Nexera x2 lc 30ad hplc system

Separation of the compounds was achieved using a Nexera X2 LC-30AD HPLC system (Shimadzu, Kyoto, Japan). The system comprises an on-line degasser, a quaternary pump, SIL-30AC autosampler (Shimadzu), CTO-20AC thermostat (Shimadzu), a column temperature controller and a SPD-M20A diode array detector (DAD). Other instruments used in the investigation were an Ultrasonic Cleaner Set (Wise Clean WUC-A06H, Witeg Labortechnik GmbH, (Wertheim Germany), Libra UniBloc AUW120D (Shimadzu Analytical Scale, Kyoto, Japan); pH-meter—Knick Electronic Battery-operated pH Meter 911 PH (Portamess, Berlin, Germany), and class A analytical vials that meet requirements of the State Pharmacopoeia of Ukraine (SPhU, 2015).

The LC/MS system was composed of a Shimadzu Nexera X2 LC-30AD HPLC system (Shimadzu, Tokyo, Japan) equipped with a LCMS-2020 mass spectrometer (Shimadzu, Tokyo, Japan). An ACE Super C18 (250 mm × 4.6 mm i.d., 3.0 µm) column (ACT, Aberdeen, UK) was maintained at 35 °C. The mobile phase of the binary gradient elution at a flow rate of 0.5 mL/min consisted of eluent A (0.1% trifluoroacetic acid in water) and eluent B (100% acetonitrile). The elution program was as follows: 0–40 min—10–30% B, 40–60 min—30–70% B, 60–64 min—70–90% B, and 64–70 min—90–10% B. The injection volume of extract was 5 µL. The flow rate was 0.5 mL/min; the column temperature was 35 °C. HPLC was run at 0.5 mL/min flow. The optimum electrospray ionization (ESI) mode conditions were set at 350 °C for interface temperature, 250 °C for DL temperature, 400 °C for heat-block temperature, 1.5 L/min for nebulising gas flow, and 10 L/min for drying gas flow. Positive and negative ion measurements were performed while switching alternately between positive and negative ionization modes. MS spectra were recorded in a range of 50 to 2000 at scan speed 15,000 µ/s, with 0.2 m/z steps.

High-performance liquid chromatography with diode array detectors (HPLC-PDA) was performed as described in previous work by Kazlauskaite et al. [36 (link)]. It was carried out using the Shimadzu Nexera X2 LC-30AD HPLC system (Shimadzu, Tokyo, Japan), equipped with an SPD-M20A diode array detector (DAD).
For the determination of polyphenols in plant extracts, an ACE 5 C18 250 × 4.6 mm column (Advanced Chromatography Technologies, Aberdeen, Scotland) was used. The mobile phase consisted of solvent A (acetic acid/methanol/deionised water) (1:10:89 v/v/v) and solvent B (acetic acid/methanol) (1:99 v/v). The linear gradient elution profile was as follows: 80% A/20% B at 0 min, 30% A/70% B at 30 min, and 90% A/10% B at 39 to 40 min. The flow rate was 1 mL/min, and the injection volume was 10 μL. Absorption was measured at 260 nm. Quantification of compounds was performed using reference standards.

The release of daidzein, genistein and biochanin A were determined using high-performance liquid chromatography (HPLC) with diode array detectors (HPLC–PDA). The analysis was conducted using the Shimadzu Nexera X2 LC-30AD HPLC system (Shimadzu, Tokyo, Japan), equipped with an SPD-M20A diode array detector (DAD). The isoflavones’ research conditions are described in the previous work of Kazlauskaite et al. [32 (link)].