Uhplc q tof

Reactions contained 20 mM β-O-4 dimer, 50 mM sodium tartrate, pH 4.0, and 330 U/L ABTS activity of FPLC-purified⁵³ PE-vpl2 heterologously produced in N. benthamiana. Coupled reactions additionally contained 0.4% w/v D-glucose. Absorbance corresponding to the formation of dehydrodimer and veratraldehyde was measured at 310 nm using a Synergy HTX plate reader and converted to an estimate of total aldehyde produced using the molar extinction coefficient for veratraldehyde (9300 1/M 1/cm). Reactions were initiated by the addition of peroxide or glucose oxidase.
After completion, 1 μl of the reaction was injected on a 6545 Agilent UHPLC Q-TOF running in positive mode with an 8-min water-acetonitrile gradient (0 min, 95% A; 0.2 min, 95% A; 5.65 min, 37.5% A; 5.66 min, 5% A; 6.11 min, 5% A; 6.15 min, 95% A; 7.18 min, 95% A; A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid; flow rate 0.8 ml/min) on an Agilent RRHD EclipsePlus 95 Å C18 column (2.1 × 50 mm, 1.8 µm, 1200 bar). EIC values were obtained as above.

The lipid species present in the plasma samples were determined by ultra-high performance chromatography coupled to quadrupole-time of flight high-resolution mass spectrometry 6550 (UHPLC-qTOF, Agilent Technologies). The ionization was performed in positive electrospray and mass calibration reference was used along all the analyses to maintain the mass accuracy below 5 ppm. Lipids were separated on a C18 reversed phase column (Kinetex C18-EVO, Phenomenex) and a ternary mobile phase (water/methanol/2-propanol) was used. The quantification of each lipid was made using one analytical standard and one deuterated internal standard for each lipid family (lysophosphatidylcholines, phosphatidylcholines, sphingomyelins, and triglycerides).

Betula alba was diluted 10 times in 95:5 H₂O:ACN and filtered through a 0.2 µm syringe filter. Empetrum nigrum was used without dilution and was filtered through a 0.2 µm syringe filter prior to analysis. Both solutions were analysed using an Agilent UHPLC/Q-ToF (1290 Infinity UHPLC and 6520 Q-ToF) with an electrospray ionization source (ESI).
The separation was performed on a Zorbax extend C₁₈ 2.1 × 150 mm, 1.8 µm column kept at 35 °C at a flow rate of 0.25 mL/min. The injection volume was 20 µL. The mobile phase consisted of H₂O + 0.1% FA (A) and ACN + 0.1% FA (B), using the following gradient: 98% A for the first 10 min which was decreased to 40% after 70 min and finally to 2% after 90 min. Negative ESI was used for BA and +ESI for EN with the following settings: gas temperature 350 °C, drying gas 8 L/min, nebulizer 40 psig and Vcap 3500 V.