Agilent 1100 hplc

After equilibration and phase separation, the aqueous phase of all samples was analysed on an Agilent HPLC 1100 (Agilent Technologies, Waldbronn, Germany) with diode array detection. Analysis was conducted with a Zorbax SB C18 column (length 150 mm, i.d. 4.6 mm, particle size 5 μm) by Agilent Technologies. 10 min chromatographic runs were performed at 25.0 ± 0.1 °C in isocratic mode using a methanol/water mixture (60/40, v/v) with addition of 0.1% of formic acid as mobile phase modifier at a flow rate of 1.0 mL/min. The mobile phase was filtered through a membrane filter, no. 66, diameter 47 mm, pore size 0.45 µm (Supelco, Bellefonte, PA, USA). The vials were placed in the rack on the autosampler at 25 °C. The injection volume was 10 µL, with a needle offset of 0.5 mm. To avoid any carryover of the analyte-containing n-octanol phase, the outside of the syringe was subsequently washed in methanol before injection. The absorbance of the analytes during a chromatographic run was collected in the spectral range 200–400 nm, and the detection wavelength for each analyte was the one providing the maximum peak height.

To study the formation of GSNO from NO₂-Ln and GSH, we incubated several concentrations of NO₂-Ln (0.1 and 1 mM) with 1 mM GSH in 50 mM phosphate buffer, pH 7.4, containing 0.1 mM DTPA (diethylenetetraminepentaacetic acid) for 1 h at RT^a with a gentle agitation. Reactions were conducted in darkness. Formation of GSNO was analyzed by LC-ES/MS (Bruker Esquire 6000, HPLC Agilent 1100) in negative ion mode. The different analytes were separated in a Waters Spherisorb ODS2 C18 column (3 mm × 125 mm, 5 μm). The mobile-phase composition was water (A) and acetonitrile (B) both with 1% of formic acid at a flow rate of 0.6 ml min⁻¹. The gradient profile was as follows: 2–5% B (0–5 min); 40–95% B (6–22 min); and 95–2% B (22–25 min). MS/MS/MS (M3) analysis from GSNO was conducted in 0.40 V (335) and 0.60 V (305). The desolvation temperature was set at 400°C. In all cases, the data were collected, analyzed, and processed using Data Analysis Mass Spectrometry Software (Bruker, Daltonics).

For inoculum preparation, Satccharomyces cervisiae was transferred from slant and cultivated in 100 mL of YPD medium at 30 °C and 150 rpm for 36 h [25 (link)].
The fermentation process was performed under anaerobic conditions using a layer of paraffin oil upon the fermented medium in 100 mL conical flasks containing 50 mL enzymatic hydrolysate supplemented with the fermentation medium components (KH₂PO₄ 1 g/L, (NH₄)₂SO₄ 5 g/L, MgSO₄ 0.5 g/L, and yeast extract 1 g/L). The prepared medium was sterilized at 121 °C for 20 min. Batch fermentation was initiated by inoculating 5% (v/v) inoculum seed (yeast) into 50 mL of the medium and covered by a paraffin oil layer in a 100 mL conical flask fitted with plugs covered by parafilm. The flasks were statically incubated at 30 °C for 72 h. After fermentation, the medium was centrifuged at 10,000 rpm for 10 min, and the supernatant was used to determine the bioethanol production yield using the chromic acid method [26 ]. The results were also emphasized using HPLC Agilent 1100 (Santa Clara, CA, USA). The carrier gas was nitrogen, with a flow of 0.2 mL/min. The used column was Hi-plexCa uspl₁₉ (Agilent) 250 $$\times$$ 4 m and operated at 80 °C. The detector was RI and operated at 55 °C.

Pure test compounds for TAC measurements were purchased from Extrasynthase (Lyon, France) and stored at 4 °C with the exception of apigenin, kaempferol-3-O-glucuronide and quercetin-3-O-glucuronide which were stored at −20 °C. These compounds were dissolved with aqueous ethanol (70 ethanol:30 water v/v) and dimethyl sulfoxide. Synthetic leaf mimics were mixed from pure test compounds following molar ratios of major compounds identified in grapevine leaves by HPLC-MS (HPLC:Agilent 1100, Waldbronn, Germany; MS: Bruker, Bremen, Germany).

LC-MS/MS analysis was performed with HPLC Agilent 1100 (AgilentTechnologies, Santa Clara, CA, USA) equipment coupled with QSTAR XL (AbSciex, Framingham, MA, USA) mass spectrometer. Electrospray ionization (ESI) was operated in a positive ion mode in order to obtain soft and efficient ionization of ivabradine and its degradation products. Kromasil 100 C8 (4.6 mm × 250 mm, 5 μm, AkzoNobel, Amsterdam, Netherlands) was used in reversed-phase mode with isocratic elution. Mobile phase composition was 65% of component A (20 mM ammonium acetate) and 35% of component B (acetonitrile). Total analysis time was 30 min. Column temperature throughout the analysis was 25°C the flow rate of the mobile phase was 1 ml/min and the injection volume was 20 μl. The high resolution Q-TOF mass spectrometer was operated in SCAN mode to obtain mass spectrum in the mass range 220–1000 m/z. MS/MS mass spectra were obtained by collision-induced dissociation of selected parent ions in Product Ion mode of mass spectrometer. Degradation studies were carried out in Thermostat CC2-K6 made by Huber company (Offenburg, Germany), photo stability studies were performed in a photostability Suntest + Atlas chamber (Accelerated Tabletop Exposure Systems) with xenon lamp. Shimadzu UV-1800 UV-Vis spectrophotometer was also used in the studies.