4.4. UHPLC-Q-Exactive Orbitrap MS Fingerprint Analysis

A sample solution of 20 μg·mL⁻¹ (calculated as crude drug) was prepared with 60% acetonitrile–water with an appropriate amount of CPPP powder. After ultrasonic treatment and centrifugation, the supernatant was filtered using a 0.22 μm microporous filter membrane, and the filtrate was used as the test solution. A mixed reference solution of about 20 μg·mL⁻¹ for each reference was prepared with 60% acetonitrile–water. The above solution was tested under the following conditions.

The CPPP extracts were analyzed using a Thermo UHPLC system connected to a Thermo Q-Exective-Orbitrap-MS system (ThermoScientific, Waltham, MA, USA). Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase comprised deionized water containing 0.1% formic acid (A) and acetonitrile (B). The gradient elution procedure was as follows: 0–4 min, 2% B; 4–5 min, 2–5% B; 5–20 min, 5–16% B; 20–50 min, 16–45% B; and 50–60 min, 45–80% B. The flow rate was set to 0.3 mL/min. The column temperature was maintained at 30 °C. The injection volume was established at 2 μL. Mass spectra were acquired in a negative mode over a mass range of m/z 120–1500. The parameters for the ESI source were as follows: the source temperature was set to 400 °C, the ion spray voltage was adjusted to 3.5 kV, and the capillary temperature was maintained at 320 °C. Both the sheath gas and auxiliary gas were nitrogen, sustained at flow rates of 40 arb and 10 arb, respectively. The collision energy gradient utilized values of 20, 40, and 60 eV. The first-level resolution achieved was 70,000, while the secondary resolution reached up to 17,500. All operations mentioned above were conducted using the Xcalibur 4.3 software.

Subsequently, the chromatographic profiles of 26 CPPP samples were imported into The Similarity Evaluation System of Chromatographic Fingerprint for Traditional Chinese Medicine (2012a version), which serves as an instrumental tool for assessing both the similarities and disparities among samples. The MCG-1 and BQZ-1 specimens were designated as the reference maps, with a “time window” width calibrated to 0.5 min. Comprehensive spectrum similarity assessments and comparative fitting analyses were conducted utilizing methods such as average calculations, multi-point corrections, and full-spectrum peak matching. Through meticulous examination of the CPPP fingerprint data, common characteristic peaks were identified for subsequent chemometric analysis.