To analyze pesticide residues, GC coupled with quadrupole-Orbitrap MS (GC-Orbitrap-MS) has become a powerful tool because of its unique characteristics of accurate mass full-spectrum acquisition, high resolution, fast acquisition rates, and overcoming matrix interference. This paper presents an efficiency evaluation of GC-Orbitrap-MS for identification and quantitation in the 352 pesticide residues analysis of chrysanthemum flowers in full-scan mode. A streamlined pretreatment approach using one-step extraction and dilution was used, which provided high-throughput processing and excellent recovery. The samples were extracted using acetonitrile. The extracted solution was purified by a Sin-QuEChERS Nano column to suppress the matrix in chrysanthemum flowers and determined by GC-Orbitrap-MS. The calibration curves for the 352 pesticides obtained by GC-Orbitrap-MS were linear in the range of 0.5–200 μg·kg−1, with the correlation coefficients higher than 0.99. The limits of detection (LODs) and the limits of quantification (LOQs) for the 352 pesticide residues were 0.3–3.0 μg·kg−1 and 1.0–10.0 μg·kg−1, respectively. The average recoveries in chrysanthemum flower at three levels were 95.2%, 88.6%, and 95.7%, respectively, with relative standard deviations (RSDs) of 7.1%, 7.5%, and 7.2%, respectively. Lastly, the validated method and retrospective analysis was applied to a total of 200 chrysanthemum flower samples bought in local pharmacies. The proposed method can simultaneously detect multipesticide residues with a good performance in qualitative and quantitative detection.

1. Introduction

Chrysanthemum flower (Dendranthema grandiflora) is one of the most common Chinese herbal medicines, and it has been consumed as food for health care and disease prevention since ancient times. It is mainly used for the treatment of respiratory and cardiovascular diseases and shows significant activities, such as antimicrobial, anti-inflammatory, and anti-cancer and neuroprotective and cardiovascular system [1]. Because of its efficacy in alleviating chronic diseases, some consumers often drink chrysanthemum tea as a health food [2]. The chrysanthemum flower, as a good product of the “integration of medicine and food” [3], has a huge consumer group.

However, in order to minimize the loss of crops during planting, pesticides are widely used to control many plant diseases and insects, such as gray mold, rust, aphids, thrips, leaf pickers, leaf folding insects, and spider mites [4]. Therefore, chrysanthemum flowers may be exposed to a variety of pesticides and contain pesticide residues. However, with the widespread use of pesticides, overuse, abuse, and misuse of pesticides also occur from time to time, which will lead to pesticide residues in chrysanthemum flowers, thus constituting a potential threat to human health and adversely affecting international trade.

Because of the potential of pesticide contamination in current agricultural products, many countries and world organizations (e.g., Codex Alimentarius Commission, European Union (EU), United States, Japan, China, Republic of Korea, Canada) have prescribed stringent stipulations for maximum residue limits (MRLs) for pesticides. For instance, there are 162,248 MRL items that cover 465 pesticides in the EU, 39,147 MRL items that cover 351 pesticides in the United States, and 51,600 MRL items that cover 579 pesticides in Japan, and the residue limit level is as low as 10 μg·kg−1, and 10,092 MRLs for 564 pesticides in 376 kinds of food was stipulated in China National standards for food safety. Implementing these laws and regulations has strengthened supervision over pesticides and ensured the standard use of pesticides to protect human health. However, laws and regulations of such a multitude of MRLs pose a new challenging issue for the monitoring and controlling of pesticide residues.

At present, the commonly used pesticide residues pretreatment methods include solid-phase extraction [5], solid-phase microextraction [6], gel permeation chromatography [7], and the QuEChERS [810]. Among them, the QuEChERS method is widely used, but in most studies, there are many kinds of purification materials with a large amount, low purification efficiency, and large matrix effect, which is not conducive to the rapid and accurate analysis of the experiment [11, 12]. Therefore, the selection of suitable purification materials is conducive to the high-throughput treatment of complex matrices. Sin-QuEChERS Nano column is a new type of rapid sample pretreatment purification column developed and optimized based on the QuEChERS method. Based on the basic principle of reversed dispersion solid-phase extraction, multiwalled carbon nanotubes (MWCNTs), PSA, and C18 solid-phase materials are filled into the column tube to achieve one-step purification. MWCNTs have the characteristics of the nanoscale hollow tubular structure and large specific surface area, small dosage, strong adsorption capacity, stability, and durability, which are suitable for the treatment of complex matrices and have better purification and adsorption effect [1315].

