C18 column

The concentration of D-phenylalanine and L-phenylalanine were detected by HPLC on a C₁₈ column (4.6 mm×75 mm, Hitachi, Japan) at 205 nm according to the method described by Fukuhara [21] . The mobile phase contained 20% (v/v) methanol and a complex of optically active L-Pro-Cu (II) (1.5 mM L-Pro and 0.75 mM CuSO₄).

To investigate the qualitative compositions of the antifungal compound, TLC was applied. TLC analysis was used to monitor the fractions from column chromatography, and spots on silica gel plates were visualized by spraying with a solution (1.5% of aluminum chloride in ethyl alcohol, ammonia vapor, and 5% sulfuric acid in ethyl alcohol pre-heated at 105°C). Next, purity of compound was measured by an UV spectrophotometer (UV-2550 Shimadzu, Japan).
HPLC analysis was performed using a Hitachi Chromaster HPLC system consisting of an 1,110 pump, DT-230 column oven, 1,430 diode array detector, and a YMC C18 column (250 × 4.6 mm, 5 μm). HPLC analysis was performed with the EZChrom Elite software. The mobile phases were water, acetonitrile, and methanol. The mass spectra were measured in a 2690-ZQ 4000 Water-Alliance LC-MS spectrometer (Applied Biosystems/MDS Sciex Concord, ON, Canada). ¹H NMR, ¹³C NMR, and 2D NMR spectra were recorded on Varian MR-400 and VNMRS-600 NMR spectrometers with TMS as an internal standard.

The level of TQ in Thymocid^® was quantified by using the high-performance liquid chromatography (HPLC) method with an Hitachi HPLC instrument (Hitachi Instruments, Inc., San Jose, CA, USA), an Alltima C₁₈ column (250 × 4.6 mm i.d., 5 µm), and a solvent system consisting of 0.1% trifluoroacetic acid in water (A) and methanol (B). A linear gradient eluting method was used as follows: 0–20 min, 50–100% B; 20–21 min, 100–50% B; 20–28 min, 50 % B with a total run time of 28 min, a flow rate of 0.75 mL/min, and an injection volume of 10 µL. Thymocid^® was dissolved in dimethyl sulfoxide (DMSO) at various concentrations (0.1–4 mg/mL) and monitored at a range of wavelengths from 200 to 400 nm with a photodiode array detector (see Supplementary Materials Figure S1). A standard curve of TQ at various concentrations (1–200 µg/mL) monitored at the wavelength of 254 nm, which is the characteristic wavelength for TQ, was constructed for its quantification in Thymocid^® (see Supplementary Materials Figure S2).