Tq s mass spectrometer

Colonic content samples (80 mg, n = 6 per group) were mixed with 500 µL of methanol/water (v/v = 1:1) mixture and shaken by vortex for 30 min. Then, 50 µL supernatant that was collected after centrifugation (14,000 rpm, 10 min) was mixed with 50 µL of internal standard (5 µg/mL propionic acid-d2). Samples were derivatized with 50 µL 3-nitrophenylhydrazine (200 μM) for 30 min, and the supernatant was collected after centrifugation (14,000 rpm, 10 min). The resulting supernatant (5 µL) was analyzed on an Acquity UPLC I-Class system equipped with a BEH C18 chromatographic column (100 mm × 2.1 mm, 1.7 µm; Waters, Milford, MA, USA) coupled to a Xevo TQ-S mass spectrometer [29 (link)].

LC/MS-MS analysis was performed using an Acquity UPLC System I-Class equipped with a Waters Acquity UPLC BEH C₁₈ column (2.1 mm i.d. × 100 mm length, 1.7 μm particle size) coupled to a Xevo TQS mass spectrometer (all from Waters, Milford, MA) used in electrospray ion positive mode.

Emvododstat-naïve, non-tumor-bearing rhesus monkeys were dosed by oral gavage with emvododstat (10 mg/kg in a lipid-based formulation). Blood samples were collected prior to dosing and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 16, 24, 32, 48, 72, and 168 hours post-dose. The blood was centrifuged, and the plasma was collected for subsequent analysis using Waters UPLC and TQ-s mass spectrometer.
Protein precipitation with an organic solvent was used to extract testing compounds from plasma before injection onto the column. In a mobile phase gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, emvododstat was separated using a Waters UPLC BEH C18 1.7 µm, 2.1x5.0 mm column and detected by multiple reaction monitoring (MRM) transition 465.6 → 127.3. The biomarkers were separated by ES-Industry Epi-Polar Column, 5 µm 120Å 10 cm x 4.6 mm, and detected by MRM transition 156.95 → 112.95 and 175.1 → 132 for DHO and N-carbamoyl-L-aspartate, respectively. Deuterated (D3)-PTC299 and deuterated (D4)-DHO were used as internal standards for emvododstat and biomarkers analysis, respectively. During sample analysis, each calibration curve spiked with emvododstat in the control plasma was analyzed with the test samples to determine the unknown plasma concentrations. The calibration curve ranged from 0.1 to 8 µg/mL for DHO and 0.002 to 2 µg/mL for emvododstat.

The Acquity UPLC system (Waters, USA), equipped with a UPLC binary solvent manager, an autosampler and column oven, was employed for the chromatography analysis. VS-5584 and IS were separated from interfering endogenous compounds via an Acquity UPLC BEH C₁₈ (2.1 × 100 mm, 1.7 μm particle diameter, Waters, USA) column under 25°C, with a mobile phase composed of acetonitrile and 0.2% formic acid in water (40 : 60) with a flow rate of 0.20 mL/min.
The mass spectrometric detection of the analytes was performed on a Waters TQ-S mass spectrometer (Waters, USA). MS-optimized parameters were listed as follows: nitrogen was used as the desolvation gas with a flow of 800 L/Hr, the desolvation temperature was set at 500°C, and capillary voltage was 2.5 kV. The MRM transitions of m/z 354.78 ⟶ 312.73 for VS-5584 and 410.64 ⟶ 366.65 for the IS were monitored under positive electrospray ion (ESI⁺) condition. The optimized cone voltage of VS-5584 and IS was 40 V, and the collision energy for VS-5584 and IS was 24 V and 32 V, respectively. Dwell time for both of the analytes was 0.025 s.

A stock solution containing 64 mg/L of compounds 1–3 was prepared in a triplicate. An 8-point serial dilution was performed using 500 µL stock and 500 µL water. A Waters Cortecs C18+ (2.1 × 100 mm, 1.6 µm) column with H-class Acquity UPLC system containing quaternary solvent manager (QSM), sampler manager (FTN), and column manager coupled to a Waters TQS mass spectrometer was used for quantification. Acidified water (0.1% formic acid, v/v) and acidified acetonitrile (0.1% formic acid, v/v) were used as mobile phases A and B, respectively. The gradient was programmed as follows: 0 min, 25% B; 1 min, 35% B; 6 min, 55% B; 7 min, 95% B; 9 min 95% B; 9.01 min, 25% B. Initial conditions were held for two minutes as an equilibration step. The column temperature was 40 °C, the column flow rate was 0.5 mL/min, and the injection volume was 2 µL. Data were collected using multiple reaction monitoring (MRM) mode using the following optimized collision energies and transitions: compound 1 (965.4 → 803.40, CE: 34v), compound 2 (983.47 → 821.57, CE: 25V), and compound 3 (981.39 → 819.39, CE 36V). The mass spectrometer was operated in negative ion mode with the following source parameters: capillary voltage 2.5 kV, cone voltage 20 V, source temperature 150 °C, desolvation gas temperature 500 °C, cone gas flow rate 150 L/h, and desolvation flow rate 1000 L/h.