In the past 10 years, most pesticide food control laboratories have shifted from GC-MS to GC-MS/MS as the preferred analytical technology for the treatment of GC amenable compounds. The main reason for this change is that the interference of eluting matrix compounds has a negative impact on single-stage GC-MS analysis. In recent years, the demand for nontargeted detection methods of LC and GC combined with full-scan (FS) MS is increasing, so as to better cover the scope of pesticides and detect them more easily. In GC-MS, FS measurement has been realized for decades, but quadrupole (Q), ion trap, nominal mass time of flight (TOF), and early-generation high-resolution TOF instruments lack sensitivity and/or selectivity. The improvement of high-resolution mass spectrometry (HRMS) in resolution and obtaining appropriate selectivity by improving mass resolution provides new opportunities for residue analysis. Initially, GC and Q-TOF instruments were coupled through the atmospheric pressure chemical ionization (APCI) interface to achieve this goal [1618], but recently, a special GC and electron ionization (EI) Orbitrap MS system was introduced. The system combines the peak capacity and chromatographic resolution of gas chromatography with the sub-ppm mass accuracy of the Orbitrap system to provide higher resolution (15,000, 30,000, 60,000, and 120,000 at half-maximum (FWHM) at m/z 200). The collected data are traceable, which is convenient for retrospective analysis and screening of more interested unknown compounds. Compared with the instrument based on APCI, EI is a more general ionization technology. Because multiple ions for quantification and identification can be obtained in one scanning event, the acquisition is also simpler.

In this study, the potential of GC-Orbitrap-MS in nontarget full-scan independent acquisition mode was evaluated for identification and quantitation purposes. A total of 352 pesticides were selected as the target analytical compounds from the 2020 edition of Chinese Pharmacopoeia. An improved QuEChERS method based on Sin-QuEChERS Nano column purification was used [19, 20]. Method validation in chrysanthemum flowers samples was carried out in terms of sensitivity, linearity, ME, and LOQ. Lastly, the validated method and retrospective analysis was applied to a total of 200 chrysanthemum flower samples bought in local pharmacies (Shijiazhuang, China). This method is suitable for the rapid screening and quantitative analysis of multipesticide residues in chrysanthemum flower and provides data and technical support for the safety evaluation of chrysanthemum flowers.

2. Materials and Methods

2.1. Instruments and Reagents

Analytical standards of the 352 pesticides (10 μg·mL−1) were purchased from Alta Scientific Ltd. (Tianjin, China). A total of 352 kinds of pesticide mixed standard stock solution were prepared with acetonitrile at a concentration of 10 μg·mL−1 and stored in the refrigerator at −18°C. HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). HPLC-grade water was from Milford Super Pure Water System (Milford, MA). Two types of traditional QuEChERS purification were purchased from Thermo Fisher Scientific (Massachusetts, USA). QuEChERS purification package (simple matrix), includes 50.0 mg PSA and 150.0 mg MgSO4; QuEChERS purification package (complex matrix), includes 50.0 mg PSA, 150.0 mg MgSO4, 50 mg C18, and 50 mg GCB. We used two kinds of salting-out for the QuEChERS method: an unbuffered salt system, including 6 g MgSO4 and 1.5 g NaCl, and an acetate buffer salt system, including 6 g MgSO4 and 1.5 g sodium acetate, purchased from Thermo Fisher Scientific Inc. (Fair Lawn, NJ). Sin-QuEChERS Nano column, including 2 g Na2SO4, 0.6 g MgSO4, 90 mg PSA, 10 mg C18, and 15 mg MWCNTs, were purchased from China Agricultural University (Beijing, China). All the chrysanthemum flower materials were purchased from local pharmacies (Shijiazhuang, China).

2.2. Sample Extraction Methods

The samples were crushed by FW100 High-Speed Universal Crusher (Tianjin Tester instrument Co. Ltd.) and mixed well. Two grams of chrysanthemum flower powder (±0.01 g) were then added into a 50 ml plug centrifuge tube; 10 ml of water was added for redissolution; the tube was vortexed for 1 min; and then the sample was allowed to fully soak and evenly disperse. Then, 10 ml acetonitrile was added, mixed well, and vortexed for 1 min. Next, an acetate buffer salt system containing 6 g anhydrous MgSO4 and 1.5 g sodium acetate was added; the tube was vortexed for 1 min, put into an ice water bath for 10 min, and centrifuged for 2 min at 4°C, 9,500 r·min−1, and the supernatant was taken for use.

