Method for detecting chemical components in artemisia annua pollen aerosol and constructing characteristic database

Abstract

The application discloses a method for detecting chemical components in Artemisia annua pollen aerosol and constructing a characteristic database, which comprises the following steps: (1) sample treatment: collecting Artemisia annua pollen aerosol, storing in a low temperature and sealing, extracting after drying and crushing to obtain a solution to be tested; (2) performing chromatography and mass spectrometry analysis on the solution to be tested; (3) data processing: including database establishment, data collection and processing, relevant literature searching, representative compound selection and cleavage rule derivation. The application adopts UHPLC-Q-Orbitrap HRMS technology, systematically separates and identifies chemical components in Artemisia annua pollen, focuses on analyzing mass spectrometry cleavage characteristics of main category compounds such as sesquiterpenes, flavones and phenylpropanoids, summarizes the cleavage rule, fills the blank of research on small molecule chemical components and mass spectrometry cleavage of Artemisia annua pollen, and provides a new thought and scientific basis for quality evaluation, sensitization mechanism analysis and resource comprehensive utilization of Artemisia annua pollen.

Description

Technical Field

[0001] This invention relates to a method for detecting chemical components, and in particular to a method for detecting chemical components in Artemisia annua pollen aerosol and constructing a characteristic database. Background Technology

[0002] Artemisia annua ( Artemisia annua L. Artemisia annua is an annual herb belonging to the genus Artemisia in the family Asteraceae. It is widely distributed in temperate and subtropical regions of the Northern Hemisphere and is a traditional medicinal plant in my country. Artemisinin can be extracted from its above-ground parts, possessing various pharmacological activities such as antimalarial and anti-inflammatory effects. As an important type of wind-pollinated pollen, Artemisia annua pollen, when released into the atmosphere, can form stable Artemisia annua pollen aerosols. These aerosols are one of the main allergens causing seasonal allergic rhinitis, asthma, and food cross-allergies. In northern my country, the positive rate of Artemisia annua pollen allergy patients has exceeded 50%, becoming a public health problem that cannot be ignored.

[0003] Current research on Artemisia annua primarily focuses on the isolation of medicinal components from its aerial parts, artemisinin extraction processes, and the quantification of pollen protein allergens. However, pollen sensitization is not solely driven by protein allergens. Naturally occurring small-molecule compounds in pollen, such as adenosine and flavonoids, can act as endogenous adjuvants, interfering with mucosal barrier function and inducing Th2 immune shifts, thereby significantly amplifying the allergic cascade response triggered by protein allergens. Therefore, identifying only protein allergens cannot fully reveal the sensitization risk of Artemisia annua pollen aerosols. Systematically analyzing its small-molecule chemical composition, especially compounds with potential adjuvant activity or hapten properties, and elucidating their mass spectrometric fragmentation patterns, is a crucial prerequisite for a deeper understanding of the sensitization mechanism of Artemisia annua pollen. However, a systematic and efficient detection method for the small-molecule chemical composition of Artemisia annua pollen aerosols is currently lacking. Summary of the Invention

[0004] The technical problem to be solved by this invention is: given that existing research only focuses on protein allergens in Artemisia annua pollen while neglecting small molecule compounds with synergistic sensitization effects or potential hapten activity, this invention provides a detection method that can comprehensively characterize the chemical fingerprint spectrum of Artemisia annua pollen aerosol, so as to accurately identify key small molecule components that may be involved in sensitization regulation, and provide chemical basis data for systematically evaluating pollen sensitization characteristics and related product quality evaluation.

[0005] The present invention provides a method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol, comprising the following steps: (1) Sample preparation: Collect Artemisia annua pollen aerosol, store it in a sealed container at low temperature, dry and pulverize it, and then extract it to obtain the test solution; (2) Perform chromatographic and mass spectrometric analysis on the test solution; (3) Data processing: including database establishment, data collection and processing, relevant literature search, selection of representative compounds and deduction of fragmentation rules.

[0006] The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosols according to the present invention, wherein: The chromatographic conditions in step (2) are as follows: The chromatographic column was equipped with an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); mobile phase: A was 0.1% formic acid aqueous solution, B was acetonitrile; flow rate was 0.3 mL / min; column temperature was 40 °C; injection volume was 6.0 µL; gradient elution program: 0–1 min, 2% B; 1–14 min, 2%–30% B; 14–25 min, 30%–100% B; 25–28 min, 100% B; 28–28.1 min, 100%–2% B; 28.1–29.5 min, 2% B.

