Method for analyzing chemical components of euphorbia macrocarpa, highland barley wine product and dairy product based on UPLC-Q-TOF-MS / MS technology

Abstract

The invention belongs to the technical field of chemical component analysis, and provides a method for analyzing chemical components of euphorbia pekinensis, a highland barley wine product thereof and a milk product based on a UPLC-Q-TOF-MSMS (Ultra Performance Liquid Chromatography-Quadrature Time of Flight Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass Spectrometry-Mass obtaining a test solution; (2) carrying out detection analysis on the test sample solution by adopting a UPLC-Q-TOF-MS / MS technology; the mobile phase A is 0.1% formic acid water, and the mobile phase B is a methanol solution; and (3) analyzing data acquired by mass spectrometry. The invention provides an important theoretical basis for quality control and clinical application of euphorbia macrocarpa and modernization improvement of a euphorbia macrocarpa processing technology.

Description

Technical Field

[0001] This invention belongs to the field of chemical composition analysis technology, and provides a method for analyzing the chemical components of Euphorbia macrocarpa, its barley wine products and dairy products based on UPLC-Q-TOF-MS / MS technology. Background Technology

[0002] Euphorbia wallichii is a perennial herbaceous plant distributed in the Himalayas, belonging to the genus Euphorbia in the family Euphorbiaceae, and possesses a certain degree of toxicity. Modern pharmacological studies have revealed that the active components in Euphorbia wallichii (such as ent-rosine and crotonine diterpenes) have significant antioxidant, anti-inflammatory, antiviral, and vascular permeability-regulating effects. Processing is a key means of reducing its toxicity and altering its medicinal properties; different processing methods can significantly affect the chemical composition of traditional Chinese medicine, thereby changing its efficacy and toxicity. Special processing methods such as barley wine processing and milk processing have unique significance in the application of Euphorbia wallichii processing in the field of ethnic medicine. However, current research on the changes in the chemical composition of processed Euphorbia wallichii is relatively scarce, and the mechanism of toxicity reduction after processing is still unclear. Therefore, studying and comparing the differences in the chemical composition of Euphorbia wallichii and its processed products is of great significance for elucidating its toxicity reduction mechanism.

[0003] The literature "Study on the Changes of Volatile Components in Euphorbia macrocarpa after Dairy and Wine Processing" (Cai Ying et al., *China Pharmacy*, 2025, Vol. 36, No. 21) discloses the use of headspace gas chromatography-mass spectrometry (HOC-GC-MS) to separate and identify volatile components in raw, dairy-processed, and wine-processed Euphorbia macrocarpa. A total of 66 volatile components were identified from the three samples, mainly alkanes, alkenes, heterocyclic compounds, and esters. Specifically, 39, 24, and 36 volatile components were identified in raw, dairy-processed, and wine-processed Euphorbia macrocarpa, respectively, totaling 10 components. However, the number of chemical components identified in the above literature is relatively small and insufficient for a more comprehensive understanding of Euphorbia macrocarpa.

[0004] Ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS / MS) has become a powerful tool for the analysis of chemical components in traditional Chinese medicine (TCM) due to its high resolution, high sensitivity, and accurate qualitative capabilities. It is widely used in the identification and differential analysis of components in various TCMs and processed products. Therefore, this invention employs UPLC-Q-TOF-MS / MS technology, combined with multivariate statistical analysis methods, to analyze the chemical components of *Euphorbia pekinensis* and its barley wine and dairy products. The aim is to clarify the differences in chemical components among different processed products, providing a scientific basis for the quality control, pharmacodynamic material basis research, and rational clinical application of processed *Euphorbia pekinensis* products. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a method for analyzing the chemical components of Euphorbia macrocarpa, its barley wine products, and dairy products based on UPLC-Q-TOF-MSMS technology, providing an important theoretical basis for the quality control, clinical application, and modernization of the processing technology of Euphorbia macrocarpa.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for analyzing the chemical components of Euphorbia macrocarpa, its barley wine products, and dairy products based on UPLC-Q-TOF-MS / MS technology is provided, including the following steps:

