Zorbax sb c18 analytical column

The raw material for AGNEX was collected from the herbarium of Kyungsung University. The extraction process was carried out in accordance with our previous study with slight modifications [46 (link)]. Briefly, dried AGN was pulverized using a grinder machine (250G New Type Pulverizing Machine, Model RT-N04-2V, Taiwan) and the powder was sieved through a 60-mesh sieve to ensure uniform particle sizes. AGN powder was mixed with a 5-fold volume of 95% ethanol, followed by a 3 h incubation at 40 °C. The mixture was then filtered through a housing filter (35 µm pore size), a 2-fold volume of water was added, and the mixture was centrifuged at 10,000 rpm for 15 min, followed by pH adjustment to 5.0. The precipitate was dissolved in a 5-fold volume of 70% ethanol and applied at 20 Brix. The quantitative determination of decursin and decursinol angelate from AGN was carried out using the Agilent 1100 HPLC system (Santa Clara, CA, USA) equipped with a ZORBAX SB-C18 analytical column (250 mm, 4.6 mm, 5 µm; Agilent, Santa Clara, CA, USA). The purity of decursin and decursinol angelate of AGNEX was approximately 78% purity. Acetonitrile and 0.1% formic acid (70:30, v/v%) were used as the mobile phase. The absorbance was detected at 329 nm using a UV monitor (Photo-Diode Array UV monitor, Agilent, Santa Clara, CA, USA) (Figure 7).

Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to 3200 QTRAP mass spectrometer (AB Sciex, Redwood City, CA, USA) was used for qualitative and quantitative analysis of flavonoids and phenolic acids in Hemerocallis extracts. The separation of analyzed compounds, injected in a 3-µL amount, was performed on a Zorbax SB-C₁₈ analytical column (2.1 × 100 mm, 1.8 µm, Agilent Technologies, Palo Alto, CA, USA) at 25 °C. Elution was carried out using solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in acetonitrile). The following gradient elution program was used: 0–2 min—20% B, 3–4 min—25% B, 5–6 min—35% B, 5–6 min—35% B, 8–12 min—65% B, 14–16 min—80% B, 20–28 min—20% B. The flow rate was 300 µL/min. The mass spectra of analyzed compounds were acquired in the negative ESI mode, and the optimum values of the source parameters were as follows: capillary temperature 450 °C, nebulizer gas 50 psi, curtain gas 30 psi, source voltage −4500 V for phenolic acids and flavonoid glycosides, and capillary temperature 550 °C, nebulizer gas 30 psi, curtain gas 20 psi, and source voltage −4500 V for flavonoid aglycones analysis. Details of LC-ESI-MS/MS analysis are presented in Table 1 and were described in our previous research [41 (link)]. The Analyst 1.5 software (AB Sciex, Redwood City, CA, USA) was used for analysis and data acquisition.

An ACQUITY UPLC detecting system (Waters Co., Milford, MA, USA) coupled to a triple time-of-flight (TOF) 5600+ mass spectrometer were employed for UPLC-Triple-TOF/MS analysis. An electrospray ionization source (AB SCIEX Co., Foster, CA, USA) and a ZORBAX-SB C18 analytical column (100 mm × 4.6 mm, Agilent Technologies Inc., Santa Clara, CA, USA) were used. Gradient elution conditions were the same as described above. MS conditions were listed as follows: negative ion scanning mode, MS scanning range of 100–1500 m/z; gas1 (GS1): 50 psi; gas2 (GS2): 50 psi; curtain gas (CUR): 35 psi; temperature of the ion source (TEM): 550 °C; voltage of the ion source(IS): −4500 V (negative); first order scanning: declustering potential (DP): 100 V; focusing voltage (CE): 10 V; second order scanning: TOF MS~Product Ion~IDA mode for data collection. CID energy: −20, −40 and −60 V. Before sample injection, CDS pump were used for mass axis correction to lower the error below 2 ppm.

Eicosanoid analysis was performed using an HPLC Agilent 1290 Infinity on a ZorBAX SB-C18 analytical column (50 × 2.1 mm, 1.8 μm) (Agilent Technologies) maintained at 40 °C. The mobile phases were composed of two solvents: solvent A, H₂O with 0.1% (v/v) HCOOH, and solvent B, ACN with 0.1% (v/v) HCOOH. The flow rate of the mobile phase was 0.35 mL.min⁻¹, and the injection volume was 5 µL. The gradient of the elution was as follows: 0% B at 0 min, 85% B at 8.5 min, 100% B at 9.5 min for 1 min.
The liquid chromatography was coupled to an Agilent 6460 triple quadrupole MS with an ESI source in negative mode. The monitoring of fragmentation was executed in Selection Reaction Monitoring (SRM) detection mode. Finally, peak detection, integration and quantitative analysis were obtained using the MassHunter Quantitative analysis software version B.09.00 (Agilent Technologies).

An Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) coupled to a 3200 QTRAP mass spectrometer (AB Sciex, Redwood City, CA, USA) was used for the analysis of phenolic acids and flavonoids in A. acutiloba various extracts. The separation of compounds was performed on a Zorbax SB-C18 analytical column (2.1 × 100 mm, 1.8 µm, Agilent Technologies, Palo Alto, CA, USA) at 25 °C. Elution was conducted using solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in acetonitrile). The following gradient elution program was used: 0–2 min—20% B, 3–4 min—25% B, 5–6 min—35% B, 5–6 min—35% B, 8–12 min—65% B, 14–16 min—80% B, 20–28 min—20% B. The flow rate was 300 µL/min. The mass spectra of analyzed compounds were acquired in the negative ESI mode, and the optimum values of the source parameters were as follows: capillary temperature 450 °C, curtain gas 30 psi, nebulizer gas 50 psi, source voltage −4500 V for phenolic acids and flavonoid glycosides, and capillary temperature 550 °C, curtain gas 20 psi, nebulizer gas 30 psi, and source voltage −4500 V for analysis of flavonoid aglycones. The other details of LC-ESI-MS/MS analysis were described in our previous research [17 (link)].