The fabrication of the Single-probe (Figure ) has been thoroughly described in our previous studies . This assembly includes three key elements: a laser-pulled dual-bore quartz needle, a solvent providing silica capillary, and a nano-electrospray ionization (nano-ESI) emitter that efficiently ionizes extracted metabolite. The fabrication of the Single-probe begins with the precise shaping of a dual-bore quartz needle (outer diameter (OD) 500 μm; inner diameter (ID) 127 μm, Friedrich & Dimmock, Inc., Millville, NJ, USA)) using a laser pipette puller (Model P-2000, Sutter Instrument CO., Novato, CA). This pulling process creates a fine, tapered structure in the quartz needle. Following this step, a fused silica capillary (outer diameter 105 μm, inner diameter 40 μm, Polymicro Technologies, Phoenix, AZ) is embedded into one bore of the pulled quartz needle to serve as the solvent delivery channel. Additionally, a nano-ESI emitter is positioned within the other bore. The nano-ESI emitter is formed by heating a similar fused silica capillary with a butane micro torch to achieve a sharp, functional tip for effective ionization. To secure both the capillary and the nano-ESI emitter within the dual-bore needle, UV-curing epoxy (Prime Dental, item No. 006.030, Chicago, IL) is applied to the glued sections and cured under a UV LED lamp.