Sil 20a hplc

For the analysis of the seven phytochemical compositions, SIL‐20A HPLC (Shimadzu) instrument was equipped with an SPDM20A detector (Shimadzu). The detection wavelengths were 209 nm (limonin), 224 nm (synephrine), 283 nm (hesperidin), and 330 nm (tangeretin, 5‐HPMF, nobiletin, and HMF). The standard solution of the above seven compounds was then diluted with methanol to appropriate concentrations to construct the calibration curves. At a temperature of 30°C with an injection volume of 10 μl and eluent flow rate of 1.0 ml·min⁻¹, separations were conducted using a Diamonsil C₁₈ column (250 mm × 4.6 mm, 5 μm). The mobile phase contained phosphoric acid water solution, pH = 3.70 (A) and methanol/acetonitrile (B, v/v = 1/1), with the following gradient variations: 0–5 min, 95% A; 5–10 min, 95%–45% A; 10–15 min, 45%–40% A; 15–20 min, 40%–35% A; 20–25 min, 35%–25% A; 25–30 min, 25%–15% A; 35–40 min, 15%–5% A. Linearity of the response, stability, precision, repeatability, and recovery verified by utilizing the HPLC–PDA method.

The HPLC condition of the MECRP was performed in a previous method described by Luo et al. [17 (link)]. SIL-20A HPLC (Shimadzu, Japan) equipped with an SPD-20A UV detector and a Diamonsil C18 column (250 × 4.6 mm, i.d. 5 μm) was used for HPLC analysis. The mobile phase was solution A (water) and solution B (acetonitrile) with the following gradient program: 0–15 min, 15–40% B; 15–35 min, 40–50% B; 35–40 min, 50–75% B; and 40–50 min, 75–85% B. Other HPLC conditions are shown as follows: flow rate: 1.0 mL/min; column temperature: 25°C; injection volume: 20 μL; and detection wavelength: 283 nm and 330 nm. The chromatograms (330 nm) of C. reticulata “Chachi” samples produced in Xinhui (S1–S12) were firstly imported into the software “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2012)” to establish the GCP fingerprint. With the median method and choosing three chromatographic peaks of hesperidin, nobiletin, and tangeretin for multipoint calibration, a GCP common pattern was generated. Next, the final fingerprint was established according to the representative chromatograms of remaining cultivars (S13–S88) together with the GCP common pattern. The similarity values of different cultivars were finally calculated based on the GCP common pattern.