Masterview

The samples were analyzed by a LC-20AD^XR UFLC-4600 Q/TOF system (Shimadzu, Japan & AB SCIEX, USA), equipped with a Shim-pack XR-ODS column (100 mm×2.0 mm, 2.2 μm; Shimadzu, Japan). Detection parameters were set according to our previous report with minor modifications [32 (link)]. The injection volume was 1 µL, and that flow rate was 0.25 mL/min. The mobile phase included water (A) and methanol (B) containing formic acid (0.1%). Gradient elution program was set as follow: 10% B at 0-0.5 min, 10–30% B at 0.5-2 min, 30–48% B at 2–15 min, 48–100% B at 15–20 min, 100% B at 20–23 min. Mass scan range were 100–1000 m/z. Data were collected by Analyst (ver. 1.7, AB SCIEX, USA), and processed with PeakView (ver. 2.2, AB SCIEX, Canada) and MasterView (ver. 1.1, AB SCIEX, Canada). Compounds were identified by Natural Products HR-MSMS Spectral Library (ver. 1.0.1, AB SCIEX, USA). Mass tolerance of the accurate molecular weight was set as ± 5 ppm. Furthermore, MS/MS fragment patterns were analyzed to verify the results.

The data analyses were performed using PeakView (v2.2) and MasterView (v1.2) programs (AB Sciex). Through comparing the mass spectrogram and retention time of an array of extracted ion chromatograms of experimental samples with the control ones, the common chromatographic peaks were eliminated and potential metabolites were detected. Based on the transformation of fragmentation patterns, mass shift and retention time, structures of the potential metabolites of Rg5 were determined.

The LC-MS data were handled using the Analyst TF 1.7 software (AB Sciex, Framingham, MA, USA) and embedded with PeakView Extra Utilities (2.2.0.11391, AB Sciex, Framingham, USA), and MasterView (1.1.1944.0, AB Sciex, Framingham, USA). In the compound identification analysis, MS and MS/MS data were both used in the “candidate search” algorithm, used to search against the TCM Library 1.0 C/D/V and TCM MS/MS library [34 ,35 ]. The following parameters were set to ensure the accuracy of each identified compound: mass error < 5 ppm, isotope ratio difference < 10%, and library score > 30.
The UPLC fingerprint analysis was performed using the “Similarity Evaluation System of Traditional Chinese Medicine Chromatographic Fingerprint” software (2012.130723 edition, Chinese Pharmacopoeia Commission, Beijing, China), peaks were manually marked, automatic corrected, and aligned, followed by reference/sample fingerprint generation. The heatmap and clustering analysis were performed using the HemI 1.0 software (Huazhong University of Science and Technology, Wuhan, Hubei Province, China). The PCA analysis was performed by using SIMCA-P 11.0 software (Umetrics, Umeå, Västerbotten, Sweden). For quantitative analysis, the peak areas were integrated using the LC solutions software (SHIMADZU, Tokyo, Japan), and linearity was calculated using the EXCEL software.