2.3. Sample Purification Methods

(1)Traditional QuEChERS purification: we tested the purification efficiency of two QuEChERS purification packages: one was a simple matrix, including 50.0 mg PSA and 150.0 mg MgSO4, and the other was a complex matrix, including 50.0 mg PSA, 150.0 mg MgSO4, 50 mg C18, and 50 mg GCB. We transferred 2 ml of the extracted supernatant into a QuEChERS purification centrifuge tube, mixed this by oscillation for 1 min, centrifuged it at 9,500 r·min–1 for 3 min, absorbed the supernatant, passed this through a 0.22 μm nylon filter membrane to an injection bottle, and waited for sample analysis by the GC-Orbitrap-MS.(2)Sin-QuEChERS Nano column purification: we tested the purification efficiency of the Sin-QuEChERS Nano column, including 2 g Na2SO4, 0.6 g MgSO4, 90 mg PSA, 10 mg C18, and 15 mg MWCNTs. The purification column of the Sin-QuEChERS Nano purification tube is vertically inserted into the 50 ml centrifuge tube containing the extract, and the top of the purification column is slowly pressed down so that the upper organic extract in the centrifuge tube passes through the water blocking filter and column filler in the purification column from bottom to top, and finally enters into the Sin-QuEChERS Nano storage tank for about 4 ml of supernatant. After mixing the purified liquid, the supernatant is sucked over a 0.22 μm nylon filter membrane to the injection bottle for analysis by GC-Orbitrap-MS.

By comparing the purification effects, total ions, and recovery rates of these three purification methods, the Sin-QuEChERS Nano column was finally selected as the purification method for method validation and real sample analysis. See Section 3.2 “Selection of Purification Conditions” for the comparison results.

2.4. Preparation of Standard Solution

The mixed standard stock solutions of 352 pesticides were diluted with the blank extract of the matrix, and a series of standard solutions with concentrations of 0.005, 0.02, 0.05, 0.1, and 0.2 μg·ml−1 were prepared. The matrix mixed standard solution was prepared and used immediately.

2.5. Instrument Conditions

We followed and optimized the methods of previous works [21, 22]. A GC-Orbitrap-MS system (Thermo Scientific, Bremen, Germany) consisting of an AI/AS 1310 TriPlus RSH™ autosampler was used. TRACE 1300 Series GC with a hot split/splitless injector, an EI source, and a hybrid quadrupole Orbitrap mass spectrometer with an HCD (higher energy collision-induced dissociation) cell was used.

GC separation was performed on a 30 m × 0.25 mm id, 0.25 μm Thermo Scientific TG-5MS column using the following temperature program: 40°C, 1.5 min; 25°C·min−1 to 90°C, 1.5 min; 25°C·min−1 to 180°C, 0 min; 5°C·min−1 to 280°C, 0 min; and 10°C·min−1 to 310°C, 3 min. Helium 5.0 (99.999%; Linde Gas, Schiedam, The Netherlands) was used as carrier gas at a constant flow of 1 mL·min−1. The transfer line was maintained at 280°C. EI was performed at 70 eV, with the source temperature set at 280°C. FS MS acquisition was done in profile mode using an m/z range of 50–550. The nitrogen gas supply for the C-trap was 5.0 grade (99.999%; Linde Gas). The resolving power was set at 60,000 (FWHM at m/z 200) to ensure high mass accuracy. The automatic gain control (AGC) target was set at 5e6 ions, with the maximum ion injection time set to 25 ms.

2.6. Establishment of Database

In this experiment, 352 pesticide compounds were selected and prepared into 1.0 μg·ml−1 mixed standard solutions. The retention time of the corresponding compounds, the accurate molecular weight, and the chemical formula of the fragment ions were obtained under the full-scan mode. Three fragment ions of each compound were selected to obtain ion information (accurate mass and chemical formula). The data were imported into TraceFinder (4.1) software, and the relevant database was established. The TraceFinder software not only can realize the rapid batch and automatic processing of data but also can set the functions of qualitative, quantitative, and method establishment. According to the established database, it can realize the rapid screening of target substances. The database mainly contains the compounds’ names, CAS registration numbers, fragment ion information, retention times, and other information (Table 1).

