The TSQ Quantum Liquid Chromatography–Mass Spectrometer (UHPLC–MS/MS) included a triple quadrupole mass analyzer, ESI ion source, and Xcalibur workstation. The liquid phase part was a Thermo Accela UHPLC, including an Accela PDA detector, Accela autosampler and Accela 1250 infusion pump, which were purchased from Thermo Fisher Scientific, Inc. XS-105DU 1/100000 and the AL204 1/10000 electronic analytical as well as the KQ-5200E type ultrasonic cleaner were both obtained from Kunshan Ultrasonic Instruments Co.

Quercetin was purchased from the China Institute of Food and Drug Control; Ferulic acid and L-epicatechin were purchased from the China Institute for Testing and Certification of Pharmaceutical and Biological Products. Chrysin, naringenin, luteolin, and cynaroside were acquired from Guizhou Dida Biotechnology Co., Ltd. Isorhamnetin was acquired from Chengdu Pfeiffer Biotechnology Co., Ltd. Quercetin was acquired from the China Institute of Food. Naringin was purchased from Chengdu Botanical Standard Pure Biotechnology Co., Ltd., Research Central Reference Materials Research Center. The purities of the 9 reference substances were all greater than 98%. The analytical purities of methanol (MeOH) and ethanol were purchased from Tianjin Comitry Co., Ltd. Acetonitrile (HPLC grade) was purchased from American TEDIA Company. Formic acid (LC/MS grade) was acquired by ROE SCIENTIFIC Corporation, United States. Food-grade distilled water was purchased from Watson’s Food and Beverage Guangzhou Co. The structural formulas of the 9 compounds are shown in Figure 1.

The majority of medicinal plants are harvested and grown in Yun Guan Shan, a state-owned forest farm in Guizhou Province, China, where they mimic wild D. officinale from Pinus massoniana Lamb. tree species.

All dry ingredients were ground up and put through a sieve with a mesh size of 60. As mentioned below, 0.1 g of samples were weighed exactly (within 0.00001), and they were then extracted once with 80% methanol (20 ml) by reflux for 1.5 h. The sample was meticulously weighed before extraction, and the weight loss was compensated once the sample solution was weighed and cooled to room temperature. All of the solutions were filtered through a 0.22 μm microporous membrane before being put on the device.

Each standard was properly weighed. The reference standards’ standard stock solutions were made by combining and evaporating in 80% methanol. The working standard solution was then made by gradient-diluting the standard stock solution with the same solvent, and both of these solutions were stored at 4°C for further analysis.

Different extraction methods were investigated by a single-factor experiment: ultrasound (100 W, 90 kHz, 60 min), reflux (60 min, 67°C), and cold soaking (60 min); different solvents: ethanol, methanol, water, different concentrations: methanol (40, 60, 80, and 100%). Then the effects of different solid–liquid ratios of 1:200, 1:300, 1:400, and 1:500 (m/v), and reflux extraction times of 0.5, 1, 1.5, and 2 h on the extraction were studied. The supernatant was examined by “2.3” chromatography after being filtered using a 0.22 μm microporous organic membrane. Nine peaks’ response values, peak areas, and peak forms were compared.

The Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) method is a multi-index decision analysis method, that calculates the multi-index as a comprehensive index, and transforms the multidimensional problem into a one-dimensional problem, which greatly reduces the interference of different types of indicators on decision-making in the analysis process, and significantly improves the scientificity and accuracy of multiobjective decision analysis (28). It has been widely used in the quality evaluation of various Chinese medicinal materials.

In this paper, 597 metabolites were detected based on the UHPLC–MS/MS detection platform and a self-built database, and 185 flavonoids were screened through primary classification. This part of the experiment was performed by Wuhan Maitville Biotechnology Co., Ltd. Therefore, the UHPLC–ESI MS/MS method was established to compare the total ion current diagram and fragment ion diagram of the D officinale extract and standard sample, and nine phenolic compounds were identified, ferulic acid (1), chrysin (2), naringenin (3), luteolin (4), L-epicatechin (5), quercetin (6), isorhamnetin (7), cynaroside (8), and naringin (9), as shown in Figure 3.

The following extraction methods (sonication, reflux, cold soak), extraction solvents (ethanol, methanol, water), solvent concentrations (40, 60, 80, 100% methanol), extraction stock ratios [1:200, 1:300, 1:400, 1:500 (m/v)], and extraction times (0.5, 1, 1.5, and 2 h) were optimized, as shown in Figure 4. Reflux extraction was chosen for the herb’s extraction because it produced much more naringenin and cynaroside than the other two extraction techniques. More phenols were extracted from methanol, while more polysaccharides were extracted from aqueous extracts that were not well filtered. The longer the extraction time was, the greater the extracted phenol content increased, but when the time was 1.5 h after the slow growth. The extraction of phenolic compounds was not significantly impacted by the material-to-liquid ratio. The feed-to-liquid ratio of 1:200 and 1.5 h of refluxing 80% methanol resulted in the best extraction conditions.

The mass concentration was used as the horizontal coordinate (X), and the peak area was used as the vertical coordinate in the linear regression (Y). The regression equation and correlation coefficient (R²) were calculated, and the maximum LOD and LOQ of the 9 compounds were 16.89 and 15 ng/ml, respectively. The result shown in Table 2, show good linearity.

The within-day and between-day variations were used to evaluate the precision of this UHPLC–MS/MS method for measuring D. officinale. The relative standard deviation RSD for the intraday and interday precision ranged from 1.60 to 7.49% and from 5.89 to 9.99%, respectively. Then, the relative standard deviation for repeatability and stability varies from 1.49 to 12.50%. Table 3 presents the outcomes. According to Table 4, the average recoveries of the spiked trials ranged from 79.87 to 99.15%, with RSDs between 7.02 and 16.98%. The experimental outcomes further showed that the analytical approach is reliable and capable of fulfilling the assay’s requirements.

The validated method is used to analyze samples from different sources. Table 5 displays the quantitative results obtained from the calibration curve. Each sample was analyzed in triplicate. A total of 9 flavonoids were detected in the D officinale samples. The ferulic acid, naringenin, and epicatechin contents were higher in each provenance than the other components, while the luteolin content was lower in each provenance. Of these, the Guangxi provenance had the highest levels of ferulic acid and L-epicatechin, at 88.27 and 109.68 μg/g, respectively. Chrysin, naringenin, quercetin, and isorhamnetin were all much more abundant in ZJ origin than they were in other compounds, while luteolin and naringenin were the least abundant of all ZJ provenances. The impacts of geographical origins and storage conditions may account for the significant variations in flavonoid composition and content between batches of D. officinale samples.

The quality of imitation wild D. officinale was properly evaluated by TOPSIS, and elevated provenances were selected based on their phenol content. D + is the distance between each treatment and the optimal index, and D − is the distance from the worst vector. The smaller the value is, the closer it is to the optimal index or the worst index. CI = D–/(D + + D–), the greater the value, the better the overall efficiency. According to the CI value of each index, the advantages and disadvantages of the 5 provenances were as follows: Zhejiang > Fujian > Guangxi > Anlong > Fanjingshan. Among them, the Anlong, Fanjingshan, and Guangxi CI values are close to the specific data shown in Table 6. The maximum CI of the ZJ provenance is 0.7296, which can be further studied and developed as a superior quality provenance (Table 6).