[0008] (1) Preparation of test solution: Take the powders of Euphorbia macrocarpa, Euphorbia macrocarpa barley wine products and Euphorbia macrocarpa dairy products respectively, extract their active ingredients with methanol solution, the material-to-liquid ratio is 0.2g:5ml, centrifuge and take the supernatant, filter through microporous membrane to obtain the test solution;

[0009] (2) The test sample solution was analyzed using UPLC-Q-TOF-MS / MS technology. The detection conditions included:

[0010] Chromatographic conditions: A Shimadzu LC-40D chromatograph was used with a C18 column. The column dimensions were 1.7 μm and 2.1 × 100 mm. Mobile phase A was 0.1% formic acid in water, and mobile phase B was methanol solution. The gradient elution program was as follows: 0–1 min, 5% B; 1–3 min, 5% B–30% B; 3–13 min, 30% B–65% B; 13–16 min, 65% B–95% B; 16–18 min, 95% B; 18–18.1 min, 95% B–5% B; 18.1–20 min, 5% B; flow rate: 0.25 mL / min. -1 Column temperature 40 ℃; injection volume 2 μL; all concentrations are volume percentages.

[0011] Mass spectrometry conditions: ESI ion source, positive / negative ion mode scanning; positive ion mode capillary voltage 5500 V, ion source temperature 500 ℃, declustering voltage 60 eV, collision energy 35±15 eV; negative ion mode capillary voltage -4500 V, ion source temperature 550 ℃, declustering voltage -60 eV, collision energy -35±15 eV; nebulizer gas: nitrogen; auxiliary gas 1 and auxiliary gas 2: 50 psi; curtain gas: 35 psi; primary mass spectrometry precursor ion scanning range: m / z 50-1500; IDA set to scan the 15 highest peaks with response values ​​exceeding 100 cps for secondary mass spectrometry; secondary mass spectrometry daughter ion scanning range: m / z 50~1500; dynamic background subtraction enabled. (3) Analyze the data collected by mass spectrometry to identify the components of Euphorbia macrocarpa, Euphorbia macrocarpa barley wine products and Euphorbia macrocarpa dairy products.

[0012] In this invention, preferably, the Euphorbia macrocarpa barley wine product is obtained by the following method: raw Euphorbia macrocarpa is crushed and placed in barley rice wine, with the mass ratio of raw Euphorbia macrocarpa to barley rice wine being 1:6. The mixture is then soaked and boiled until the wine is nearly dry, removed, washed, and dried to obtain the Euphorbia macrocarpa barley wine product.

[0013] In this invention, preferably, the Euphorbia macrocarpa dairy product is obtained by the following method: raw Euphorbia macrocarpa is crushed and placed in fresh milk, with the mass ratio of raw Euphorbia macrocarpa to fresh milk being 1:6. The mixture is then boiled until the milk is nearly dry, removed, washed, and dried to obtain the Euphorbia macrocarpa dairy product.

[0014] In this invention, preferably, the volume concentration of the methanol solution in step (1) is 50%.

[0015] In this invention, preferably, the extraction in step (1) is performed by ultrasonic extraction, with a temperature of 40°C, a power of 150W, a frequency of 40kHz, and an extraction time of 40min.

[0016] In this invention, preferably, the centrifugation in step (1) is performed at a rotation speed of 12,000 r·min. −1 Centrifuge for 10 min.

[0017] In this invention, preferably, the pore size of the microporous filter membrane in step (2) is 0.22 μm.

[0018] In this invention, preferably, the specific method for analysis and identification in step (3) is as follows:

[0019] First, data on compounds related to *Euphorbia macrocarpa* were obtained through searches of PubChem, PubMed, TCMSP, and the China Traditional Chinese Medicine Database, along with domestic and international literature reports. The English and Chinese names, molecular formulas, relative molecular weights, and secondary fragment information of these compounds were input into the software to establish a chemical composition database. Second, positive and negative ion chromatogram data acquired by mass spectrometry were imported into MSDIAL software for peak extraction and peak matching data processing. Finally, based on the established database, chemical components were compared according to their retention time and secondary ion fragments. Components with secondary ion fragments in the *Euphorbia macrocarpa* ion chromatogram that matched the database and had a score greater than 0.7 were selected. The final components were then obtained by manually comparing the secondary fragments using the PubChem database, thus identifying the chemical components of *Euphorbia macrocarpa*, *Euphorbia macrocarpa* barley wine products, and *Euphorbia macrocarpa* dairy products.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects:

