Markerlynx application manager

The morphology and Energy Dispersive X-Ray (EDX) spectroscopies of MNPs were obtained by the scanning electron microscopy (SEM, JSM-6460LV, Japan). Fourier transform infrared spectroscopy (FT-IR, IFS 66 V/S, Germany) was performed to identify the materials with KBr. Other characteristics were measured by vibrating sample magnetometry (VSM, MPMS XL, USA) at room temperature and a D8 advance X-ray diffractometer (XRD, D8, Germany) with Cu Kα radiation (λ = 1.5406 Å). The UPLC–MS/MS analyses were performed by a Waters ACQUITY UPLC system (Waters Corp., Milford, USA) coupled to a triple quadrupole (TQ) mass spectrometer equipped with the electrospray ionization (ESI) source in multiple reactions monitoring (MRM) mode. A Q-TOF SYNAPT G2-S High Definition Mass Spectrometer coupled to ESI (Waters Crop., Manchester, UK) was adopted for detection of ginsenosides metabolites before and after treatment with Fe₃O₄@SiO₂@PDA NPs. The C18 column (50 mm × 2.1 mm, 1.9 μm; Thermo scientific) was employed in the entire experiments. MassLynxV4.1 and MarkerLynx Application Manager (Waters Corporation, Milford, USA) and UNIFI 1.8 (Waters, USA) were carried out to process the collected data. And all detailed steps of mass spectrometry analyses were described in Supplementary data (Methods section), including UPLC–MS/MS analysis, Q-TOF analysis and mass spectral data analysis with UNIFI^TW.

All the mass data were imported and recorded to MarkerLynx Application Manager and MassLynx V4.1 software (Waters Corp., Milford, USA) for peak detection and alignment. The retention time and m/z data for each peak also were determined. The resultant data matrices were introduced to EZinfo software 2.8 for the principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and orthogonal projection to latent structures (OPLS) analysis. PCA is used for variable reduction and separation into classes. To maximize class discrimination and biomarkers, the data should be further analyzed by OPLS-DA. S-plots were then calculated to visualize the relationship between covariance and correlation within the OPLS-DA results.

The UPLC-MS data were processed using the MarkerLynx Application Manager (Waters Corp.). After UPLC/ESI-QTOF/MS measurement, the raw data were imported into the MassLynx software for peak detection and alignment. The intensity of each ion was normalized with respect to the total ion count to generate a data matrix that consisted of the retention time, m/z value, and the normalized peak area. The multivariate data matrix was analyzed by EZinfo software 2.0 (Waters Corp., Milford, USA). All the variables were mean-centered and Pareto-scaled prior to PCA and PLS-DA. If a separation between the control and the treatment groups was observed in the PCA scores plot, PLS-DA was performed to highlight the differences between the groups. After the analysis of all samples was finished, low-molecular weight metabolites were presented as chromatographic peaks in the base peak intensity (BPI) chromatograms.