3. Results and Discussion

3.1. Optimization of Extraction Conditions

According to the list of pesticides involved in the 2020 edition of Chinese Pharmacopoeia, combined with pesticides, herbicides, and fungicides that may be used in chrysanthemum flower planting, 352 pesticides were selected as the target analytical compounds. Because it contains many pesticides, including organophosphorus, organochlorine, pyrethroids, triazoles, carbamates, and other insecticides, there are many kinds and polarity differences. At the same time, chrysanthemum flower contains pigments, amino acids, and volatile components, so it is particularly important to choose the appropriate extraction solvent. The QuEChERS method uses acetonitrile as the extraction solvent, which is due to the good solubility, permeability, and versatility of acetonitrile and high extraction efficiency for most pesticides. The results showed that the recovery rate of some pesticides with poor stability was low by adding ordinary salt, which was related to the pH value of the matrix; the recovery of 280 pesticides was between 70% and 120%; 38 pesticides were less than 70%; and 34 pesticides were more than 120%. Because carbamates are sensitive to pH value, they are more stable under acidic conditions and easily to decompose under alkaline conditions. Therefore, adding acetate buffer salt makes the sample extract weak acidic, thus improving the recovery rate of acid-base-sensitive pesticides. The recovery rate of all pesticides is between 70% and 125%.

Using the QuEChERS method, adding the appropriate amount of water is conducive to the full contact between organic solvent and sample, improves the extraction efficiency, and helps achieve better recovery. However, adding too much water will lead to the dissolution of water-soluble pigment and other soluble matrix components. The effects of 0, 10, and 15 ml of water on the recovery of the target were compared, and the extraction efficiency of 10 ml water was higher than that of the other two groups. There were only 34 pesticides with a recovery rate of more than 120% in the nonwater group. Therefore, in this method, 10 ml water was added.

Some organophosphorus pesticides (such as parathion and fenitrothion) are unstable in chemical properties and easy to decompose at high temperatures. Because there is anhydrous MgSO4 in the acetic acid buffer salt system, a lot of heat will be released in the process of water absorption. Therefore, after adding acetic acid buffer salt, we put the centrifuge tube of extracting sample into an ice water bath for 10 min to improve the recovery rate of pesticides with poor thermal stability.

3.2. Selection of Purification Conditions

It is important to select suitable purification adsorption materials for the efficient purification of complex substrates. The ideal purification adsorption material should achieve the purification effect required by the experiment and ensure that it does not adsorb the target analyte in the extraction solvent. In this experiment, the purification effects of QuEChERS purification and Sin-QuEChERS Nano column were compared (Figures 1 and 2). Mixed reference materials (10 μg·kg−1) were added to the chrysanthemum flower sample and then extracted. The extracts were purified by QuEChERS purification (simple matrix), QuEChERS purification (complex matrix), and Sin-QuEChERS Nano column. It can be seen from Figure 1 that the color of samples purified by QuEChERS purification (simple matrix) is dark, the color of samples purified by Sin-QuEChERS Nano column is lighter, and QuEChERS purification (complex matrix) is almost colorless. It can be seen from the total ions in Figure 2 that the samples purified by QuEChERS purification (simple matrix) have more impurities and greater interference, while the samples purified by the Sin-QuEChERS Nano column are less interfered with, and the peak of QuEChERS purification (complex matrix) is less after 25 min, which is due to the adsorption of the target substance with late peak, making it look cleaner. Meanwhile, the recovery rates of target compounds were 72.7–118.9% in QuEChERS purification (simple matrix), 72.8–123.4% with the Sin-QuEChERS Nano column, and 62.4–120.7% by QuEChERS purification (complex matrix). The results showed little difference in the recovery rate between QuEChERS purification (simple matrix) and Sin-QuEChERS Nano column, but the recovery rate of QuEChERS purification (complex matrix) was relatively low. Primary-secondary amine (PSA), which plays the main role in QuEChERS purification (simple matrix), is a weak anion exchange adsorbent. It can effectively remove polar pigments, organic acids, sugars, fatty acids, and other components that are easy to form hydrogen bonds in the sample, but its adsorption capacity is limited. In addition to PSA, QuEChERS purification (complex matrix) also contains graphitized carbon black (GCB). GCB can remove pigments from chrysanthemum flower, such as chlorophyll, radish-like hormone, and sterol, but the strong adsorption force will absorb the target of a benzene ring, which leads to a low recovery rate. In addition to PSA, 15 mg MWCNTs (particle size length: 10–50 μm, outer diameter: 30–60 nm, and specific surface area: 280 m2·g−1) was added to the Sin-QuEChERS Nano column. MWCNTs are nano hollow tubes with high mechanical strength, strong acid-base resistance, stronger adsorption, and purification capacity but do not affect the recovery rate of the target substance [2325]. This experiment shows that the combination of PSA and MWCNTs can effectively remove impurities in the sample, reduce the interference to the target substance, improve the recovery rate of the target substance, and protect the analytical instrument from pollution and damage. At the same time, the high-resolution mass spectrometer can detect low concentration pesticide residues in a complex matrix, so the Sin-QuEChERS Nano column was selected for purification.