[0021] This invention utilizes UPLC-Q-TOF-MS to conduct preliminary chemical component activity analysis on *Euphorbia pekinensis*, its barley wine products, and dairy products. By optimizing the extraction, chromatographic, and mass spectrometric conditions for the active ingredients, a highly efficient method for separating multiple active ingredients was established under positive and negative ion modes. Through activity matching with a database, the chemically active components of the samples were qualitatively analyzed, identifying over 191 compounds; approximately 110 compounds were detected in pressure-processed *Polygonatum sibiricum*; approximately 90 compounds were detected in nine-times-processed *Polygonatum sibiricum*; 86 common components were identified; and 31 differentially identified components were found. This method allows for a more comprehensive identification and characterization of the chemical components in *Euphorbia pekinensis*, its barley wine products, and dairy products, providing data support for further research on the pharmacokinetics and pharmacodynamic material basis of this drug, laying a theoretical foundation for clinical application, and providing a theoretical basis for improving the identification of the pharmacodynamic material basis and quality control standards of processed *Euphorbia pekinensis*. Attached Figure Description

[0022] Figure 1 This is a positive ion chromatogram of Euphorbia macrocarpa;

[0023] Figure 2 This is a positive ion chromatogram of Euphorbia pekinensis barley wine products;

[0024] Figure 3 This is a positive ion chromatogram of Euphorbia macrocarpa dairy products;

[0025] Figure 4 This is a negative ion flow graph of Euphorbia macrocarpa;

[0026] Figure 5 This is a negative ion flow graph of Euphorbia pekinensis barley wine products;

[0027] Figure 6 This is a negative ion flow graph of Euphorbia pekinensis dairy products. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] I. Instruments and Materials

[0030] 1.1 Instruments: Chromatographic column (2.1×100 mm, 1.7 μm, Phenomenex); Ultra-high performance liquid chromatography system (LC-40D X3, Shimadzu Nexera Series); High resolution mass spectrometer (ZenoTOF 7600 System, AB Sciex); 0.001 g electronic balance (BSA224S, Sartorius, Germany); Automatic double-purified water distiller (SZ-93, Shanghai Yarong Biochemical Instrument Factory); Ultrapure water purification system (Milli-Q Synthesis, Millipore, USA); High-speed centrifuge (TGL-16B, Shanghai Anting Scientific Instrument Factory); CNC ultrasonic cleaner (KQ-3200DE, Kunshan Ultrasonic Instrument Co., Ltd.).

[0031] 1.2 Materials: Euphorbia macrocarpa was collected from Mozhugongga County, Lhasa, Tibet; Euphorbia macrocarpa barley wine products (Tibet University of Tibetan Medicine); Euphorbia macrocarpa dairy products (Tibet University of Tibetan Medicine); methanol (Sigma-Aldrich, mass spectrometry grade); formic acid (Maclean, chromatographic grade).

[0032] In an embodiment of the present invention, the Euphorbia macrocarpa barley wine product is obtained by the following method: raw Euphorbia macrocarpa is crushed and placed in barley rice wine, with the mass ratio of raw Euphorbia macrocarpa to barley rice wine being 1:6. The mixture is then soaked and boiled until the wine is nearly dry, removed, washed, and dried to obtain the Euphorbia macrocarpa barley wine product.

[0033] In an embodiment of the present invention, the Euphorbia macrocarpa dairy product is obtained by the following method: raw Euphorbia macrocarpa is crushed and placed in fresh milk at a mass ratio of 1:6. The mixture is then boiled until the milk is nearly dry, removed, washed, and dried to obtain the Euphorbia macrocarpa dairy product.

