, Shim) was employed for chromatography separation, and 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.4 mL/min was used to establish the gradient elution (17% B for 0–2.0 min, 17%–35% B for 2.0—3.0 min, 35%–44% B for 3.0–7.0 min, 44%–55% B for 7.0–9.0 min, 55%–60% B for 9.0–14.0 min, 60%–63% B for 14.0–17.0 min, and 63%-68% B for 17.0–19.0 min). The column temperature was set at 40 °C. The sample injection volume was 5 μL. The detection wavelengths ranged from 190 nm to 800 nm. The following parameters were used for analytical MS: interface voltage, 4.5 kV; ESI voltage, 1.6 kV; nebulizing gas (N₂) flow rate, 1.5 L/min; ion trap pressure, 1.8 × 10⁻² Pa; interface temperature, 200 °C; drying gas pressure, 100 kPa; ion accumulation time, 30 ms. Ultra-high purity argon was applied for collision-induced dissociation (CID). The above-mentioned standard solution of 1 g/ml XFZY was centrifuged for 10 min (16,000 rpm, 4 °C). The upper layer was filtered through a 0.22-μm nylon filter. The leachate was diluted twice with distilled water and then injected into the LCMS-IT-TOF.

A Shimadzu UHPLC Nexera X2 attached to a Shimadzu (Tokyo, Japan) LCMS-9030 QTOF with ESI source was used. Once samples were filtered (0.45 μm nylon filter, Sigma-Aldrich, St. Louis, MO, USA), betalains were separated using a Shim pack XR–ODS C18 column (150 × 2 mm, 2.2 μm particle size), at 7500 psi maximum working pressure and maximum flow rate of 0.4 mL/min, and using 1% formic acid in water (v/v, eluent A) and a mixture of acetonitrile/water/formic acid (80:19:1) (eluent B) (Sigma-Aldrich, St. Louis, MO, USA). The volume injection was 5 μL. The chromatographic method started with 100% of A, followed by a linear gradient from 0 to 20% of B in 35 min and then a linear gradient from 20 to 100% of B in 5 min [6 (link)]. To re-establish the initial conditions, a linear gradient from 100% of B to 100% of A was used for 10 min. The identification of the individual betalains was performed in positive mode using a sweeping range of m/z 100 to 1000 u. An amount of 4.5 mL/min of nitrogen was used as the drying gas, the temperature was set at 300 °C, the nebulizer gas flow was 3 L/min, the interface temperature was 526 °C, the sampling rate was 5 µ/s, the heater gas flow was 10 L/min, and the detector voltage was 0.20 kV.

UPLC-MS analysis was performed using a Shimadzu LCMS-8050 mass spectrometer coupled with a UPLC chromatograph. Chromatographic separation was achieved using a Shim-pack XR-ODS C18 column (2.0 × 75 mm, 1.6 μm). The parameters used are as follows: capillary voltage, 15 kV; spray flow, 3 L/min; heating gas flow, 10 L/min; interface temperature, 350°C; DL temperature, 250°C; heating block temperature, 400°C; and drying airflow rate, 10 L/min. Gradient elution was performed using acetonitrile and water (2%–98%) for 120 min. The MS spectra of the compounds were imported into the natural compound database for comparison, and the results with a similarity of more than 90% were taken for LCMS comparison analysis and qualitative analysis with the corresponding standards. The LC-MS/MS chromatograms of the 45 compounds in QGHXR are shown in Figure 1.