Thermo c18 column

Dried plant powder (0.5 g) was sonicated for 30 min in 10 mL of methanol and re-extracted for another two times from the residue. After centrifugation at 5,000 g for 10 min, the combined supernatants were rotary evaporated at 50 °C. Methanol was added to a constant volume of 1 mL. The mixture was filtered with 0.22 µm Millipore membrane for qualitation and quantification.
Identification of salidroside and tyrosol was based on ¹H-NMR and UPLC-Q/TOF/-MS according to our previous methods⁴⁴ . In brief, dried plant extracts were dissolved in methanol-d₄ (CD3OD) to elucidate the structure through comparison of parameters, such as the chemical shift of standard salidroside and tyrosol (bought from National Institute for Food and Drug Control, Beijing), using an AVANCE 300 MHz spectrometer (Bruker, Switzerland). Molecular masses of salidroside and tyrosol were confirmed through comparison of parameters with the standard based on Waters Xevo G2Q-TOF (Micromass MS Tech., UK). The detection of salidroside and tyrosol content was conducted with HPLC (Agilent 1200, Agilent Tech., USA) equipped with a diode array detector and a Thermo-C18 column (250 mm × 4.6 mm, 5 µm) at 30 °C. The mobile phase was methanol: H₂O₂ (32:68 v/v) with a flow rate of 0.8 µL min⁻¹. The monitoring wavelength was 277 nm.

SA quantification was performed using RP-HPLC (Agilent 1200, Agilent technology, USA) equipped with Thermo-C18 column (250 mm × 4.6 mm, 5 µm) and fluorescence detector under 294 nm of excitation wavelength and 426 nm of emission wavelength. The injection volume was 10 µL, and flow rate was 0.5 mL min⁻¹ at 25 °C. The mobile phase was sodium acetate buffer (0.2 M):methanol (9:1 v/v).
The SA extraction method was modified according to that of Yuan et al.^{16 (link)}. Leaf samples (1 g) were ground in liquid nitrogen and extracted ultrasonically in 1 mL of methanol. After centrifugation at 10,000 g for 15 min, the residue was re-extracted two times as above. The supernatants were combined and freeze dried. The concentrate was dissolved in 0.5 mL of trichloroacetic acid. After oscillation for 2 min, the mixture was extracted twice with 0.8 mL of acetic acid ester:cyclohexane (1:1 v/v). Organic phases were combined and freeze dried. The concentrate was dissolved with 0.6 mL of HPLC mobile phase and filtered with 0.22 µm Millipore membrane for detection.

An Agilent 1260 series HPLC instrument (Santa Clara, CA, USA) equipped with an Agilent DAD detector (All Tech, Lexington, KY, USA) was used to obtain HPLC fingerprints. A Thermo C₁₈ column (250 mm × 4.6 mm, i.d., Agilent) was used for analyses. For separation, a column temperature of 30°C, a flow rate of 1.0 mL/min, and an injection volume of 10 μL were used.
The optimized elution conditions were as follows: (C) acetonitrile and (D) 0.3% formic acid in water were used as the mobile phase. A linear gradient of 0%–7% (C) (0–15 min), 7%–20% (C) (15–20 min), and 20%–43% (C) (20–40 min) with wavelength conditions of 273 nm (0–20 min), 360 nm (21–30 min), and 270 nm (31–40 min) was applied.