Acquity uplc beh phenyl column

Extraction of P. notoginseng was prepared and quantitative analysis of PNE and PNS was performed as stated in our previous article [23 (link)]. In brief, the dried P. notoginseng powder was extracted with 95% ethanol and the ethanol was removed by rotary evaporation. Finally, the extract was freeze-dried to obtain PNE. PNS was obtained from Yunnan Yunke Pharmaceutical Co. Ltd. (China). The component analysis of PNS and PNE was determined by Waters ACQUITY-UPLC CLASS system (Waters Corp., USA) with an ACQUITY UPLC BEH phenyl column (150 mm × 2.1 mm, 1.7 μm) maintained at 45 °C to quantify the contents of notoginsenoside R1, ginsenoside Rb1, ginsenoside Re, ginsenoside Rg1, and ginsenoside Rd.

After each preparative or semi-preparative purification step, the resulting fractions were analysed by UPLC-DAD. Sample analyses was carried out with an Acquity UPLC system (Waters Corporation, Milford, MA, USA) coupled with a DAD detector. The separation was performed on 100 × 2.1 mm i.d., 1.7 μm, Acquity UPLC BEH Phenyl column (Waters Corporation, Wexford, Ireland). The eluent flow rate was set to 0.5 mL/min. The elution profile used two solvents, acetonitrile (A) and 0.1% aqueous formic acid (B): 0–0.5 min, 0.1% A in B; 0.5–5.0 min, 0.1–30% A in B (linear gradient); 5.0–5.1 min, 30–90% A in B (linear gradient); 5.1–8.5 min, column wash and stabilization. HPLC/UV chromatogram was collected for the first 6 min of the separation.

GDP profiling was performed as previously reported [17 (link)]. Briefly, α-dicarbonyls were derivatized with o-phenylenediamine yielding their respective quinoxaline derivatives. The pH of the double-chamber bag model was adjusted to 5.5 prior to derivatization. A Thermo Fisher UltiMate 3000RS liquid chromatography system consisting of a pump with degasser, autosampler, column compartment, and diode array detector equipped with an ACQUITY UPLC® BEH phenyl column (1.7 μm particle size; 2.1 × 100 mm, Waters, Eschborn, Germany) was used for the chromatographic separation of the quinoxalines. The system was controlled by Chromeleon 6.80 (Thermo Fisher Scientific, Dreieich, Germany). The quinoxaline derivatives were analyzed between 120 and 650 min after adding the derivatizing reagent.

Sample
analysis was carried
out with an Acquity UPLC system (Waters Corporation, Milford, MA,
U.S.A.). The UPLC system consisted of a sample manager, a binary solvent
manager, a column, and a diode array detector. The column used was
a 100 × 2.1 mm inner diameter, 1.7 μm, Acquity UPLC BEH
Phenyl column (Waters Corporation, Wexford, Ireland). The flow rate
of the eluent was 0.5 mL min^–1. The elution profile
used two solvents, acetonitrile (A) and 0.1% aqueous formic acid (B):
0–0.5 min, 0.1% A in B; 0.5–5.0 min, 0.1–30%
A in B (linear gradient); 5.0–5.1 min, 30–90% A in B
(linear gradient); and 5.1–8.5 min, column wash and stabilization.
Ultraviolet (UV) data (190–500 nm) were collected from 0 to
6 min.

All of the eicosanoid standards including HETEs, EETs, and dihydroxy‐eicosatrienoic acids (DHETs) were purchased from Cayman Chemical (Arbor, MI, USA). AA metabolites in the striatum were detected as previously described.
²³ (link) Briefly, samples were homogenized and extracted with an extraction solvent containing 2,6‐di‐tert‐butyl‐4‐methylphenol and formic acid. The stable isotope probes, 2‐dimethylaminoethylamine (DMED) and d4‐DMED were added for derivatization. A Shimadzu LC‐30 AD UPLC (Tokyo, Japan) equipped with an Acquity UPLC BEH phenyl column (2.1 mm × 50 mm, 1.7 m, Waters) was used for UPLS analysis. The separation was performed with the mobile phase consisting of (A) FA in water (0.1%, v/v) and (B) ACN/MeOH (7/3, v/v). An ABI/SCIEX 4500 Triple Quad™ equipped with a Turbo V ion source was used for mass spectrometry analysis. Samples were detected using multiple reaction monitoring mode.

The thawed filtrates were derivatized and analyzed by UHPLC-DAD, as described before [53 (link)]. Following this method, 80 µL of sample containing α--dicarbonyls were reacted with 20 µL of derivatizing reagent containing o-PD and 2,3-dimethylquinoxaline, as the internal standard. After at least 2 h, the formed quinoxalines were subsequently separated, applying a multi-step gradient, consisting of ammonium formate buffer (pH 2.8) and methanol on an ACQUITY UPLC^® BEH phenyl column (2.1 × 100 mm, 1.7 µm particle size; Waters, Eschborn, Germany) equipped with a pre-column of the same material, detected by DAD at 316 nm (3-DG_Qx and 3-DGal_Qx) and 335 nm (3,4-DGE_Qx), and quantified via external calibrations. The applied UHPLC-DAD system (Ultimate 3000RS, Thermo Fisher Scientific, Dreieich, Germany) comprised a pump with degasser, autosampler, column oven, and DAD. The software, Chromeleon 6.80 (Thermo Fisher Scientific, Dreieich, Germany), was used for system control and data analysis. The areas of the two 3,4-DGE–GSH monoadducts were summed up and quantified via the 3-DG calibration curve because of their similar absorption behavior.