Acquity uplc 1 class plus system

After the sample preparation (as described above, the same brain extracts have been used for targeted and for the untargeted analyses), untargeted lipidomics was performed by UPLC-ESI-MS/MS equipped with the ACQUITY UPLC I-Class PLUS system (Waters, Milford, MA, United States). The LC conditions were as described in the previous section. The mass analyzer (Vion IMS QTof; Waters, Israel) was equipped with an ESI source and parameters were as follows: the source and de-solvation temperatures were maintained at 120°C and 450°C, respectively. The capillary voltage was set to 3.0 kV and 2.0 kV for positive and negative ionization mode, respectively; cone voltage was set for 40 V. Nitrogen was used as the de-solvation and cone gas at a flow rate of 800 L/h and 30 L/h, respectively. The mass spectrometer was operated in full scan HDMS^E resolution mode over a mass range of 50–2000 Da. For the high-energy scan function, a collision energy ramp of 20–80 eV was applied and for the low energy scan function, −4 eV was applied. Data processing was performed with Progenesis QI software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom). The lipids were identified by comparing the masses and the fragments to databases: HMDB (Human Metabolome Database) (Wishart et al., 2022 (link)), ChemSpider (Pence and Williams, 2010 (link)), and LipidBlast (Kind et al., 2013 (link)).

UPLC-MS was used for quantitative analysis of DHD and valerosidate present in the extract of PV. The equipment used was a Waters ACQUITY UPLC I-Class PLUS System consisting of a binary high-pressure pump with vacuum degasser, a single quadrupole mass detector (SQD2), an autosampler and the Acquity UPLC^® BEH C₁₈ column (2.1 mm × 50 mm, 1.7 μm; Waters, MA, United States). Gradient elution was utilized in the chromatographic separation method under the following conditions: column temperature, 30°C; injection volume, 1 μL; flow rate, 0.5 mL/min. Mobile phases A and B were 0.1% v/v formic acid (FA) in water and 0.1% v/v FA in acetonitrile, respectively. Elution in UPLC used the following linear gradient: 0–2 min, 5% B; 2–2.5 min, 5%–13% B; 2.5–6.5 min, 13% B; 6.5–7 min, 13%–100% B; 7–9 min, 100% B; and 9–11 min, 5% B. The mass spectrometric parameters were set as below: the ion source was electrospray ionization (ESI); the mass spectra were recorded in positive mode; nitrogen nebulizing gas was used for desolvation at a flow rate of 700 L h⁻¹ at 400°C; the capillary voltage was 3000 V; the temperature of the ionization source was 150°C; and the cone voltage was 50 V. Data were acquired in single ion recording (SIR) mode with unit resolution. Data were generated and analyzed on the Masslynx v 4.2 software.

The UPLC-PDA system used the Waters ACQUITY UPLC I-class plus system with the ACQUITY UPLC PDA eλ detector. An ACQUITY UPLC^®HSS C18 SB column (1.8 μm, 2.1 mm × 100 mm) equipped with an ACQUITY UPLC^®HSS C18 VanGuard^TM pre-column (1.8 μm, 2.1 mm × 5 mm) was used for the separation. The column oven was set at 30 °C. The PDA detection wavelength range was from 200 to 780 nm. Formaldehyde-DNPH, acetaldehyde-DNPH, furfural-DNPH, and HMF-DNPH were detected at 360 nm. Glyoxal-bis-DNPH, methylglyoxal-bis-DNPH, and KDG-bis-DNPH were detected at 430 nm. The mobile phase containing 0.1% formic acid in water was designated as mobile phase A and acetonitrile containing 0.1% FA was designated as mobile phase B. The gradient mode was as follows: 0–0.5 min, 5% B; 0.5–7 min, 5%–100% B; 7–8 min, 100% B min; 8–8.5 min, 100%–5% B; and 8.5–9 min, 5% B. The flow rate was 0.6 mL/min and the injection volume was 5 μL. Data acquisition and processing were carried out by Empower 3 software.

LC was performed on an ACQUITY UPLC I-Class Plus System (Waters, Manchester, UK). Roughly 2 μL of the filtered supernatant described above was directly loaded into an ACQUITY UPLC BEH C18 Column (130 A, 1.7 µm, 2.1 mm × 50 mm; Waters, Manchester, UK) pre-warmed to 35 °C. Separation was performed with a mobile phase consisting of buffer A (0.1% formic acid in water) and buffer B (0.1 formic acid in acetonitrile), at a constant flow rate of 0.4 mL/min. The gradient of B is as follows: 1% at 0 min, 1% to 30% over 1 min, 30% to 60% over 5 min, 60% to 99% over 2 min, and finally 99% to 1% over 2 min.