3.4. LC-qTOF-MS Conditions

The LC-qTOF-MS method consisted of a chromatographic pump system (Shimadzu CBM-20A/LC-20AD, Shimadzu Corporation, Columbia, MD, USA), an auto-sampler system (Eksigent CTC HTS PAL, LEAP Technologies, Carrboro, NC, USA), and a quadrupole time-of-flight mass spectrometer (TripleTOFTM 5600, Sciex, Foster City, CA, USA) with an ion source (Duospray™, Sciex, Foster City, CA, USA).

A Kinetex XB-C18 analytical column (2.1 mm × 50 mm, 2.6 μm; Phenomenex) was used for bioanalytical sample quantification and a Hydro-RP analytical column (2 mm × 100 mm, 2.5 μm; Phenomenex) was used for MetID. Security Guard Cartridge (4 mm × 2 mm; Phenomenex) was placed on the upstream of the analytical column. The LC mobile phase was DW containing 0.1% formic acid for the mobile phase A and ACN containing 0.1% formic acid for the mobile phase B. The LC gradient is shown in Table 9. The flow rate was 0.4 mL/min and the injection volume was 10 μL.

Mobile phase conditions for LC gradient.

For the TOF-MS scan, the scan range was m/z 100–1000. For the product ion scan, the parent ion of α-amanitin was m/z 919.36 ([M+H]⁺) and a product ion at m/z 901.35 was used as a quantitative ion (DP: 10 and CE: 31). The parent ion of verapamil (internal standard) was m/z 455.3 ([M+H]⁺), and a product ion at m/z 165.1 was used as a quantitative ion (DP: 125 and CE: 30). Gas sources 1 and 2 were set to 50 psi and the curtain gas flow was 30 L/min. The ion spray voltage (ISVF) was 5500 V, and the source temperature was 500 °C.

The high-resolution TOF full scan was used for metabolite profiling. The scan range was m/z 50–1300 (DP: 50 and CE: 10). In product ion mode, α-amanitin was set to m/z 919.36 (DP: 10 and CE: 31), and the suspected metabolite was set to m/z 548.20 (DP: 10 and CE: 10) to determine the metabolite. Gas sources 1 and 2 were set to 50 psi and the curtain gas flow was 35 mL/min. The ISVF was 5500 V and the source temperature was 500 °C. The MetID analysis was done by the Information-Dependent Analysis (IDA) method which includes both a real time multiple mass defect filtering (MDF) function and a dynamic background subtraction function.