A method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS
Patent Information
- Application Number
- CN202611061872.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-16
- Publication Date
- 2026-09-01
AI Technical Summary
[0003]然而，现行《贵州省中药材、民族药材质量标准》（2019年版）中仅收录了抓地虎的性状与鉴别项，相关系统性研究文献尚属空白
1.本研究建立了基于UHPLC-MS/MS技术同时测定抓地虎中对羟基苯甲醛、绿原酸、芹菜素等18种成分含量的分析方法，并对13批次抓地虎样品进行了含量测定。研究中优化了色谱与质谱条件，采用Waters ACQUITY UPLC BEH C18色谱柱，以0.1%甲酸乙腈-0.1%甲酸水为流动相进行梯度洗脱，加热电喷雾电离源（H-ESI）正模式扫描，选择反应监测（SRM）模式检测。方法学验证结果显示，18种成分在各自线性范围内线性关系良好，专属性良好，精密度、重复性、稳定性和加样回收率的RSD均小于3%，表明该方法可靠、准确，满足于抓地虎的多成分定量分析要求。
Smart Images
Figure CN122671584A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug content determination technology, specifically relating to a method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS. Background Technology
[0002] Sorghum vlgare Pers., a plant of the Poaceae family, is the root and rhizome of the sorghum plant. It is a medicinal herb used by ethnic minorities in Guizhou Province, possessing properties of clearing heat and detoxifying, cooling the blood and stopping bleeding. It is used for conditions such as bone-steaming fever, cough with hemoptysis, burns, and scalds, and valuable clinical experience has been accumulated in its application. As a local specialty medicinal resource, Sorghum vlgare has significant development and utilization value.
[0003] However, the current "Quality Standards for Traditional Chinese and Ethnic Medicinal Materials of Guizhou Province" (2019 edition) only includes the characteristics and identification of *Gnaphalium affine*, and there is a lack of relevant systematic research literature. Existing quality control methods are unable to comprehensively and objectively reflect the integrity and complexity of its internal components, which seriously restricts the scientific establishment of quality standards for this medicinal material and the in-depth development of its subsequent products.
[0004] Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) combines ultra-high separation efficiency, high sensitivity, and strong specificity, making it a key technique for the simultaneous quantification of multiple components in complex systems of traditional Chinese medicine (TCM). This technique is particularly suitable for TCM samples with complex chemical compositions, unclear active ingredients, or low content, enabling efficient and accurate determination of various active ingredients or biomarkers, thus providing reliable technical support for the systematic evaluation of TCM quality.
[0005] Therefore, this patent proposes a multi-component UHPLC-MS / MS quantitative analysis method for *Gnaphalium affine* medicinal materials. The establishment and application of this method is expected to fill the gap in modern quality control technology for *Gnaphalium affine*, providing key technical support for improving its quality standards and developing high-value-added products, and will also have a positive impact on promoting the protection and industrialization of Guizhou's distinctive ethnic medicinal resources. Summary of the Invention
[0006] The purpose of this invention is to provide a method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS. This method can simultaneously detect the content of 18 components, including p-hydroxybenzaldehyde, chlorogenic acid, and apigenin. It is reliable and accurate, meeting the requirements for quantitative analysis of multiple components in *Gnaphalium affine*.
