Zorbax rx c8

The analyses were performed with a Waters™ alliance 2690 separations module, which was equipped with an autosampler unit, a gradient pump, a column oven and a sample heater. The chromatographic device was connected via Peek capillary (0.18 mm ID) to a Waters™ triple quadrupole mass spectrometer (Micromass Quattro Micro). The final optimized method used a Waters XSelect CSH Phenyl-Hexyl-Column (130 Å, 150 mm length, 2.1 mm ID, 3.5 μm particle size) and a column guard of the same material. Further tested HPLC columns were zorbax RX-C8 (Agilent, 150 mm length 2.1 mm ID, 5 μm particle size) and Nucleodur π² (Macherey Nagel, Düren, Germany, 250 mm length 3 mm ID, 5 μm particle size). The 2 mL clear glass vials for the extracts were purchased from neoLab Migge (Heidelberg, Germany).

Optical rotation measurements were obtained on a Perkin-Elmer 241 Polarimeter calibrated using a Rudolph Quartz Control Plate Calibration Standard at sodium D line (at +11.502°). Ultraviolet spectra were obtained on a UV-visible Molecular Devices SpectraMax M5 spectrophotometer using 1-ml cuvettes with 1.0 cm path lengths at room temperature in solvent methanol (MeOH). Spectrophotometric assays were performed on Molecular Devices SpectraMax M5 384 variable wavelength spectrometer. All NMR spectra were acquired on a Varian INOVA 600 MHz and a Varian INOVA 700 MHz spectrometer at the NMR Facility, Department of Chemistry, University of Michigan. HRESIMS spectra were measured at the University of Michigan core facility in the Department of Chemistry using an Agilent 6520 Q-TOF mass spectrometer equipped with an Agilent 1290 HPLC system. RP-HPLC was performed using Econosil C18 10 μm 22 × 250-mm column and Agilent ZORBAX RX-C8 5 μm 9.4 × 250-mm column and a solvent system of MeCN and H₂O. The LC-MS analysis of HPLC fractions was performed on a Shimadzu 2010 EV APCI spectrometer.

The HPLC analysis of FXE was performed using HPLC-DAD (Agilent 1200, Germany) equipped with a Zorbax RXC8 (Agilent, USA) analytical column with a 5 μm particle size and a 25 ml capacity using the previously reported method by Zu et al.^{23 (link)} Each sample was diluted with HPLC grade methanol. The mobile phase consisted eluent A, (acetonitrile–methanol–water–acetic acid/5 : 10 : 85 : 1)-and eluent B (acetonitrile–methanol–acetic acid/40 : 60 : 1). The gradient (A : B) utilized was as follows: 0–20 min (0 to 50% B), 21–25 min (50 to 100% B), 26–30 min (100% B) and 31–40 (100 to 0% B) at a flow rate of 1 ml min⁻¹. The standards and samples were prepared in HPLC grade methanol (1 mg ml⁻¹), filtered through a 0.45 μm-membrane filter and 20 μl was injected for the analysis. Myricetin, quercetin, and kaempferol were investigated at the wavelength of 368 nm, gallic acid and rutin acid were analyzed at 257 nm, catechin was examined at 279 nm and caffeic acid was evaluated at 325 nm. Before each column was run the column was reconditioned for 10 min and the analysis was performed in triplicate for each one of the FXE samples. By using the external standard method, we have fully assimilated the peaks for the quantification of samples. All work was carried out at ambient temperature.

Commercial reagents and anhydrous solvents were used without further purification. Flash chromatography was performed on the Reveleris^® X2 system, Buchi. ¹H-NMR and ¹³C-NMR spectra were obtained using a Bruker Avance spectrometer at 400 MHz proton frequency (AV-III-HD, 400, Rheinstetten, Germany). All data were processed with TOPSPIN (version 3.5pl5, Bruker Biospin, Spring, TX, USA) software. Conventional abbreviations used for signal shape are as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. LC-MS analysis (1260 Infinity Series HPLC, 6120 Quadrupole MSD, Agilent Technologies Inc.; Richardson, TX, USA) was carried out with the use of a reverse phase column, Zorbax RX-C8 (5 µm, 250 × 4.6 mm, Agilent Technologies, Santa Clara, CA, USA), maintained at 40 °C. Chromatograms were integrated and analyzed using OpenLAB Chemstation (version M8301AA, Revision C.01.07 Agilent Technologies Inc., Richardson, TX, USA). Mobile phase A was water, mobile phase B was acetonitrile and mobile phase C was glacial acetic acid. The mobile phases A, B and C were mixed at a ratio of 67.5:25.0:7.5 (v/v).