Gemini nx rp 18 column

Ultraviolet lamp (UVP, LLC, USA) was used for visualization of spots on thin layer chromatograms at 254 and/or 365 nm. UV spectra of different samples were acquired using a Spectronic^® Genesys^TM 2PC UV spectrophotometer (Shimazdu, Japan) as solutions in methanol as well as with different diagnostic UV shift and complexing reagents for flavonoids [20 ]. ¹H (400 MHz) as well as ¹³C NMR (100 MHz) and distortionless enhancement by polarization transfer (DEPT-Q; 100 MHz) spectra were recorded on Bruker Avance 400 MHz instruments in DMSO-d₆ and CD₃OD. Chemical shift values (δ) were recorded in ppm units and coupling constants (J) in Hz. Solvent signals of DMSO-d₆ (δ_H 2.5 ppm and δ_C 39.5 ppm) and CD₃OD (δ_H 3.3 ppm and δ_C 49.0 ppm) were considered as the internal reference signals for calibration. Electrospray ionization mass spectrometry (ESI‒MS) spectra were obtained using a Synapt G2 HDMS QTOF (quadrupole time-of-flight)-mass spectrometer (Waters, Germany). HPLC separations and purifications were performed on KNAUER HPLC (smart line pump 1000, degasser, diode array detector) with UV Detector, using semi-prep RP-18 column (5 μm, 10 × 250 mm; Waters XBridge, Germany), while an analytical Gemini-NX RP-18 column (5 μm, 4.60 × 100 mm; Phenomenex, Germany) was used for analytical purposes.

The analyses were performed employing a previously validated HPLC-PDA method [6 ]. Briefly, a Gemini-NX RP-18 column (250 × 4.6 mm i.d., 5 μm) (Phenomenex, USA) protected by a RP-18 guard column was used. The mobile phase consisted of ammonium acetate buffer 10 mM (pH 7.0) (A) and acetonitrile (B) in a linear gradient program. The flow rate and temperature were kept constant at 1.0 mL/min and 23 ± 1°C, respectively, the detection being performed at 245 nm. The total pentacyclic oxindole alkaloids (POA) and tetracyclic oxindole alkaloids (TOA) contents were calculated by sum of individual alkaloid contents, namely, speciophylline, uncarine F, mitraphylline, isomitraphylline, pteropodine, and isopteropodine for POA and rhyncophylline and isorhyncophylline for TOA. The results were expressed as μg/mL of extractive solution by mean value of three determinations using the mitraphylline (Phytolab, batch 2946, Germany) as external standard.