Acquity tqd

In this study, high-performance liquid chromatography-tandem mass spectrometry (HP-LC-MS/MS) was used to test the norepinephrine (NE) content in the liver. First, 10.0mg of NE (Zhongshan Golden Bridge, Beijing, China) was added to 0.1% ascorbic acid solution (1mg ascorbic acid dissolved in 1ml of methanol). The pretreatment of samples was conducted according to a previous study (Tsunoda et al., 2002 (link)). Then, the 250-μl sample was dried with a nitrogen gun and reconstituted with 50μl of acetonitrile: water (85:15) solution (containing 2% formic acid), and 20μl of sample and NE standard were added to the LC/MS vial. High-performance liquid chromatography (ACQUITY UPLC, Waters, United States) and mass spectrometry (ACQUITY TQD, Waters, United States) were used for analysis. The MS conditions were as follows: cone gas flow rate of 150L/h, desolvation gas flow rate of 900L/h, source temperature of 150°C, desolvation temperature of 550°C, and ionization mode ESI+. Finally, the samples were subjected to gradient elution as follows: mobile phase A (5:95 acetonitrile containing 30mM formic acid: Milli-Q water) and mobile phase B (85:15 acetonitrile containing 30mM formic acid: Milli-Q water).

The investigated extracts were also subjected to UHPLC-ESI-MS analyses, using an ACQUITY UPLC chromatographic system (Waters, Milford, MA, USA), coupled with a PDA detector and a triple quadrupole mass detector (ACQUITY TQD, Waters). Saponins were separated on an ACQUITY HSS C18 column (2.1 × 100 mm, 1.8 µm; Waters), maintained at 40 °C; the injection volume was 2.5 µL. The elution method was: 0.0–0.5 min, 1% of solvent B (acetonitrile with 0.1% formic acid) in solvent A (0.1% formic acid in MilliQ water); 0.5–25.5 min, a linear gradient to 50% B. The column was then washed with 99% B (2 min) and re-equilibrated with 1% B (1.95 min) to return to the initial gradient. The flow rate was 0.400 mL min⁻¹. The mass detector operated in negative ion mode. Capillary voltage was 3.0 kV; cone voltage was 40 V; source temperature was 140 °C; desolvation temperature was 350 °C; desolvation gas flow was 800 L h⁻¹; cone gas flow was 100 L h⁻¹. Full-scan spectra were acquired in the range from 150 to 1600 m/z.

The NeoBase™ nonderivatized MS/MS kit is a product of the PerkinElmer Company, and the series mass spectrometry analysis system (ACQUITY TQD) is a product of the Waters Company. The cutoff values were initially set with reference to the worldwide collaborative project and other screening centers (Niu et al., 2010 (link)), and as the number of samples increased, they were adjusted over time.

The rate of MPS was determined by measuring the incorporation of the injected [²H₅]Phe into triceps muscle proteins, using a procedure described previously [25 (link)]. Briefly, frozen triceps muscle samples (565–785 mg) were weighed and homogenized in ice-cold 3% ((w/v)) perchloric acid. After centrifugation, the supernatants were collected and the pellets were further washed with distilled water and hydrolyzed with hydrochloric acid. The enrichment of [²H₅]Phe in intramuscular free amino acids was measured in the supernatants and the muscle protein-bound [²H₅]Phe enrichment was measured in the hydrolyzed muscle protein pellets using liquid chromatography tandem-mass spectrometry (ACQUITY TQD, Waters Corporation, Milford, MA, USA). We calculated the FSR, defined as the percentage of muscle protein renewed each day, of triceps muscle proteins according to the formula:

where Eb is the protein-bound Phe enrichment, Ea is the Phe enrichment in the free intramuscular pool, and t is the time interval between the injection of the tracer and the cooling of the muscle sample, expressed in days.

The mass spectrometric analysis was performed using Waters Ultra Performance LC® system (Waters 3100 series, USA), including; binary solvent delivery system, autosampler, Waters Acquity TQD (Triple-Quad detector). Data was processed and acquired using Mass Lynx V4.1 software.

Drug concentrations in the samples were determined using an ultra-performance liquid chromatography (UPLC) system (Acquity^® UPLC, Waters, MA, USA) equipped with a tandem mass spectrometer (Acquity^® TQD, Waters, MA, USA). A reversed-phase Waters Acquity UPLC BEH C18 analytical column (50 mm × 2.1 mm, 1.7 µm particle size) was used, with a mobile phase consisting of 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). The initial mobile phase was 98% solvent A and 2% solvent B, pumped at a 0.3 mL/min flow rate. The percentage of solvent B increased linearly to 95% for 1.0 min and was then maintained for 1.0 min. Between 2.01 and 2.5 min, the percentage of solvent B decreased linearly to 2% and was maintained until the end of the run time of 3.0 min. All samples were injected at 5 µL into the UPLC system. The molecular mass of the analytes was monitored in positive ionization mode. For naftopidil, the m/z values of the precursor and production ions were 393.39 and 190.09, respectively. For pitavastatin, they were 423.26 and 275.22, respectively. For memantine, they were 180.18 and 163.18, respectively.