[0007] The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosols according to the present invention, wherein: The mass spectrometry conditions in step (2) are as follows: Analysis was performed using a Q Exactive quadrupole / electrostatic field orbital trap high-resolution mass spectrometer equipped with an electrospray ionization source. The spray voltages in positive and negative ion modes were 3.7 kV and 3.5 kV, respectively; the capillary temperature was 320 °C; the sheath gas pressure was 30 psi, the auxiliary gas pressure was 10 psi, the auxiliary heating temperature was 300 °C, both the sheath gas and the auxiliary gas were nitrogen, and the collision gas was nitrogen at a pressure of 1.5 mTorr; Full MS / dd-MS was used. 2 Data acquisition mode, Full MS parameters: resolution 70000, automatic gain control (AGC) target value 1 × 10⁻⁶. 6 The maximum injection time is 50 ms, and the scanning range is... m / z 100~1500; dd-MS 2 Parameters: Resolution 17500, AGC target value 1 × 10 5 The maximum injection time is 50 ms, a maximum of 10 ions can be selected for secondary fragmentation ion scanning, dynamic exclusion is enabled, and the isolation window is 2.0. m / z The normalized collision energy (NCE) is set to 10, 30, and 60, and the intensity threshold is set to 1 × 10⁻⁶. 5 .

[0008] The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosols according to the present invention, wherein: The data processing procedure in step (3) is as follows: 1) Establish a database: Search online databases such as PubMed, Web of Science, CNKI, MassBank and related literature for the names, molecular formulas, precise relative molecular masses, adduct ion types, characteristic fragment ion information and compound categories of possible chemical components in Artemisia annua pollen, and use this as a self-built database of chemical components in Artemisia annua pollen; 2) Data acquisition was performed using Xcalibur 4.1 software to comprehensively record the primary and secondary mass spectrometry information of the compounds; 3) The acquired raw mass spectrometry data were processed using Progenesis QI 3.0 software, including raw data import, peak extraction, and deconvolution. High-resolution mass spectrometry parameters were set as follows: mass accuracy of 5 ppm, retention time window of 0.1–0.3 min, and peak intensity threshold of 3 times the background noise for peak extraction; retention time deviation ≤ 0.05 min and mass-to-charge ratio deviation ≤ 5 ppm for deconvolution. The preprocessed data were compared with the TCM Pro 2.0 reference database and a self-built database. Compound identification was performed based on the precise molecular weight, retention time, isotope distribution, and fragment ion information, with a mass error ≤ 5 ppm and at least two characteristic fragment ions matched in the secondary mass spectrometry. 4) Relevant literature search, selection of representative compounds, and deduction of fragmentation patterns: Based on the above automatic identification, according to the precise molecular weight, molecular formula, characteristic fragment ions, and retention time range of the compound, a combined search is performed in PubMed, Web of Science, and CNKI databases. Priority is given to using keywords such as "compound name + plant source" or "molecular formula + characteristic fragments." One to three references are located by comparing the measured secondary mass spectra with the fragmentation patterns in the literature. In the total ion chromatogram, chromatographic peaks with high intensity and good resolution are selected as the main components. Representative compounds are selected, and the main fragment ions in their secondary mass spectra are manually analyzed. Possible fragmentation pathways are deduced, including the location of chemical bond breakage, rearrangement reactions, neutral loss, and the generation mechanism of characteristic fragment ions. By systematically summarizing the fragmentation patterns of this type of compound, the database comparison results can be effectively verified or supplemented, improving the reliability of structural identification.

[0009] The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosols according to the present invention, wherein: The sample processing procedure in step (1) is as follows: Airborne Artemisia annua pollen aerosols were collected using a combination of natural sedimentation and an impactor sampler. The collected aerosols were immediately sealed and stored at low temperature. The frozen Artemisia annua pollen was dried, pulverized, sieved, and thoroughly mixed. The powder was then mixed with methanol and water, and extracted using an ultrasonic-microwave synthetic extractor. The extract was cooled, weighed, and any lost mass was replenished. After thorough mixing, the supernatant was collected by centrifugation. An equal volume of the solution was mixed with water and placed in a sample vial for analysis.

[0010] The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosols according to the present invention, wherein: Sample processing specifically includes the following steps: Airborne Artemisia annua pollen aerosols were collected using a combination of natural sedimentation and an impactor sampler. The collected aerosols were immediately sealed and stored at -20°C. The frozen pollen was dried, pulverized, and passed through a 40-mesh sieve. 1.0 g of the sample powder was accurately weighed and placed in a three-necked flask. 40 mL of 80% methanol aqueous solution was added, and ultrasonic / microwave-assisted extraction was performed for 45 min. The extraction temperature was set at 45°C, the ultrasonic power was adjusted to 340 W, and the microwave power was adjusted to 300 W. After cooling, the extract was weighed, and the lost mass was replenished with 80% methanol aqueous solution. The mixture was then centrifuged at 14000 r / min for 10 min at 10°C. 100 μL of the supernatant was collected, added to 100 μL of ultrapure water, mixed, and then placed in a sample vial for analysis.