3.3. Optimization of Instrument Resolution

As a high-resolution mass spectrometer, Orbitrap mass spectrometer can fully scan acquisition and collect data in the range of m/z 50–550, ensuring the retrospective data analysis. Resolution is an important parameter in high-resolution mass spectrometry. In the presence of matrix interference, the resolution will affect the accuracy of quality measurement. Therefore, the key to qualitative analysis is to choose the appropriate resolution. High resolution can improve the accuracy of mass determination and can effectively identify compounds with very close accurate mass. In the experiment, the content of trifloxystrobin in chrysanthemum flower was 10 μg ·kg−1, which was determined at three different resolutions (15,000, 30,000, and 60,000). In Figure 3, the qualitative ion m/z 186.05251 is the qualitative ion of trifloxystrobin, and m/z 186.06752 is the interference ion. Only when the resolution is 60,000 or above, the two ions with the same mass can be clearly distinguished. At the same time, the accurate qualitative and quantitative analysis can be carried out, and the screening accuracy will be greatly improved; the quality accuracy is less than 2.0 ppm; and the high sensitivity can still be maintained, so it fully meets the requirements of pesticide residue detection in chrysanthemum flower. Also, the accurate mass number and deviation, retention time window, isotopic distribution, and isotopic abundance information were used simultaneously in this method to realize the rapid and accurate screening of target substances.

3.4. Matrix Effect

Matrix effects (MEs) are very common in GC-MS/MS and should be assessed at the method validation stage. MEs were estimated via the ratio of the calibration curve slopes of matrix to solvent. Studies recommend that MEs can be ignored when the ME values are in the range of 0.9–1.1 [15]. If the ME cannot be ignored, using a matrix-matched standard is the most effective way to compensate for MEs.

The MEs in this study are listed in Table S1. The MEs of the Sin-QuEChERS Nano method were in the range of 1.01–1.86; the MEs of the QuEChERS (simple matrix) and QuEChERS (complex matrix) method ranged between 1.05 and 2.38 and between 1.08 and 2.89, respectively. As for the matrix suppression or enhancement effect, QuEChERS (simple matrix) was the strongest, while Sin-QuEChERS nano was the weakest. This indicated that the Sin-QuEChERS Nano method reduced the matrix effect more efficiently than QuEChERS (simple matrix) and QuEChERS (complex matrix).

3.5. Linear Range, Limit of Detection, Limit of Quantitation, and Recovery

The blank matrix standard solution is prepared according to the pretreatment method; the standard curve was drawn with the mass concentration of the compound as abscissa and the corresponding peak area as ordinate. The linear range of 352 compounds was 0.5–200 μg ·kg−1, and the correlation coefficient (R) was greater than 0.99. The LODs and LOQs of the method were investigated by adding blank samples. The LODs were three times the signal-to-noise ratio (S/N = 3), and the LOQs were 10 times the signal-to-noise ratio (S/N = 10). The mixed standard solutions of 352 compounds were added to the negative samples of chrysanthemum flower at the levels of 10, 50, and 100 μg·kg−1, respectively, and each level was repeated six times. The results showed that the detection limits of 352 pesticides were 0.3–3 μg·kg−1, and the quantification limits were 1–10.0 μg·kg−1, which met the requirements of pesticide residue detection [26]. The average recovery rates of 352 compounds at three levels were 73.2–110.3%, 72.8–112.6%, and 77.6–123.4%, respectively. The average RSDs of 352 compounds at three levels were 3.2–9.6%, 4.0–9.7%, and 4.0–11.3%, respectively. The results showed that the method could be used for the determination of pesticide residues in chrysanthemum flower. The correlation coefficients, limits of detection, limits of quantitation, spiked recovery rates, and relative standard deviations of 352 compounds in chrysanthemum flower are shown in Table S1.

