Turboionspray ion source

The instruments used were a SCIEX X-500R quadrupole time-of-flight mass spectrometer (AB SCIEX, USA), a TurboIonSpray ion source (AB SCIEX, USA), and a Waters ACQUITY I-Class Plus UPLC ultraperformance liquid chromatography system (Waters, USA). The chromatographic conditions were as follows: ACQUITY UPLC BEH C18 (150 × 2.1 mm², 1.7 μm) column, mobile phase, and 0.1% formic acid acetonitrile (A)–0.1% formic acid water (B). The gradient-elution program was as follows: 0~4 min, 99%–87% B; 4–14 min, 87%–80% B; 14–20 min, 80%–50% B; 20–24 min, 50–10% B–24 min, 50%–10% B; 24–24.5 min, 10%–1% B; 24.5–28 min, and 1% B. The flow rate was 0.3 mL/min, the injection-tray temperature was 8°C, the column temperature was 40°C, and the injection volume was 2 μL. Time-of-flight mass spectrometry was performed with a TurboIonSpray ion source and the ESI positive- and negative-ion scanning mode.
SCIEX OS software was used to acquire and process the data. The screening of the target compounds can be completed without standards by searching the secondary database of the TCM MS/MS Library (containing more than 1000 secondary data of TCM compounds) configured by SCIEX OS based on the exact mass number of the compounds at the primary level, isotope-distribution ratio, and MS/MS.

The instruments and reagents used for mass spectrometry were as follows: SCIEX X-500R Quadrupole Time of Flight Mass Spectrometer (AB SCIEX, USA); TurboIonSpray ion source (AB SCIEX); Waters ACQUITY I-Class Plus UPLC Ultra-High-Performance Liquid Chromatography System (Waters); Thermo ST40R Low-temperature high-speed centrifuge (Thermo Fisher); IKA Miniature scroll mixing instrument (IKA Germany); AUW220D electronic balance (Shimadzu Company, Japan). Methanol, acetonitrile and formic acid (Merck, Germany), Milli-Q ultra-pure water (Millipore, USA); and other reagents were obtained in analytically pure form.

After the biosynthesis of metabolites, the metabolite solution was analyzed by LC-MS/MS to identify oxidation metabolites. LC analysis was accomplished using a Shimadzu Nexera X2 UHPLC system (Columbia, MD, USA) coupled with a 4000 QTRAP^® triple quadrupole mass spectrometer system equipped with a Turbo Ion Spray ion source (AB Sciex. Redwood City, CA, USA) using the instrument conditions optimized in Section 3.3 The mass spectrometer strategy used Q1MI, product ion (MS2), and Neutral Loss (NL) to identify metabolites.
Based on the data obtained from the product ion spectra of OJT007, a neutral loss scan was performed using the OJT007 microsome sample to screen metabolites. The first neutral loss (NL1) scan conducted corresponded to the major product ion of OJT007. The second neutral loss scan conducted was based on NL1+16 in order to screen for possible hydroxylated metabolites. As a final confirmation step, a product ion scan (MS2) was performed. The product ion scan of m/z 341, indicating mono-hydroxylated metabolites, was conducted.