[0011] The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol of this invention differs from existing technologies in that: This invention provides a method for the detection and characterization database construction of chemical components in Artemisia annua pollen aerosols. The method employs ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q / Orbitrap HRMS) to establish a systematic identification method suitable for the chemical components of Artemisia annua pollen. Sample pretreatment utilizes ultrasonic / microwave-assisted extraction, and the resulting extract is separated using an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). Mass spectrometry analysis is performed in positive and negative ion modes using an electrospray ionization source, employing a full scan-data-dependent secondary mass spectrometry mode (Full MS / dd-MS). 2Data was collected. Raw mass spectrometry data were processed using Progenesis QI 3.0 software, and combined with comparison to a self-built database, literature search, and fragment ion analysis to comprehensively identify the compounds. A total of 150 compounds were identified, covering structural types such as flavonoids and their glycosides, terpenes, organic acids, amino acids, phenylpropanoids, and nucleosides. Simultaneously, the mass spectrometry fragmentation patterns of several representative compounds were analyzed to further elucidate their characteristic fragmentation pathways. This study provides a reliable analytical method for the identification of chemical components in Artemisia annua pollen and also provides a reference for its quality evaluation and sensitization-related mechanism research.

[0012] In this invention: Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS) boasts advantages such as high separation efficiency, high detection sensitivity, and accurate qualitative analysis. It has been widely applied in the identification of chemical components in complex systems such as plant pollen and medicinal plants, and is particularly suitable for the rapid identification and structural analysis of trace components. The study of mass spectrometry fragmentation patterns can clarify the structural characteristics and fragmentation pathways of compounds, providing theoretical support for the structural identification of similar unknown compounds, and simultaneously providing a basis for the selection of characteristic ions for the quantitative analysis of chemical components.

[0013] This invention employs UHPLC-Q-Orbitrap HRMS technology to systematically separate and identify chemical components in Artemisia annua pollen. It focuses on analyzing the mass spectrometry fragmentation characteristics of major compounds such as sesquiterpenes, flavonoids, and phenylpropanoids, summarizing their fragmentation patterns, and filling the gap in research on small molecule chemical components and mass spectrometry fragmentation of Artemisia annua pollen. This provides new ideas and scientific basis for the quality evaluation, sensitization mechanism exploration, and comprehensive resource utilization of Artemisia annua pollen.

[0014] The method for detecting chemical components and constructing a feature database in Artemisia annua pollen aerosol of the present invention will be further described below with reference to the accompanying drawings. Attached Figure Description

[0015] Figure 1 This is the total ion chromatogram of Artemisia annua pollen in the positive (A) and negative (B) ion modes of the method of the present invention; Figure 2 The image shows the secondary mass spectrum (a) of luteolin in the negative ion mode of the method of the present invention and the possible mass spectrometry fragmentation pathway (b). Figure 3 The image shows the secondary mass spectrum (a) of artemisinin in positive ion mode and the possible mass spectrometry fragmentation pathway (b) in the method of this invention. Figure 4 The image shows the secondary mass spectrum (a) of ferulic acid in positive ion mode and the possible mass spectrometry fragmentation pathway (b) in the method of this invention. Figure 5The image shows the secondary mass spectrum (a) of chlorogenic acid in negative ion mode and the possible mass spectrometry fragmentation pathway (b) in the method of the present invention. Figure 6 The image shows the secondary mass spectrum (a) of arginine in positive ion mode and the possible mass spectrometry fragmentation pathway (b) in the method of this invention. Figure 7 The image shows the secondary mass spectrum (a) of guanosine in the negative ion mode of the method of the present invention and the possible mass spectrometry fragmentation pathway (b). Figure 8 The image shows the secondary mass spectrum (a) of scopolamine in negative ion mode in the method of this invention and the possible mass spectrometry fragmentation pathway (b). Detailed Implementation

[0016] 1. Experimental Section

[0017] 1.1 Main Instruments and Apparatus

[0018] Vanquish Flex ultra-high performance liquid chromatograph and Q Exactive quadrupole / electrostatic field orbit trap high-resolution mass spectrometer: products of Thermo Fisher Scientific, USA; UWave-2000 multifunctional microwave synthesis and extraction system: products of Shanghai Xinyi Microwave Chemical Technology Co., Ltd.; KQ-3200D ultrasonic cleaner: product of Kunshan Ultrasonic Instrument Co., Ltd.; Mikro 220R high-speed refrigerated centrifuge: product of Hettich, Germany.

[0019] 1.2 Samples and Reagents

[0020] Pollen samples of Artemisia annua were collected in Song County, Luoyang City, Henan Province; methanol, acetonitrile, and formic acid (mass spectrometry grade): products of Thermo Fisher Scientific, USA; ultrapure water: prepared by a Milli-Q IQ 7005 ultrapure water system.

[0021] 1.3 Experimental Conditions

[0022] 1.3.1 Chromatographic conditions: An ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used; mobile phase: A was 0.1% formic acid aqueous solution, B was acetonitrile; flow rate was 0.3 mL / min; column temperature was 40 °C. Injection volume was 6.0 µL. Gradient elution program: 0–1 min (2% B), 1–14 min (2%–30% B), 14–25 min (30%–100% B), 25–28 min (100% B), 28–28.1 min (100%–2% B), 28.1–29.5 min (2% B).