3.6. Determination of Actual Samples

Two hundred samples were analyzed by the established method. Among them, 137 samples were detected with pesticide residues, and the chemical substances with a high detection rate were profenofos, procymidone, metalaxyl, chlorfenapyr, difenoconazole, dimethomorph, cypermethrin, tebuconazole, propiconazole, and pyrimethanil, among others (Table 2). Figure 4 is the mass spectrum of profenofos in the standard and chrysanthemum flower positive samples. The fragment ions (338.96369, 205.91286, and 207.91063) can be detected, and the ion ratio is highly matched. The results show that the method was also suitable for detecting 352 pesticide residues in chrysanthemum flowers, such as calendula and chamomile. The results showed that the nontarget rapid screening method established in this study could rapidly screen potential pesticide residues in chrysanthemum flower with high throughput.

3.7. Retrospective Analysis

GC-Orbitrap-MS often collects the full spectrum, which can collect data more comprehensively. The data collection has no relationship with the number of compounds in the database, so the data can be reviewed and reanalyzed to expand the target range. In the analysis of samples, we added the retention time, molecular formula, accurate relative molecular weight, and CAS number of new compounds pentachlorobenzonitrile, simazine, and simetone into 352 databases and verified them with actual samples. It was found that the linearity of these three compounds in each matrix was greater than 0.99, the average recovery rates were 72.8–123.4%, and the average RSD values were 3.2–11.3% at three levels (10, 50, and 100 μg·kg−1, respectively), which met the requirements of detection. Among 200 chrysanthemum flower samples, 5 chrysanthemum flower samples were detected with pentachlorobenzonitrile with a detection value range of 0.048–0.22 mg·kg−1; 3 chrysanthemum flower samples were detected with simetone, with a detection value range of 0.032–0.051 mg·kg−1; and no samples were detected with simazine. Retrospective analyses can expand and analyze target compounds without recollecting data, which is flexible and is convenient for high-throughput screening and quantitative analysis of pesticide residues. It is the development direction of chrysanthemum flower risk monitoring technology in the future.

4. Conclusion

The work presented a method that had been developed and validated for the simultaneous determination of 352 pesticide residues in chrysanthemum flower by GC-Orbitrap-MS, which was established based on the purification of the Sin-QuEChERS Nano column. The Sin-QuEChERS Nano column simplifies the pretreatment process and effectively improves the purification efficiency. After systematic validation for linearity, precision, accuracy, stability, and matrix effects, the developed method was successfully applied for qualitative confirmation and quantitative detection of 352 pesticide residues in 200 chrysanthemum flower samples bought from local pharmacies. No saturation phenomena were experienced in any case. The developed and validated method has proved to be robust and appropriate in sensitivity, mass accuracy, and quantification in full-scan mode and provide good results in the analysis of real samples. These good results show the advantages of full-scan analysis, which is applicable to other compounds that do not appear in selective and retrospective evaluation and easier range management than GC-MS/MS. This method has the advantages of simple pretreatment, high purification efficiency, high throughput, and accurate analysis. It can effectively reduce the amount of standard substances in the detection of multipesticide residues in chrysanthemum flowers, which provides technical support for rapid screening and analysis of potential pesticide residues in the chrysanthemum flower.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no potential conflicts of interest.

Authors’ Contributions

Yuanyuan Wang and Zhijuan Meng contributed equally to this work.


This work was supported by the Program of Traditional Chinese Medicine Scientific Research foundation in Hebei Administration of Traditional Chinese Medicine (2019091, Hebei, China), the Project of Basic Scientific Research of Provincial Universities (JYZ2020003), Excellent Young Teacher Fundamental Research (YQ2020009), and Doctoral Foundation (BSZ2018008) of Hebei University of Chinese Medicine.

Supplementary Materials

Table S1 is included in the supplementary file. (Supplementary Materials)