*2.5. LVDI-UHPLC-MS*/*MS Analysis*

The generic layout of instrumentation setup of the LVDI-UHPLC-MS/MS was conducted on a Shimadzu UHPLC system consisting of two LC-20ADXR pumps, a DGU-20A3R degasser, and a CBM-20A controller (Kyoto, Japan) with a SCIEX 4500 QTRAP mass spectrometer mounted with an electron spray ionization (ESI) interface as well as an electronic 6-port/2-channel valve (Foster City, CA, USA). Analyst software package (version 1.6.2, SCIEX) was implemented to control and synchronize the entire system, and also for data acquisition and processing. A single analytical run was fragmented into two phases, namely, loading and elution phases, by switching the electronic valve (Figure 2). At the loading phase, the valve was maintained at A-channel. The specimen aliquot of large volume delivered from the auto-sampler was captured onto a guard column. Meanwhile, the pumps were responsible for delivering the mobile phase at a high flow rate, aiming to dilute the sample solvent, thus facilitating the candidate constituents to concentrate on the pre-guard column. Then, the valve was automatically switched to the B-channel to trigger the elution phase. The trapped components were flushed from the pre-guard column into an analytical column, and underwent multiple reaction monitoring (MRM) analysis on the optimized LC gradients. The detailed information of LVDI-UHPLC-MS/MS analysis for pharmacokinetic and cocktail studies is shown in the Supplementary Materials section.

**Figure 2.** Connectivity sketch of the six-port/two-channel switching valve controlling the large volume direct injection ultra-high performance liquid chromatography–tandem mass spectrometry (LVDI-UHPLC-MS/MS) system. At the loading phase, the valve was maintained at A-channel. The sample delivered from the auto-sampler was captured onto a pre-guard column. Meanwhile, the pumps delivered the mobile phase at a high flow rate. Then, the valve was automatically switched to the B-channel to trigger the elution phase. The trapped components were flushed from the pre-guard column into an analytical column.
