*4.4. Preparation of Four Di*ff*erent OTA-Fluorescein Tracers*

OTA has an active group that can be coupled with commonly used fluorescein and directly reacted with FITC to become a tracer OTA-EDF (or OTA-BDF, OTA-AMF, and OTA-HDF) as previously reported [39,40]. In brief, 250 μL (80 μmol/mL) NHS and 250 μL (80 μmol/mL) EDC were dissolved in 0.2 mL DMF, then 2 mg (5 μmol) OTA was added. The solution was stirred for 2 h and subsequently reposed overnight at room temperature. For the synthesis of OTA-EDF, 2 mg (4 μmol) EDF was added into 200 μL (2 μmol) activated OTA solution, followed by 10 h incubation at room temperature. For OTA-BDF and OTA-HDF, 1 mg (2 μmol) BDF (or HDF) was added into 100 μL (1 μmol) activated OTA solution, followed by 10 h incubation at room temperature and an aliquot. For OTA-AMF, 0.5 mg (1 μmol) AMF was added into 100 μL (1 μmol) activated OTA solution after 10 h reaction at room temperature.

An aliquot of the mixture was separated and purified by TLC. The silica gel plates were activated at 110 ◦C for 30 min before use. Toluene ± acetic acid (99:1 *v*/*v*) was chosen as the spotting solvent. Toluene ± ethyl acetate ± 88% formic acid (6:3:1 *v*/*v*/*v*) was regarded as the suitable solvent for eluting samples on TLC plates. The TLC plates were examined visually under UV light at 365 nm [36].

The OTA-AMF and other tracers were dissolved in 2 mL and 1 mL methanol, respectively, for separation and purification. A primary band at Rf <sup>1</sup> <sup>4</sup> 0.9 and Rf <sup>1</sup> <sup>4</sup> 0.7 were collected and eluted with methanol. The methanol tracer solutions were stored at −20 ◦C.

Dye-labeled tracers were identified by mass spectrometry (SHIMADZU, LCMS-8060, liquid chromatograph-mass spectrometer). The instrument was corrected with raffinose before the experiment. The OTA-AMF, OTA-BDF, OTA-EDF, and OTA-HDF were diluted to 1 μg/mL with 1 mL methanol, and then injected into the mass spectrometer through autosampler. The parameters of the mass

spectrum were adjusted to real-time at the beginning of data collection. The parameters of the compound ion mass spectrum were obtained and analyzed for their chemical structures.
