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Centrifuge QCs and plasma samples at 2,750 × g for 5 min at 4 °C. Add 10 μL of the ISTD mix to each well on the Kit plate except A1. Pipette 10 μL of PBS / Cal / QC / sample according to the plate layout generated by MetIDQ. Dry for 30 min under nitrogen. After derivatization, prepare the extraction solvent and add 300 μL to each well. Shake for 30 minutes at 450 rpm, then centrifuge for 2 minutes at 500 × g or use a pressure manifold. Verify that the fill levels are equal in all wells; if they are not, repeat centrifugation at a higher g-force or apply higher pressure.

Mobile Phase Preparation: Mobile Phase A: 1000 mL water + 2 mL formic acid (FA), Mobile Phase B: 475 mL acetonitrile + 25 mL water + 1 mL formic acid (FA) Install the column. Wash the column with 95% Mobile Phase B, then condition to 100% Mobile Phase A. Install the acquisition method from the provided USB stick. Enter autosampler parameters and check overall system cleanliness. System Check & Calibration: Load the prepared LC plate from the starter kit. Inject blank to confirm system cleanliness. Inject Cal5 standard three times using method: "KIT2-LC_5xxxx_adjustRT.dam". Dwell time: 15 msec. Injection volume: 5 μL for UHPLC. Column oven temperature: 50 °C

The LC–MS analysis was performed on a QTRAP 5500 triple quadrupole mass spectrometer (SCIEX, Canada) equipped with an electrospray ionization (ESI) source and controlled using Analyst® software. Data were acquired in positive ion mode using a full MS scan (Q1, EMS) without fragmentation, with the collision gas (CAD) turned off. The LC column oven was maintained at 50 °C, and the mobile phase composition followed the manufacturer’s kit recommendations. An injection volume of 5 µL was used for UHPLC analysis. The source and ionization parameters were set according to the recommended starting values, and mass spectra were recorded over an m/z range of 50–1,000. Additional information is decribed in the manual provided by Biocrates.