[0023] 1.3.2 Mass Spectrometry Conditions A Q Exactive quadrupole / electrostatic field orbital trap high-resolution mass spectrometer equipped with an electrospray ionization source was used for analysis. The spray voltages in positive and negative ion modes were 3.7 kV and 3.5 kV, respectively; the capillary temperature was 320 °C; the sheath gas pressure was 30 psi, and the auxiliary gas pressure was 10 psi; the auxiliary heating temperature was 300 °C; both the sheath gas and auxiliary gas were nitrogen; the collision gas was nitrogen at a pressure of 1.5 mTorr. Full MS / dd-MS was used. 2 Data acquisition mode. Full MS parameters: resolution 70000, automatic gain control (AGC) target value 1 × 10⁻⁶. 6 The maximum injection time is 50 ms, and the scanning range is... m / z 100~1500; dd-MS 2 Parameters: Resolution 17500, AGC target value 1 × 10 5 The maximum injection time is 50 ms, a maximum of 10 ions can be selected for secondary fragmentation ion scanning, dynamic exclusion is enabled, and the isolation window is 2.0. m / z The normalized collision energy (NCE) is set to 10, 30, and 60, and the intensity threshold is set to 1 × 10⁻⁶. 5 .

[0024] 1.4 Sample processing methods

[0025] Airborne Artemisia annua pollen aerosols were collected using a combination of natural sedimentation and an impactor sampler. The collected aerosols were immediately sealed and stored at -20°C. The frozen pollen was dried, pulverized, and passed through a 40-mesh sieve. 1.0 g of the sample powder was accurately weighed and placed in a three-necked flask. 40 mL of 80% methanol aqueous solution was added, and ultrasonic / microwave-assisted extraction was performed for 45 min. The extraction temperature was set at 45°C, the ultrasonic power was adjusted to 340 W, and the microwave power was adjusted to 300 W. After cooling, the extract was weighed, and the lost mass was replenished with 80% methanol aqueous solution. The mixture was then centrifuged at 14000 r / min for 10 min at 10°C. 100 μL of the supernatant was collected, added to 100 μL of ultrapure water, mixed, and then placed in a sample vial for analysis.

[0026] 1.5 Data Processing

[0027] 1) Establish a database: Search online databases such as PubMed, Web of Science, CNKI, and MassBank, as well as relevant literature, for the names, molecular formulas, precise relative molecular masses, and adduct ion types (e.g., [M+H]) of possible chemical components (such as flavonoids, terpenes, and phenolic acids) in Artemisia annua pollen. + [MH] - [M+Na]+ Based on information such as characteristic fragment ions and compound categories, a database of chemical components of Artemisia annua pollen was built. 2) Data acquisition was performed using Xcalibur 4.1 software to comprehensively record the primary and secondary mass spectrometry information of the compounds; 3) The acquired raw mass spectrometry data were processed using Progenesis QI 3.0 software, sequentially completing raw data import, peak extraction, and deconvolution. Standard high-resolution mass spectrometry parameters were set as follows: for peak extraction, mass accuracy was 5 ppm, retention time window was 0.1–0.3 min, and peak intensity threshold was 3 times the background noise; for deconvolution, retention time deviation was ≤0.05 min, and mass-to-charge ratio deviation was ≤5 ppm. The preprocessed data were compared with a reference database (TCM Pro 2.0, Beijing Hexin Technology Co., Ltd.) and a self-built database. Compound identification was performed based on the precise molecular weight, retention time, isotope distribution, and fragment ion information, requiring a mass error ≤5 ppm and at least two characteristic fragment ions matched in the secondary mass spectrometry. 4) Relevant Literature Search, Representative Compound Selection, and Derivation of Fragmentation Patterns: Based on the above automatic identification, according to the compound's precise molecular weight (error ≤ 5 ppm), molecular formula, characteristic fragment ions, and retention time range, a combined search is performed in databases such as PubMed, Web of Science, and CNKI. Priority is given to using keywords such as "compound name + plant source" or "molecular formula + characteristic fragments." One to three references are located by comparing the measured secondary mass spectra with literature fragmentation patterns. In the total ion chromatogram, chromatographic peaks with high intensity and good resolution are selected as the main components. Representative compounds are selected, and the main fragment ions in their secondary mass spectra are manually analyzed. Possible fragmentation pathways are deduced, including the location of chemical bond breakage, rearrangement reactions, neutral loss (such as H2O, CO2, glycosyl groups, etc.), and the generation mechanism of characteristic fragment ions. By systematically summarizing the fragmentation patterns of this type of compound, the database comparison results can be effectively verified or supplemented, improving the reliability of structural identification.