[0034] II. Implementation Examples

[0035] A method for analyzing the chemical components of Euphorbia macrocarpa, its barley wine products, and dairy products based on UPLC-Q-TOF-MS / MS technology includes the following steps:

[0036] (1) Preparation of test solution: Take powders of Euphorbia macrocarpa, Euphorbia macrocarpa barley wine products, and Euphorbia macrocarpa dairy products respectively, and extract their active ingredients with a 50% (v / v) methanol solution. The material-to-liquid ratio during extraction is 0.2 g: 5 ml. Ultrasonic extraction is used at a temperature of 40 °C, a power of 150 W, a frequency of 40 kHz, and an extraction time of 40 min at a rotation speed of 12000 r·min. −1 Centrifuge for 10 min, collect the supernatant, and filter it through a 0.22 μm microporous membrane to obtain the test solution;

[0037] (2) The test sample solution was analyzed using UPLC-Q-TOF-MS / MS technology. The detection conditions included:

[0038] Chromatographic conditions: A Shimadzu LC-40D chromatograph was used with a C18 column. The column dimensions were 1.7 μm and 2.1 × 100 mm. Mobile phase A was 0.1% formic acid in water, and mobile phase B was methanol solution. The gradient elution program was as follows: 0–1 min, 5% B; 1–3 min, 5% B–30% B; 3–13 min, 30% B–65% B; 13–16 min, 65% B–95% B; 16–18 min, 95% B; 18–18.1 min, 95% B–5% B; 18.1–20 min, 5% B; flow rate: 0.25 mL / min. -1 Column temperature 40 ℃; injection volume 2 μL; all concentrations are volume percentages.

[0039] Mass spectrometry conditions: ESI ion source, positive / negative ion mode scanning, positive ion mode capillary voltage 5500V, ion source temperature 500℃, declustering voltage 60 eV, collision energy 35±15 eV; negative ion mode capillary voltage -4500 V, ion source temperature 550℃, declustering voltage -60 eV, collision energy -35±15 eV; nebulizing gas is nitrogen, auxiliary gas 1 and auxiliary gas 2 are both 50 psi, curtain gas is 35 psi; primary mass spectrometry parent ion scanning range m / z 50-1500, IDA is set to 15 peaks with response values ​​exceeding 100cps for secondary mass spectrometry scanning, secondary mass spectrometry daughter ion scanning range m / z 50~1500, dynamic background subtraction is enabled; (3) Data analysis: the specific analysis and identification methods described in step (3) are:

[0040] First, data on compounds related to *Euphorbia macrocarpa* were obtained through searches of PubChem, PubMed, TCMSP, and the China Traditional Chinese Medicine Database, along with domestic and international literature reports. The English and Chinese names, molecular formulas, relative molecular weights, and secondary fragment information of these compounds were input into the software to establish a chemical composition database. Second, positive and negative ion chromatogram data acquired by mass spectrometry were imported into MSDIAL software for peak extraction and peak matching data processing. Finally, based on the established database, chemical components were compared according to their retention time and secondary ion fragments. Components with secondary ion fragments in the *Euphorbia macrocarpa* ion chromatogram that matched the database and had a score greater than 0.7 were selected. The final components were then obtained by manually comparing the secondary fragments using the PubChem database, thus identifying the chemical components of *Euphorbia macrocarpa*, *Euphorbia macrocarpa* barley wine products, and *Euphorbia macrocarpa* dairy products.

[0041] As a result, this invention identified a total of 191 compounds from *Euphorbia macrocarpa* and its processed products (see Table 1). Of these, 121 were identified from raw *Euphorbia macrocarpa*, 132 from barley wine products, and 129 from dairy products, totaling 69 compounds. These included various chemical components such as sesquiterpenes, diterpenes, flavonoids, triterpenes, alkaloids, terpenes, coumarins, and organic acids. The total ion chromatogram is shown in [Table 1]. Figure 1-6 The ion chromatograms of the QC samples showed stable responses to the major chromatographic peaks and good reproducibility of retention times, indicating that the instrument system's stability meets the analytical requirements.

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] This invention, through analysis and identification, reveals differences in the chemical composition of Euphorbia macrocarpa and its processed products, providing a scientific basis for the material basis and research of the chemical composition of Euphorbia macrocarpa before and after processing, and further revealing the important influence of the processing process on the transformation of chemical components.

