2.4 Sample Preparation and HPLC Analysis

According to the CP (2020 Edition) (Chinese Pharmacopoeia Commission, 2020), all the processed or raw samples were oven-dried at 50°C for 24 h. Then, the dried samples were finely powered and passed through a 50-mesh sieve. Each sample powder (2.0 g) was weighed and then extracted by ultrasonication with 3 ml of ammonia TS and 50 ml of isopropanol–ethyl acetate (1/1, v/v) for 30 min (300 w, 40 kHz). Following extraction, isopropanol–ethyl acetate (1/1, v/v) was added to compensate for the lost weight during extraction and then filtered. 25 ml of filtrate was accurately measured and evaporated under reduced pressure at 40°C to dryness. Three milliliters of 0.05 M hydrochloric acid–methanol solution was precisely added to dissolve the residue. The supernatants were used as the sample solution after being centrifuged at 15,000 rpm for 30 min. The concentration of aconitine (AC, 40), mesaconitine (MAC, 22), hypaconitine (HAC, 36), benzoylaconine (BAC, 34), benzoylmesaconine (BMAC, 32), and benzoylhypaconine (BHAC, 29) in every sample was calculated on the basis of established calibration curves.

The filtered sample was analyzed using Agilent Technology 1260 Infinity (Agilent Technologies, Santa Clara, CA, United States) equipped with a binary pump, mobile phase degasser, temperature-controlled autosampler, column thermostat, and diode array detector (1260-DAD). HPLC separation was performed on an Agilent Eclipse XDB C₁₈ column (4.6 mm × 250 mm, 5 μm) and maintained at 30°C. The mobile phase consisted of 0.04 M of ammonium acetate solution (solvent A, adjusting pH to 8.5 with an ammonia solution) and acetonitrile (solvent B) using the following gradient: 20%–28% B for 0–10 min, 28%–30% B for 10–20 min, 30%–35% B for 20–50 min, and 35%–38% B for 50–60 min. The flow rate was kept at 1 ml/min, and the injection volume was 10 μl. The UV detection wavelength was set at 235 nm.

HPLC-DAD was validated with regard to linearity, stability, recovery, repeatability, and precision. The storage solutions of six compounds were used in the regression equations between the peak areas and concentrations of six compounds. The limit of detection (LOD) and quantification (LOQ) were determined at signal-to-noise ratios of 3 and 10, respectively.