[0028] 2 Results and Discussion

[0029] The acquired mass spectrometry data were processed using Progenesis QI 3.0 software, and qualitative analysis of the chemical components in Artemisia annua pollen was performed by combining a self-built database comparison, literature search, and fragment ion analysis. The results showed that a total of 150 chemical substances were identified, including 55 flavonoids, 52 terpenoids, 12 organic acids, 8 amino acids, 5 nucleoside compounds, 8 phenylpropanoids, and 10 other compounds. The total ion chromatograms of Artemisia annua pollen in positive and negative ion modes are shown below. Figure 1A and 1B. Some representative compounds are listed in Table 1.

[0030] Table 1. Identification of chemical components in Artemisia annua pollen

[0031] 2.1 Identification of Flavonoids in Artemisia annua Pollen

[0032] Flavonoids are common and important secondary metabolites in plants, widely distributed within the plant body, and possess various physiological activities such as antioxidant, anti-inflammatory, antibacterial, antitumor, free radical scavenging, and immunomodulatory effects. Their basic structural characteristic is a C6–C3–C6 skeleton, consisting of two benzene rings (A and B rings) connected by an oxygen-containing six-membered heterocycle (C ring). The benzene rings often bear substituent groups such as hydroxyl, methyl, and methoxy groups. Most flavonoids often combine with glycosyl groups such as glucose, rhamnose, and rutin to form corresponding glycosides, further enhancing their water solubility and bioavailability. A total of 55 flavonoids were identified from Artemisia annua pollen, including flavones, flavonols, dihydroflavones, biflavones, and their glycosides, among other subclasses. The cleavage mechanisms of flavonoids mainly include glycosidic bond cleavage, reverse Diels-Alder reaction (RDA) cleavage, loss of neutral small molecules such as CO, CO2, H2O, and CH2O2, and removal of free radicals such as CH3· and HCO· (Cui Xinyuan, Ji Yujie, Ding Zhaoqi, Liu Borui, Wei Yiling, Wang Xinyi, Liu Yuanyuan. Study on chemical constituents and mass spectrometric cleavage rules of roasted Xanthium sibiricum based on UPLC-Q-Orbitrap MS [J]. Journal of Mass Spectrometry, 2026, 47(02): 187-96.). Taking luteolin as an example, its quasi-molecular ion peak can be seen in negative ion mode. m / z 447.0935 [M–H] - The chromatographic retention time was 11.50 min, suggesting its molecular formula is C. 21 H 20 O 11 Its secondary mass spectrum shows... m / z 285.0399 [M–H–C6H 10 O5] - 151.0015 [M–H–C 14 H 16 O7] - 107.0013 [M–H–C 14 H 16 O7–CO2] -The presence of characteristic fragment ions indicates that glycosyl removal occurs first, followed by further skeletal fragmentation of the flavonoid core. The possible mass spectrometric fragmentation pattern is shown in... Figure 2 This is basically consistent with the literature report (Zhu Xinyi, Yang Chunguo, Tian Haitao, Hou Miao, Han Liwen, Deng Zhipeng. Characterization and identification of chemical components of Sedum sarmentosum based on UHPLC-Q-Exactive Orbitrap-MS technology [J]. Shandong Science, 2024, 37(05): 10-6.).

[0033] 2.2 Identification of Terpenoids in Artemisia annua Pollen

[0034] Terpenoids are a class of lipid-soluble components in Artemisia annua pollen with significant biological activity. They possess various pharmacological activities such as anti-inflammatory, antitumor, antimalarial, antibacterial, and immunomodulatory effects. Their chemical structure is characterized by isoprene units (C5) linked head-to-tail or head-to-head. Based on the number of isoprene units, they can be classified into monoterpenes, diterpenes, sesquiterpenes, etc. A total of 52 terpenoids were identified from Artemisia annua pollen. The cleavage mechanisms of terpenoids mainly include C–C bond breaking, ring cleavage, substituent loss, and the loss of neutral small molecules such as H2O, CO, and C2H4, as well as the removal of free radicals such as CH3· (Chen Shaoying, Lan Wei. Analysis of parsley components based on UPLC-QE-Orbitrap-MS technology [J]. Chinese Journal of Hospital Pharmacy, 2025, 45(04): 410-7.). Taking artemisinin as an example, its quasi-molecular ion peak can be seen in positive ion mode. m / z 283.1534[M+H] + The chromatographic retention time was 20.70 min, suggesting its molecular formula is C. 15 H 22 O5. It appears in its secondary mass spectrometry. m / z 265.1433 [M+H–H2O] + 247.1326 [M+H–2H2O] + And 237.1478 [M+H–HCOOH] + Characteristic fragment ions indicate that the fragmentation process is mainly accompanied by dehydration and loss of neutral small molecules. The possible mass spectrometric fragmentation pattern is shown in... Figure 3 This is basically consistent with the literature report (Wang Yueyue. Pharmacokinetic study of Artemisia annua antimalarial pill in rats [J]. Dalian University of Technology, 2018.).