[0007] This invention is achieved using the following technical solution: a method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS, which is carried out according to the following steps: (1) Preparation of standard stock solutions: Accurately weigh p-hydroxybenzaldehyde standard, 4-methoxysalicylaldehyde standard, isovanillin standard, p-coumaric acid standard, p-hydroxycinnamic acid standard, methyl ferulic acid standard, genistein standard, apigenin standard, naringenin standard, luteolin standard, chlorogenic acid standard, apigenin-7-O-β-D-glucoside standard, isovitexin standard, luteolin standard, shamfotaside standard, isovitexin standard, kaempferol-3-O-rutin glycoside standard and vitexin glucoside standard, dissolve them in methanol and make up to volume to obtain 18 standard stock solutions with a mass concentration of 0.5-1.5 mg / mL; (2) Preparation of mixed standard solution: Accurately pipette the 18 standard stock solutions in step (1) and dilute them to 40-60 μg / mL intermediate stock solutions. Then accurately pipette the above 18 intermediate stock solutions and dilute them to volume with 50% methanol to obtain the mixed standard solution. (3) Preparation of test solution: Accurately weigh 0.3-0.7g of *Gnaphalium affine* and add 40-60mL of 50% methanol solution. After weighing, reflux extract at 70-90℃ for 1-2h. Cool to room temperature, add weight, mix well, and centrifuge at 12000-16000r / min at 3-5℃ for 5-15min. Take the supernatant to obtain the test solution. (4) Chromatographic and mass spectrometric conditions: Chromatographic conditions: Column: The chromatographic column specifications were 2.1 mm × 100 mm, 1.7 µm; the guard column was a Waters Van Guard BEH C18 (2.1 mm × 5 mm, 1.7 µm); the column temperature was 40 °C; the injection volume was 5 µL; mobile phase A was 0.1% formic acid acetonitrile, and mobile phase D was 0.1% formic acid water; the gradient elution program was as follows: ;
[0008] Mass spectrometry conditions: Heated electrospray ionization source (H-ESI) was used in positive mode. Ion spray voltage: 3500 V; Sheath Gas: 20 Arb; Aux Gas: 5 Arb; Sweep Gas: 10 Arb; Ion Transfer Tube Temp: 325°C; Vaporizer Temp: 275°C; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CIDGas: 2 mTorr; Selected reaction monitoring (SRM) was used for scanning; Xcalibur workstation was used for mass spectrometry data acquisition and processing. The mass spectrometry conditions for the 18 components are as follows:
[0009] (5) Standard curve plotting: Take the mixed standard solution and inject it according to the chromatographic and mass spectrometric conditions in step (4). Plot the standard curve with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of each component. (6) Content detection: Take the test solution and inject it according to the chromatographic and mass spectrometry conditions in step (4). Calculate the content of 18 components in the test solution according to the regression equation of the standard curve in step (5).
[0010] In step (1) above, the preparation of the mixed standard solution involves accurately weighing the following standards: p-hydroxybenzaldehyde standard, 4-methoxysalicylaldehyde standard, isovanillin standard, p-coumaric acid standard, p-hydroxycinnamic acid standard, methyl ferulic acid standard, genistein standard, apigenin standard, naringenin standard, luteolin standard, chlorogenic acid standard, apigenin-7-O-β-D-glucoside standard, isovitexin standard, luteolinoside standard, and shamotene standard. Standards for isosorbide dimethylbenzyl, kaempferol-3-O-rutin, and vitexin glucoside were dissolved in methanol and diluted to volume to obtain stock solutions of p-hydroxybenzaldehyde (1.054 mg / mL), 4-methoxysalicylaldehyde (1.089 mg / mL), isovanillin (1.042 mg / mL), p-coumaric acid (0.931 mg / mL), and 1.121 mg / mL... p-Hydroxycinnamic acid standard stock solution, methyl ferulic acid standard stock solution (1.022 mg / mL), genistein standard stock solution (1.015 mg / mL), apigenin standard stock solution (0.662 mg / mL), naringenin standard stock solution (1.004 mg / mL), luteolin standard stock solution (1.054 mg / mL), chlorogenic acid standard stock solution (1.022 mg / mL), apigenin-7 standard stock solution (0.835 mg / mL). O-β-D-glucoside standard stock solution, 0.967 mg / mL isovitexin standard stock solution, 1.031 mg / mL luteolin standard stock solution, 1.005 mg / mL shampodoside standard stock solution, 0.952 mg / mL isovitexin standard stock solution, 0.975 mg / mL kaempferol-3-O-rutin standard stock solution, and 0.965 mg / mL vitexin glucoside standard stock solution.
[0011] In step (2) above, the preparation of the mixed standard solution is as follows: Accurately pipette the 18 standard stock solutions from step (1) and dilute them to 40-60 μg / mL as intermediate stock solutions. Then, accurately pipette 2300-2500 μL of p-hydroxybenzaldehyde intermediate stock solution, 190-210 μL of 4-methoxysalicylaldehyde intermediate stock solution, 70-90 μL of isovanillin intermediate stock solution, 900-1100 μL of p-coumaric acid intermediate stock solution, 900-1100 μL of p-hydroxycinnamic acid intermediate stock solution, 19-21 μL of methyl ferulic acid intermediate stock solution, 190-210 μL of genistein intermediate stock solution, and 190-210 μL of apigenin intermediate stock solution. Transfer 1700-1800 μL of naringenin intermediate stock solution, 35-45 μL of luteolin intermediate stock solution, 90-110 μL of chlorogenic acid intermediate stock solution, 70-90 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 70-90 μL of isovitexin intermediate stock solution, 70-90 μL of luteolin intermediate stock solution, 790-810 μL of succinyl benzoate intermediate stock solution, 35-45 μL of isovitexin intermediate stock solution, 2-4 μL of kaempferol-3-O-rutin intermediate stock solution, and 2-4 μL of vitexin glucoside intermediate stock solution into a 10 mL volumetric flask, dilute to volume with 50% methanol, and obtain the mixed standard solution.
