Acquity uplc flr detector

Ten microlitres (10 μL) of reconstituted released N-glycans were injected into an ACQUITY H-Class UPLC (Waters Corporation, United States) coupled to a Xevo G2S QTof mass spectrometer (Waters Corporation, United States). Samples were separated using an ACQUITY UPLC Glycan BEH amide column (130 Å, 1.7 μm, 2.1 mm × 150 mm, Waters Corporation, United States) at 60°C and 400 μL/min, with a 40 min gradient from 25 to 49% of 50 mM Ammonium Formate (mobile phase A). 100% ACN was used as mobile phase B. RFMS-labelled glycans were excited at 265 nm and measured at 425 nm with an ACQUITY UPLC FLR detector (Waters Corporation, United States). The MS1 profile scans of m/z 400–2,000 were acquired using the Xevo G2S-QTof in positive mode with an acquisition rate of 1 Hz. The electrospray ionisation capillary voltage was set at 2.75 kV, cone voltage at 15 V, desolvation gas flow at 800 L/h, ion source temperature and desolvation temperature were kept at 120°C and 300°C, respectively. Glu1-fibrinopeptide B (Waters Corporation, United States) was used as the LockSpray compound for real-time mass correction. RapiFluor-MS Dextran Calibration ladder (Waters Corporation, United States) was also injected into LC-MS to calibrate the retention time of sample peaks. The retention times were normalised using the dextran calibration curve to Glucose Units (GU).

(S)-Warfarin 7-hydroxylation by CYP2C9 was measured as previously reported, with several modifications [23 (link)]. The reaction mixture, in a total volume of 150 μL, consisted of the following components: the microsomal fraction (25 μg), (S)-warfarin (0.2, 0.5, 1, 2, 5, 10, 20, or 40 μM), and 100 mM potassium phosphate buffer (pH 7.4). Following pre-incubation at 37 °C for 3 min, reactions were initiated by the addition of 10 mM NADPH, with incubation at 37 °C for 60 min. Reactions were terminated by adding 150 μL of acetonitrile containing 25 nM 7-ethoxycoumarin as an internal standard. After protein removal by centrifugation at 15,400× g for 10 min, 10 μL of the supernatant was injected into an ultra-high performance liquid chromatography (UPLC)-fluorescence system consisting of an ACQUITY UPLC H-Class PLUS (Waters, Milford, MA, USA), ACQUITY UPLC FLR Detector (Waters), and an ACQUITY UPLC HSS C18 column (2.1 × 50 mm, 1.8-μm particle size; Waters) maintained at 40 °C. The mobile phase was a mixture of acetonitrile and water (40:60, v/v) containing 0.1% formic acid at a flow rate of 0.5 mL/min. (S)-7-Hydroxywarfarin content was measured at an excitation wavelength of 320 nm and an emission wavelength of 415 nm. Standard curves were constructed in the 12.5–6400 nM range using metabolite standards, with a quantification limit of 10 nM for (S)-7-hydroxywarfarin.