[0035] 2.3 Identification of organic acid compounds in Artemisia annua pollen

[0036] Organic acids are an important class of water-soluble active components in Artemisia annua pollen. Containing a carboxyl group, they exhibit both acidity and polarity, encompassing common subclasses such as quinic acids and phenolic acids. They typically possess a variety of pharmacological activities, including antioxidant, anti-inflammatory, hepatoprotective, cardiovascular protective, and antibacterial effects. Twelve organic acids were identified in Artemisia annua pollen. For example, ferulic acid exhibits a quasi-molecular ion peak in positive ion mode. m / z 195.0653 [M+H] + The chromatographic retention time was 16.88 min, suggesting its molecular formula is C. 10 H 10 O4 appears in the secondary mass spectrum. m / z 177.0546 [M+H–H2O] + 149.0597 [M+H–HCOOH] + 163.0389 [M+H–CH3OH] + ,145.0286 [M+H–CH3OH–H2O] + Characteristic fragment ions, and their possible mass spectrometric fragmentation patterns are shown in Figure 4 This is basically consistent with the literature report (Hu Hanwen, Zhao Yongyan, Yang Tianlong, Zheng Zhenxing, Peng Teng, Deng Fang. Chemical composition analysis of Citrus medica based on UPLC-Q-Orbitrap HRMS [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2020, 26(07): 148-55.). Taking chlorogenic acid as an example, its quasi-molecular ion peak can be seen in negative ion mode. m / z 353.0876 [M–H] - The chromatographic retention time was 7.30 min, suggesting its molecular formula is C. 16 H 18 O9. Appears in the secondary mass spectrum. m / z 191.0546 [M–H–C9H6O3] - ,173.0437 [M–H–C9H6O3–H2O] - 179.0334 [M–H–C7H 10 O5] - 135.0448 [M–H–C7H 10 O5–CO2] - Characteristic fragment ions, and their possible mass spectrometric fragmentation patterns are shown in Figure 5 This is basically consistent with the literature report (Su Rina, Luo Weizao, Wei Rongrui, Ao Wuliji, Zhong Guoyue. Rapid identification of chemical components of Tibetan medicine, plateau nettle, based on UPLC-ESI-Q-TOF-MS / MS technology [J]. Chinese Journal of Traditional Chinese Medicine, 2019, 44(08): 1607-14.).

[0037] 2.4 Identification of amino acid compounds in Artemisia annua pollen

[0038] Amino acids are the basic building blocks of proteins and an important nutrient in Artemisia annua pollen. They play a role in regulating metabolism, enhancing immunity, and maintaining physiological stability. Their chemical structure is characterized by the presence of both amino and carboxyl groups in the molecule, with the amino and carboxyl groups attached to the same carbon atom. The side chain groups determine the types and properties of amino acids. Eight amino acid compounds were identified from Artemisia annua pollen. The cleavage of amino acid compounds mainly includes the loss of amino groups, decarboxylation of carboxyl groups, breakage of side chain groups, and loss of neutral small molecules such as H2O, CO2, and NH3 (Cui Weiheng, Deng Xiaolan, Wang Siqin. Rapid identification of chemical components of Isatis indigotica by high-resolution mass spectrometry using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap [J]. Chemical Research, 2023, 34(04): 313-8.). Taking arginine as an example, its quasi-molecular ion peak can be seen in positive ion mode. m / z 175.1190 [M+H] + The chromatographic retention time was 0.72 min, suggesting its molecular formula is C6H. 14 N4O2. Appears in the secondary mass spectrum. m / z 158.0925 [M+H–NH3] + 130.0977 [M+H–NH3–CO] + ,116.0711 [M+H–NH3–CH2N2] + ,70.0664 [M+H–NH3–CH2N2–HCOOH] + 60.0571 [M+H–C5H9NO2] + Characteristic fragment ions, and their possible mass spectrometric fragmentation patterns are shown in Figure 6 This is basically consistent with the literature report (Yu Quanlin, Song Jiajia, Li Hanbing, Li Minfeng, Tan Shizu, Jiang Qiaoping, Wang Tianshu, Cai Chengke, Wang Hongfei. Study on chemical composition of extract of *Vibrio wiltii* based on UPLC-Q-Exactive-MS and multiple technologies [J]. Drug Evaluation Research, 2024, 47(06):1285-94.).

