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One hundred microliters of plasma sample w … One hundred microliters of plasma sample were extracted with 300 uL of methanol or 1 mL of 75% MeOH (aq). The mixture was mixed and centrifuged. The supernatant was collected for analysis.
LC-MS was performed using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) coupled to an LTQ Orbitrap XL-MS system (Thermo Fisher Scientific, Inc., San Jose, CA), equipped with an electrospray source operating in either positive- or negative-ion modes. The spray voltage and capillary temperature were 4 kV and 300°C, respectively. The analysis consisted of 2 scan events. Scan event 1 was a full mass type (Analyzer, FTMS; Resolution, 60,000). Scan event 2 was an MS/MS type (analyzer, Ion Trap MS; act type, collision-induced dissociation; normalized collision energy, 35.0%). An aliquot of the extracted sample (5 µL) was injected into a TSK gel ODS-100V reversed-phase column (column size, 3.0 × 50 mm; particle size, 5.0 µm; Tosoh Corp., Tokyo, Japan). The column temperature was set at 40°C. Mobile phases A (0.1% formic acid) and B (acetonitrile with 0.1% formic acid) were used with a gradient of 3% to 97% B from 0 to 15 min, 97% B from 15 to 20 min, 97% to 3% B from 20 to 20.1 min, and 3% B for 4.9 min before the next injection, at a flow rate of 400 µL/min.
Data were acquired with Xcalibur software (Thermo Fisher Scientific Inc., San Jose, CA). rmo Fisher Scientific Inc., San Jose, CA).
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