Xevo tqd mass spectrometer

Gb3 isoforms and analogs were determined using a Xevo TQD mass spectrometer with an Acquity UPLC system (Waters). An Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7-μm particle size; Waters) was used for the chromatographic separation of Gb3 isoforms/analogs. The flow rate was 0.5 ml/min, and the column temperature was 60 °C using mobile phase A (H₂O-ACN (95:5) + 0.1% FA) and mobile phase B (ACN-IPA-MeOH (40:40:20) + 0.1% FA). The column was equilibrated with 30% mobile phase A and 70% mobile phase B. The elution was started with a linear gradient from 70 to 100% mobile phase B in 1 min, followed by 2 min of using 100% mobile phase B, and then an immediate return to initial conditions for 2 min. MS/MS methods were set as Table S1. Retention time on the LC and basic mass condition for the detection of each Gb3 isoform and analog are summarized in Table 3. The cone voltage and collision energy were set as 120 and 60 V, respectively, for Gb3 in basic mass conditions. The peak areas corresponding to the Gb3 isoforms and analog/isoforms and the internal standard in the MRM chromatogram were calculated using MassLynx software (Waters).

U251 cells (2 × 10⁶) seeded in six-well plates were collected in six replicates, mixed with 1 ml chilled methanol/water (4:1, v-v) and 200 μl chloroform, and were further ultrasonically broken in an ice bath for 3 min. After adding internal standards, the solution was sonicated for 20 min and deproteinized by centrifugation at 4 °C (13,000 rpm, 10 min). Finally, 1 ml of the supernatant was evaporated, reconstructed with 200 μL methanol/water (1:4, v-v), further incubated for 2 h at −20 °C, and centrifugated prior to analysis. Metabolic extracts were analyzed using a Nexera UPLC-MS/MS system (Shimadzu, Japan). Chromatographic separation was conducted on a Waters ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm) at a flow rate of 0.35 mL/min, with acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid). The electrospray ionization (ESI) operation of the Waters Xevo TQD mass spectrometer (Waters Milford, USA) was used in the positive (ESI+) and negative (ESI–) ion modes; the negative and positive modes were 3.0 and 3.5 kV, respectively, and the drying gas (N2) flow rate was 10.0 L/min 320 °C, Data were recorded from 50 to 1000 m/z.

The MCFA levels in the plasma and intestinal samples were determined following a previously described protocol with modifications (17 (link)). Briefly, the samples containing an internal control (C19:0) were homogenized in methanol and mixed with chloroform and water to extract the lipids. The samples were centrifuged at 2,000 × g at 17 °C for 10 min. The supernatant containing MCFAs was collected and dried. The samples were resuspended in chloroform:methanol (1:3, v/v) and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis using an ultra-performance LC system (UPLC, Waters, Milford, MA, USA) equipped with an Acquity UPLC system coupled to a Waters Xevo TQD mass spectrometer (Waters). The samples were separated on an ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 μm; Waters) using a methanol gradient in 10 mM ammonium formate aqueous solution.

A Waters ACQUITY UPLC system (Waters, Milford, MA, USA) was connected to a Waters Xevo TQD mass spectrometer (Waters) operated in the positive electrospray ionization mode. M20.7 was separated on a Polaris 5 μm C18-A 50 × 2.0-mm column (25 °C) (Agilent Technologies, Amstelveen, The Netherlands) with a MetaGuard 2.0 mm Polaris 5 μm C18-A guard column (Agilent Technologies). The mobile phase of A/B (1:3, v/v) was used, where A was methanol/10 mM ammonium acetate, pH 8.0 (5:95, v/v), and B was acetonitrile/methanol (10:90, v/v). The flow rate was adjusted to 0.2 mL/min, and an eluent between 0 and 5 min was introduced into the mass spectrometer. The sample was analyzed during the multiple reaction monitoring mode of the mass spectrometer at a dwell time of 0.1 s per channel using m/z 323.1 > 173.3 as the transition. The ionization conditions were as follows: capillary voltage, 3.4 kV; cone voltage, 42 V; collision energy, 18 V; source temperature, 150 °C; desolvation temperature, 200 °C; collision gas, argon. Data acquisition, instrument control and data handling were performed with MassLynx Software (version 4.1; Waters).

Metabolic profiling of aerial parts and rhizomes of Egyptian and Japanese I. pseudacorus extracts was carried out on a Waters Xevo TQD mass spectrometer with UPLC Acquity mode (Milford, CT, USA) at the Center for Drug Discovery Research and Development, Faculty of Pharmacy, Ain Shams University. Extracts were liquefied in diluted methanol and injected directly into the UPLC-ESI-MS system. Both negative and positive ESI ionization modes were applied under the following conditions: A gradient of water and acetonitrile (ACN) with 0.1% was applied from 2 to 100% ACN in 60 min at 30 ◦C. The flow rate was 0.5 mL/min. The injection volume was 20 µL. The capillary voltage of MS (10 V), the ions were noticed within a mass range from 50 to 2000 m/z with collision energy (35 eV).

¹H and ¹³C (APT) NMR analyses were done using a Bruker Ascend 400/R spectrometer (Burker Avance III, Fallanden Switzerland) at the Center for Drug Discovery, Research and Development, Faculty of Pharmacy, Ain Shams University using 400 and 100 MHz the operating frequencies. Chemical shifts were reported in δ ppm and were related to that of the solvents. Dissolution of the tested samples was done using various deuterated solvents (Sigma Aldrich, Germany) in 3 mm NMR tubes (Bruker). Spectra were recorded at 25 °C; δ ppm relative to tetramethylsilane (Me₄Si) as the internal standard. Two-dimensional (2D) NMR experiments (¹H, ¹H-¹H COSY; ¹H-¹³C HSQC; ¹H-¹³C HMBC) were done using the pulse sequences from the Bruker user library. Waters Xevo TQD mass spectrometer supplied with UPLC Acquity mode (Milford, USA) was employed to carry out ESI-MS analysis. Normal phase column chromatography was done using silica gel (Kieselgel 60, 70–230, and 230–400 mesh, Merck KGaA, Darmstadt, Germany). TLC analysis was done utilizing normal phase silica gel precoated plates F₂₅₄ (Merck, Germany). Detection of TLC spots was done using UV light at 254 nm and 365 nm as well as by spraying with 10% H₂SO₄ with subsequent heating on a hot plate at 100 °C.

Before the analysis, the dried residues were reconstituted in 70 μL of acetonitrile-water, 95:5 (v/v) and vortex-mixed. A hydrophilic interaction liquid chromatography (HILIC)—MS/MS method [31 (link)] was applied, focusing on 110 key hydrophilic metabolites. The separation was performed on an ACQUITY UPLC H-Class system using an ACQUITY UPLC BEH Amide column (Waters Ltd., Elstree, UK). A Xevo TQD mass spectrometer (Waters Corporation, Millford, MA, USA) was operated in MRM mode in both positive and negative electrospray ionization (ESI) modes. The mobile phase consisted of (A) acetonitrile-water, 95:5 (v/v) and (B) acetonitrile-water, 30:70 (v/v), both containing 10 mM ammonium formate at pH 6.