Triple quad mass spectrometer

A LC/MS/MS method was utilized to determine the DS concentration in plasms. The system was composed of a Triple Quad Mass Spectrometer (Agilent Corp.) and a HPLC column (Durashell C18, 2.1 mm × 50 mm, 1.8 μm). Mass spectrometry was performed using a negative ionization mode (ESI‑) electrospray ionization interface and quantitative analysis was performed using multiple reaction monitoring (MRM). The ion pairs used for monitoring were 315.1→200.04 (DS) and 229.1.00→185.06 (internal standard, resveratrol), respectively. The mobile phase consisted of solvent A (0.1% aqueous formic acid Solution) and solvent B (0.1% aqueous formic acid in methanol). Analytes were eluted with the following gradient program: gradient elution started at 5% B in 1.5 min and linearly increased to 95% from 1.5 min to 8 min, and then decreased to 5% from 11.5 min to 15 min. The flow rate was 0.25 mL/min and the column temperature was 35 °C. The injection volume was 20 μL.

LC–MS equipment (LC–MS QqQ-6410B Agilent Technologies) comprised a chomatographic system (1260 Infinity Agilent Technologies) coupled with an Agilent Triple Quad mass spectrometer fitted with an ESI source. MS conditions were the following: MS range 100–1200 Da, MSn spectra were obtained using both positive and negative modes, nebulizer gas 45 Psi, gas temperature 325 °C, capillary voltage 4000 V.
HPLC analysis was carried out by an Agilent 1260 Infinity series. A Chomolith RP-18e column (4.6 mm ID, 50 mm length) (Merck) was used. Mobile phase consisted of (A) aqueous formic acid (0.1%) and (B) methanol. Gradient condition was; 0–8 min, linear gradient from 12 to 25% of B; 8–12 min, isocratic conditions at 25% of B; 12–16 min, linear gradient from 25 to 40% of B; 16–40 min, linear gradient from 40 to 50% of B, 40–50 min, linear gradient from 50 to 100% of B. Flow rate: 1 mL/min.

Lipidomics analysis was performed as previously described (Admasu et al., 2018 (link)). Briefly, we used an Agilent 1260-Ultra Performance Liquid chromatography (UPLC) system coupled to Triple Quad Mass spectrometer (Agilent 6,490) with dynamic multiple reaction monitoring (dMRM) for lipid quantification. The UPLC system was equipped with a Waters ACQUITY BEH C18 column (1.0 × 100 mm). Solvent A was acetonitrile/H₂O (60:40) with 10 mM ammonium formate and 1% formic acid. Solvent B was isopropanol/acetonitrile (90:10) containing 10 mM ammonium formate and 1% formic acid. Gradient elution was performed initially from 40 to 100% solvent B over 14 min at a flow rate was 0.13 ml/min and a column temperature of 60°C. After 3 min at 100%, solvent B was decreased rapidly back to 40% in 1 min and this was then maintained until the end of the run at 20 min. The eluent was directed to the ESI source of the mass spectrometer operated in the positive ion mode. The MS conditions were as follows: For ESI: gas temperature, 300 °C; gas flow, 10 L/min; sheath gas temperature, 350 °C; sheath gas flow, 8 l/min; and capillary voltage, 3,500 V.