[0039] 2.5 Identification of nucleoside compounds in Artemisia annua pollen

[0040] Nucleoside compounds are compounds formed by the linkage of bases (purines or pyrimidines) with ribose or deoxyribose through glycosidic bonds. In vivo, they can be further phosphorylated to form nucleotides, which are the basic units that make up nucleic acids (DNA, RNA) (RESEARCH DE. Establishment of LC detection method for 16 nucleosides and nucleotides and study on mass spectrometry fragmentation rules [J]. Chinese Journal of Modern Applied Pharmacy, 2022, 39(20): 2614-21.). Five nucleoside compounds were identified from Artemisia annua pollen. The fragmentation modes of nucleoside compounds mainly include glycosidic bond breakage, base removal, ribose ring cleavage, and loss of neutral small molecules such as H2O and CO. Taking guanosine as an example, its quasi-molecular ion peak can be seen in negative ion mode. m / z 282.0843 [M–H] - The chromatographic retention time was 1.59 min, suggesting its molecular formula is C. 10 H 13 N5O5. Appears in the secondary mass spectrum. m / z 150.0399 [M–H–C5H8O4] - , 133.0132 [M–H–C5H8O4–NH3] - Characteristic fragment ions, and their possible mass spectrometric fragmentation patterns are shown in Figure 7 This is basically consistent with the literature report (Hu Jing, Xu Jianqin, Chen Zhiyong, Ren Hui, Zhang Xiaofeng. Chemical composition analysis of Tangniaokang capsules based on UHPLC-Q-Orbitrap HRMS technology [J]. Central South Pharmacy, 2023, 21(07):1800-8.).

[0041] 2.6 Identification of Phenylpropanoid Compounds in Artemisia annua Pollen

[0042] Phenylpropanoids are natural products derived from phenylalanine or tyrosine, with a core structure containing a phenylpropane unit, encompassing subclasses such as lignans and coumarins (Ye Xinyuan, Wu Jianming, Yang Jie, FAHSAI K, WARUNEE K, Zeng Jing. Research progress on the chemical constituents and mass spectrometry fragmentation characteristics of representative components of Panax notoginseng [J]. Chinese Traditional and Herbal Drugs, 2021, 52(21): 6687-700.). Eight phenylpropanoids were identified from Artemisia annua pollen. The fragmentation modes of phenylpropanoids mainly include glycosidic bond breakage, ring skeleton cleavage, ester bond hydrolysis, and benzene ring substituent removal, accompanied by the loss of neutral fragments such as H2O, CH3, and CO. Taking scopolamine as an example, its quasi-molecular ion peak can be seen in negative ion mode. m / z 191.0335 [M–H] - The chromatographic retention time was 10.91 min, suggesting its molecular formula is C. 10H8O4 appears in the secondary mass spectrum. m / z 176.0097[M–H–CH3] - ,148.0144 [M–H–CH3–CO] - , 120.0197 [M–H–CH3–CO–CO] - ,104.0242 [M–H–CH3–CO2–CO] - Characteristic fragment ions, and their possible mass spectrometric fragmentation patterns are shown in Figure 8 This is basically consistent with the literature report (Wang Weihai, Yan Pingzhen, Yang Bin. Study on the fragmentation law of coumarin compounds in Notopterygium incisum by UPLC-Q-TOF mass spectrometry [J]. Chinese Journal of Traditional Chinese Medicine, 2021, 46(05): 1179-90.).

[0043] 3. Conclusion

[0044] This invention employs UHPLC-Q / Orbitrap HRMS technology in both positive and negative ion modes to systematically analyze and identify the chemical components in Artemisia annua pollen. By comprehensively analyzing the precise mass numbers, isotope peak distributions, and secondary mass spectrometry fragmentation characteristics of the compounds, combined with database comparison, literature search, and fragment ion analysis, a total of 150 compounds were identified, covering seven major categories: flavonoids, organic acids, terpenes, phenylpropanoids, nucleosides, amino acids, and other compounds. For each category, the mass spectrometric fragmentation characteristics of representative compounds were summarized, and their possible fragmentation patterns were analyzed. Among them, terpenoids are numerous and exhibit strong mass spectrometric responses, making them an important category of components worthy of attention in Artemisia annua pollen; flavonoids and phenylpropanoids also showed good mass spectrometric responses. This invention provides fundamental data for further research on the quality evaluation, sensitization-related aspects, and resource development and utilization of Artemisia annua pollen, and also provides a referable technical route for the rapid analysis and identification of chemical components in medicinal pollen samples.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol, characterized in that: Includes the following steps: (1) Sample preparation: Collect Artemisia annua pollen aerosol, store it in a sealed container at low temperature, dry and pulverize it, and then extract it to obtain the test solution; (2) Perform chromatographic and mass spectrometric analysis on the test solution; (3) Data processing: including database establishment, data collection and processing, relevant literature search, selection of representative compounds and deduction of fragmentation rules.

2. The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol according to claim 1, characterized in that: The chromatographic conditions in step (2) are as follows: The chromatographic column was equipped with an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); mobile phase: A was 0.1% formic acid aqueous solution, B was acetonitrile; flow rate was 0.3 mL / min; column temperature was 40 °C; injection volume was 6.0 µL; gradient elution program: 0–1 min, 2% B; 1–14 min, 2%–30% B; 14–25 min, 30%–100% B; 25–28 min, 100% B; 28–28.1 min, 100%–2% B; 28.1–29.5 min, 2% B.

