Acquity uplc 1 class system

All measurements with the poly(dT) ladder were acquired on an ACQUITY UPLC I-Class system (Waters Corporation, Milford, MA, USA) coupled with a VION IMS qTOF mass spectrometer (Waters Corporation, Milford, MA, USA), measurements with the 24 nt thiolated oligonucleotide were acquired on a BioAccord LC-MS System with ACQUITY Premier (Waters Corporation, Milford, MA, USA). Source parameters were set as: 100 V source offset, 120 °C source temperature, 400 °C desolvation temperature, 800 L/h (L/h) desolvation gas flow and 50 L/h cone gas flow. The scan range of full MS was set to m/z 500.00–2000.00 at a scan rate of 1 Hz (1 s⁻¹). Data acquisition and processing were performed with UNIFI^TM (1.9.13.9 and 3.0.0.15). Calibration of MS data was achieved throughconstant infusion of leucine enkephalin calibration solution by Waters (SKU: 186006013) at a flow rate of 10 µL/min.

The chromatographic separation was carried out using an Acquity UPLC I-Class system (Waters, Milford, CT, USA) coupled to a triple quadrupole mass analyzer (QqQ; Xevo TQ-S) with a ZSpray ESI ion source (Waters, Milford, CT, USA). The analytical column ACQUITY UPLC HSS T3 (100 × 2.1 mm, 1.7 μm) was connected to an ULTRA C18 guard column (20 × 2.1 mm). The mobile phase consisted of water with 0.1% (v/v) acetic acid (A) and methanol (B). The initial mobile phase composition was 100% A, held for 1 min, and then decreased to 15% A in 2.5 min, increased to 100% A in 0.5 min and held for another 1 min, for a total run time of 5 min. The flow rate was kept at 0.2 mL/min. The temperature in the autosampler was set at 10 °C. The injection volume was 2 µL, while the column temperature was set at 40 °C. Electrospray ionization under positive ionization mode (ESI+) was used for the analysis. The MS/MS detector parameters were set as follows: 150 °C for source temperature, 500 °C for desolvation temperature, 1000 L/h for desolvation gas flow, 150 L/h for cone gas flow, 0.15 mL/min for collision gas flow, 6 bar for nebulizer gas flow, and +2.5 kV for capillary voltage.

For untargeted metabolomics, reverse phase (RP) separation was performed by an Acquity UPLC I-Class system from Waters Corporation (Milford, MA, USA) while detection used MaXis Impact qTOF-MS instrumentation from Bruker Daltonics (Bremen, Germany). The qTOF-MS instrument was operated in positive electrospray ionization mode (ESI+) using a capillary voltage of 4.0 kV and in negative electrospray ionization mode (ESI−) with a capillary voltage of 2.5 kV. The nebulizing gas pressure was 4 bar, and the drying gas flow and temperature were 11 L/min and 220 °C. HyStar, (Bruker Daltonics, Bremen, Germany) was used as a common platform to control both systems.

Serum samples and haematoma fluids were extracted with ethyl acetate, and cell samples were processed with acetonitrile for protein precipitation before LC-MS/MS. Ultra-high-performance liquid chromatography (UPLC) coupled with triple quadrupole MS was performed to detect ATO, its two active metabolites ortho-hydroxy-atorvastatin (o-ATO) and para-hydroxy-atorvastatin (p-ATO), and DEX in haematoma fluids, serum samples, and cultured cells. A Waters ACQUITY UPLC I-Class system (Waters, USA) equipped with an Acquity UPLC BEH C18 column (1.7 μm, 100×2.1 mm, Waters, USA) was coupled online to a Waters Xevo TQD IVD triple quadrupole mass spectrometer (Waters, Ireland) with electrospray ionization and selective reaction monitoring in positive ion mode. Solvent A (acetonitrile with 0.01% formic acid) and solvent B (0.01% aqueous formic acid) were used with a flow rate of 0.4 mL/min and the following solvent (time [min], vol% solvent B): 0.0, 80%; 1.0, 60%; 4, 10%; 4.5, 90%; 4.51, 80%; and 5, 80%. The injection volume was 10 μL.
Drugs in cells, including those binding to the cell membrane, were extracted in a lysis buffer. Some cells were treated with 0.25% trypsin-EDTA (25200-056, Gibco, New Zealand) to remove the surface-bound drugs before being lysed.

The separation process was performed by the Waters ACQUITY UPLC I-Class system (Waters Corporation, Milford, MA, USA) with the controlled software of Masslynx V4.1. The mobile phase consisted of solvent A (HCOOH : H₂O = 0.1 : 100, v/v) and solvent B (CH₃CN); the gradient eluting procedure was as follows: 0-1 min, 10%B; 1–14 min, 10%–100%B; and 14–17 min, 100% B at a flow rate of 0.3 mL/min. The volume of the sample solution injected into the chromatographic system was 1 μL, and all the separations were performed at 40°C.