Acquity uplc ms tqd system

Freshly prepared 10
mM MeCN stock solution of test compound was diluted 50-fold with DMSO/H₂O (1:1) and incubated at 37 °C for 2 h with mouse plasma
added in 5% DMSO (preheated at 37 °C for 10 min). The final concentration
was 2 μM. At each time point (0, 5, 15, 30, 60, and 120 min),
50 μL of incubation mixture was diluted with 200 μL of
cold MeCN spiked with 200 nM internal standard followed by centrifugation
at 3300 × g for 20 min. The supernatant was
further diluted with H₂O (1:1) for analysis. The concentration
of test compound was quantified by LC/MS/MS on a Waters ACQUITY UPLC/MS
TQD system consisting of a TQD MS equipped with an ESI interface.
The analyses were run on an ACQUITY UPLC BEH C18 (50 × 2.1 mm
ID, particle size 1.7 μm) with a VanGuard BEH C18 precolumn
(5 × 2.1 mm ID, particle size 1.7 μm) at 40 °C. For
each compound, the appropriate mobile phase was chosen. ESI was applied
in positive mode. The response factors, calculated on the basis of
the internal standard peak area, were plotted over time. When possible,
response vs time profiles were fitted with Prism (GraphPad Software,
Inc., USA) to estimate compound t_1/2 in
plasma. Values are the mean of at least two independent experiments
performed in two technical replicates.

10 mM DMSO stock solution of test compound was pre-incubated at 37 °C for 15 min with mouse liver microsomes added 0.1 M Tris–HCl buffer (pH 7.4). The final concentration was 4.6 µM. After pre-incubation, the co-factors (NADPH, G6P, G6PDH and MgCl₂ pre-dissolved in 0.1 M Tris–HCl) were added to the incubation mixture and the incubation was continued at 37 °C for 1 h. At each time point (0, 5, 15, 30, 60 min), 30 µL of incubation mixture was diluted with 200 µL cold CH₃CN spiked with 200 nM of internal standard, followed by centrifugation at 3500 g for 15 min. The supernatant was further diluted with H₂O (1:1) for analysis. The concentration of test compound was quantified by LC/MS–MS on a Waters ACQUITY UPLC/MS TQD system consisting of a TQD (Triple Quadrupole Detector) Mass Spectrometer equipped with an Electrospray Ionization interface. The analyses were run on an ACQUITY UPLC BEH C18 (50 × 2.1mmID, particle size 1.7 µm) with a VanGuard BEH C18 pre-column (5 × 2.1mmID, particle size 1.7 µm) at 40 °C, using 0.1% HCOOH in H₂O (A) and 0.1% HCOOH in CH₃CN (B) as mobile phase. Electrospray ionization (ESI) was applied in positive mode. The percentage of test compound remaining at each time point relative to t = 0 was calculated. The half-lives (t_½) were determined by an one-phase decay equation using a non-linear regression of compound concentration versus time.

10 mM DMSO stock solution of test compound was diluted 50-fold with DMSO-H₂O (1:1) and incubated at 37 °C for 2 h with mouse plasma added 5% DMSO (pre-heated at 37 °C for 10 min). The final concentration was 2 µM. At each time point (0, 5, 15, 30, 60, 120 min), 50 µL of incubation mixture was diluted with 200 µL cold CH₃CN spiked with 200 nM of internal standard, followed by centrifugation at 3500 g for 20 min. The supernatant was further diluted with H₂O (1:1) for analysis. The concentration of test compound was quantified by LC/MS–MS on a Waters ACQUITY UPLC/MS TQD system consisting of a TQD (Triple Quadrupole Detector) Mass Spectrometer equipped with an Electrospray Ionization interface. The analyses were run on an ACQUITY UPLC BEH C18 (50 × 2.1mmID, particle size 1.7 µm) with a VanGuard BEH C18 precolumn (5 × 2.1mmID, particle size 1.7 µm) at 40 °C, using 0.1% HCOOH in H₂O (A) and 0.1% HCOOH in CH₃CN (B) as mobile phase. Electrospray ionization (ESI) was applied in positive mode. The response factors, calculated on the basis of the internal standard peak area, were plotted over time. When possible, response vs. time profiles were fitted with Prism (GraphPad Software, Inc., USA) to estimate compounds half-life in plasma.

10 mM DMSO stock solution of test compound was diluted 50-fold with DMSO-H2O (1:1) and incubated at 37°C for 2 h with mouse plasma added 5% DMSO (pre-heated at 37°C for 10 min). The final concentration was 2 μM. At each time point (0, 5, 15, 30, 60, 120 min), 50 μL of incubation mixture was diluted with 200 μL cold CH₃CN spiked with 200 nM of internal standard, followed by centrifugation at 3.750 rpm for 20 min. The supernatant was further diluted with H₂O (1:1) for analysis. The concentration of test compound was quantified by LC/MS-MS on a Waters ACQUITY UPLC/MS TQD system consisting of a Triple Quadrupole Detector (TQD) Mass Spectrometer (MS) equipped with an Electrospray Ionization (ESI) interface. The analyses were run on an ACQUITY UPLC BEH C18 column (50 × 2.1 mmID, particle size 1.7 μm) with a VanGuard BEH C18 pre-column (5 × 2.1 mmID, particle size 1.7μm) at 40°C, using H₂O + 0.1% HCOOH in H2O (A) and CH3CN + 0.1% HCOOH (B) as mobile phase. ESI was applied in positive mode. The response factors, calculated on the basis of the internal standard peak area, were plotted over time. Response factor versus time profiles were fitted with Prism (GraphPad Software, Inc., USA) to estimate compounds half-life (t½) in mouse plasma.