3. The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol according to claim 2, characterized in that: The mass spectrometry conditions in step (2) are as follows: Analysis was performed using a Q Exactive quadrupole / electrostatic field orbital trap high-resolution mass spectrometer equipped with an electrospray ionization source. The spray voltages in positive and negative ion modes were 3.7 kV and 3.5 kV, respectively; the capillary temperature was 320 °C; the sheath gas pressure was 30 psi, the auxiliary gas pressure was 10 psi, and the auxiliary heating temperature was 300 °C; both the sheath gas and auxiliary gas were nitrogen; the collision gas was nitrogen at a pressure of 1.5 mTorr; and Full MS / dd-MS was used. 2 Data acquisition mode, Full MS parameters: resolution 70000, automatic gain control (AGC) target value 1 × 10⁻⁶. 6 The maximum injection time is 50 ms, and the scanning range is... m / z 100~1500; dd-MS 2 Parameters: Resolution 17500, AGC target value 1 × 10 5 The maximum injection time is 50 ms, a maximum of 10 ions can be selected for secondary fragmentation ion scanning, dynamic exclusion is enabled, and the isolation window is 2.

0. m / z The normalized collision energy (NCE) is set to 10, 30, and 60, and the intensity threshold is set to 1 × 10⁻⁶. 5 .

4. The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol according to claim 1, characterized in that: The data processing procedure in step (3) is as follows: 1) Establish a database: Search online databases such as PubMed, Web of Science, CNKI, MassBank and related literature for the names, molecular formulas, precise relative molecular masses, adduct ion types, characteristic fragment ion information and compound categories of possible chemical components in Artemisia annua pollen, and use this as a self-built database of chemical components in Artemisia annua pollen; 2) Data acquisition was performed using Xcalibur 4.1 software to comprehensively record the primary and secondary mass spectrometry information of the compounds; 3) The acquired raw mass spectrometry data were processed using Progenesis QI 3.0 software, including raw data import, peak extraction, and deconvolution. High-resolution mass spectrometry parameters were set as follows: mass accuracy of 5 ppm, retention time window of 0.1–0.3 min, and peak intensity threshold of 3 times the background noise for peak extraction; retention time deviation ≤ 0.05 min and mass-to-charge ratio deviation ≤ 5 ppm for deconvolution. The preprocessed data were compared with the TCM Pro 2.0 reference database and a self-built database. Compound identification was performed based on the precise molecular weight, retention time, isotope distribution, and fragment ion information, with a mass error ≤ 5 ppm and at least two characteristic fragment ions matched in the secondary mass spectrometry. 4) Relevant literature search, selection of representative compounds, and deduction of fragmentation patterns: Based on the above automatic identification, a combined search is conducted in PubMed, Web of Science, and CNKI databases according to the precise molecular weight, molecular formula, characteristic fragment ions, and retention time range of the compounds. "Compound name + plant source" or "molecular formula + characteristic fragments" are prioritized as keywords. One to three references are located by comparing the measured secondary mass spectra with the fragmentation patterns in the literature. High-intensity, well-resolved chromatographic peaks are selected as the main components in the total ion chromatogram. Representative compounds are selected, and the main fragment ions in their secondary mass spectra are manually analyzed. Possible fragmentation pathways are deduced, including the location of chemical bond breakage, rearrangement reactions, neutral loss, and the generation mechanism of characteristic fragment ions. By systematically summarizing the fragmentation patterns of this type of compound, the database comparison results can be effectively verified or supplemented, improving the reliability of structural identification.

5. The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol according to claim 1, characterized in that: The sample processing procedure in step (1) is as follows: Airborne Artemisia annua pollen aerosols were collected using a combination of natural sedimentation and an impactor sampler. The collected aerosols were immediately sealed and stored at low temperature. The frozen Artemisia annua pollen was dried, pulverized, sieved, and thoroughly mixed. The powder was then mixed with methanol and water, and extracted using an ultrasonic-microwave synthetic extractor. The extract was cooled, weighed, and any lost mass was replenished. After thorough mixing, the supernatant was collected by centrifugation. An equal volume of the solution was mixed with water and placed in a sample vial for analysis.

6. The method for detecting chemical components and constructing a characteristic database in Artemisia annua pollen aerosol according to claim 5, characterized in that: Sample processing specifically includes the following steps: Airborne Artemisia annua pollen aerosols were collected using a combination of natural sedimentation and an impactor sampler. The collected aerosols were immediately sealed and stored at -20°C. The frozen pollen was dried, pulverized, and passed through a 40-mesh sieve. 1.0 g of the sample powder was accurately weighed and placed in a three-necked flask. 40 mL of 80% methanol aqueous solution was added, and ultrasonic / microwave-assisted extraction was performed for 45 min. The extraction temperature was set at 45°C, the ultrasonic power was adjusted to 340 W, and the microwave power was adjusted to 300 W. After cooling, the extract was weighed, and the lost mass was replenished with 80% methanol aqueous solution. The mixture was then centrifuged at 14000 r / min for 10 min at 10°C. 100 μL of the supernatant was collected, added to 100 μL of ultrapure water, mixed, and then placed in a sample vial for analysis.

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