[0052] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for analyzing the chemical components of Euphorbia macrocarpa, its barley wine products, and dairy products based on UPLC-Q-TOF-MS / MS technology, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the powders of Euphorbia macrocarpa, Euphorbia macrocarpa barley wine products and Euphorbia macrocarpa dairy products respectively, extract their active ingredients with methanol solution, the material-to-liquid ratio is 0.2g:5ml, centrifuge and take the supernatant, filter through microporous membrane to obtain the test solution; (2) The test sample solution was analyzed using UPLC-Q-TOF-MS / MS technology. The detection conditions included: Chromatographic conditions: A Shimadzu LC-40D chromatograph was used with a C18 column. The column dimensions were 1.7 μm and 2.1 × 100 mm. Mobile phase A was 0.1% formic acid in water, and mobile phase B was methanol solution. The gradient elution program was as follows: 0–1 min, 5% B; 1–3 min, 5% B–30% B; 3–13 min, 30% B–65% B; 13–16 min, 65% B–95% B; 16–18 min, 95% B; 18–18.1 min, 95% B–5% B; 18.1–20 min, 5% B; flow rate: 0.25 mL / min. -1 Column temperature 40 ℃; injection volume 2 μL; all concentrations are volume percentages. Mass spectrometry conditions: ESI ion source, positive / negative ion mode scanning; positive ion mode capillary voltage 5500 V, ion source temperature 500 ℃, declustering voltage 60 eV, collision energy 35±15 eV; negative ion mode capillary voltage -4500 V, ion source temperature 550 ℃, declustering voltage -60 eV, collision energy -35±15 eV; nebulizer gas is nitrogen, auxiliary gas 1 and auxiliary gas 2 are both 50 psi, curtain gas is 35 psi; primary mass spectrometry precursor ion scanning range m / z 50-1500; IDA is set to scan the 15 highest peaks with response values ​​exceeding 100 cps for secondary mass spectrometry; secondary mass spectrometry daughter ion scanning range m / z 50~1500; dynamic background subtraction is enabled. (3) Analyze the data collected by mass spectrometry to identify the components of Euphorbia macrocarpa, Euphorbia macrocarpa barley wine products and Euphorbia macrocarpa dairy products.

2. The method according to claim 1, characterized in that, The Euphorbia macrocarpa barley wine product is obtained by the following method: raw Euphorbia macrocarpa is crushed and placed in barley rice wine. The mass ratio of raw Euphorbia macrocarpa to barley rice wine is 1:

6. The wine is soaked and boiled until the liquid is almost dry. The wine is then removed, washed, and dried to obtain the Euphorbia macrocarpa barley wine product.

3. The method according to claim 1, characterized in that: The Euphorbia macrocarpa dairy products are obtained by the following method: raw Euphorbia macrocarpa is crushed and placed in fresh milk at a mass ratio of 1:

6. The mixture is then boiled until the milk is almost dry, removed, washed, and dried to obtain the Euphorbia macrocarpa dairy products.

4. The method according to claim 1, characterized in that: The volume concentration of the methanol solution in step (1) is 50%.

5. The method according to claim 1, characterized in that: The extraction method described in step (1) is ultrasonic extraction, with a temperature of 40℃, a power of 150W, a frequency of 40kHz, and an extraction time of 40min.

6. The method according to claim 1, characterized in that: The centrifugation described in step (1) is performed at a rotation speed of 12,000 r·min. −1 Centrifuge for 10 min.

7. The method according to claim 1, characterized in that: The pore size of the microporous filter membrane in step (2) is 0.22 μm.

8. The method according to claim 1, characterized in that: The specific analytical and identification method described in step (3) is as follows: First, data on compounds related to *Euphorbia macrocarpa* were obtained through searches of PubChem, PubMed, TCMSP, and the China Traditional Chinese Medicine Database, along with domestic and international literature reports. The English and Chinese names, molecular formulas, relative molecular weights, and secondary fragment information of these compounds were input into the software to establish a chemical composition database. Second, positive and negative ion chromatogram data acquired by mass spectrometry were imported into MSDIAL software for peak extraction and peak matching data processing. Finally, based on the established database, chemical components were compared according to their retention time and secondary ion fragments. Components with secondary ion fragments in the *Euphorbia macrocarpa* ion chromatogram that matched the database and had a score greater than 0.7 were selected. The final components were then obtained by manually comparing the secondary fragments using the PubChem database, thus identifying the chemical components of *Euphorbia macrocarpa*, *Euphorbia macrocarpa* barley wine products, and *Euphorbia macrocarpa* dairy products.

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