[0012] Specifically, in step (2) above, the preparation of the mixed standard solution involves accurately pipetting the 18 standard stock solutions from step (1) and diluting them to 50 μg / mL intermediate stock solutions. Then, accurately pipetting 2400 μL of p-hydroxybenzaldehyde intermediate stock solution, 200 μL of 4-methoxysalicylaldehyde intermediate stock solution, 80 μL of isovanillin intermediate stock solution, 1000 μL of p-coumaric acid intermediate stock solution, 1000 μL of p-hydroxycinnamic acid intermediate stock solution, 20 μL of methyl ferulic acid intermediate stock solution, 203 μL of genistein intermediate stock solution, and 200 μL of apigenin intermediate stock solution... 1781 μL of naringenin intermediate stock solution, 40 μL of luteolin intermediate stock solution, 102 μL of chlorogenic acid intermediate stock solution, 80 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 80 μL of isovitexin intermediate stock solution, 82 μL of luteolin intermediate stock solution, 799 μL of shampodoside intermediate stock solution, 40 μL of isovitexin intermediate stock solution, 3 μL of kaempferol-3-O-rutin intermediate stock solution, and 3 μL of vitexin glucoside intermediate stock solution were transferred into a 10 mL volumetric flask and diluted to volume with 50% methanol to obtain the mixed standard solution.
[0013] In step (3) above, the preparation of the test solution is as follows: accurately weigh 0.4-0.6g of *Gnaphalium affine* and add 45-55mL of 50% methanol solution. After weighing, reflux extract at 75-85℃ for 1.2-1.8h, cool to room temperature, add weight, mix well, centrifuge at 13000-15000r / min at 3-5℃ for 8-12min, and take the supernatant to obtain the test solution.
[0014] In step (4) above, the preparation of the test solution is as follows: accurately weigh 0.5g of *Gnaphalium affine* and add 50mL of 50% methanol solution. After weighing, reflux extract at 80℃ for 1.5h, cool to room temperature, add weight, mix well, centrifuge at 14000r / min at 4℃ for 10min, and take the supernatant to obtain the test solution.
[0015] Specifically, in the aforementioned step (6), the 18 components are p-hydroxybenzaldehyde, 4-methoxysalicylaldehyde, isovanillin, p-coumaric acid, p-hydroxycinnamic acid, methyl ferulic acid, genistein, apigenin, naringenin, luteolin, chlorogenic acid, apigenin-7-O-β-D-glucoside, isovitexin, luteolin, shampodoside, isovitexin, kaempferol-3-O-rutin, and vitexin glucoside.
[0016] Beneficial effects of this invention: 1. This study established an analytical method based on UHPLC-MS / MS technology for the simultaneous determination of 18 components, including p-hydroxybenzaldehyde, chlorogenic acid, and apigenin, in *Cephalotaxus fortunei*. The content of these components was determined in 13 batches of *Cephalotaxus fortunei* samples. The chromatographic and mass spectrometric conditions were optimized. A Waters ACQUITY UPLC BEH C18 column was used, with gradient elution of 0.1% formic acid-acetonitrile-0.1% formic acid-water as the mobile phase. Heated electrospray ionization (H-ESI) was used for positive mode scanning, and reaction monitoring (SRM) mode was selected for detection. Method validation results showed that the 18 components exhibited good linearity within their respective linear ranges, good specificity, and RSDs for precision, repeatability, stability, and recovery were all less than 3%, indicating that the method is reliable and accurate, meeting the requirements for multi-component quantitative analysis of *Cephalotaxus fortunei*.
[0017] 2. Among the 18 components determined in this invention, chlorogenic acid exhibits pharmacological activities such as antioxidant and anti-inflammatory effects; flavonoids such as luteolin, apigenin, and genistein possess antibacterial, antiviral, and immunomodulatory effects; and benzaldehydes such as p-hydroxybenzaldehyde and isovanthan have also been confirmed to have certain biological activities. The content determination results of these components not only reveal the pharmacological material basis of *Gnaphalium affine*, but also provide a chemical basis for its effectiveness in clinical application. Furthermore, the detection and quantification of flavonoid glycosides such as shampooside and isoshampooside enrich the research on the chemical composition of *Gnaphalium affine*, providing a reference for the formulation of quality standards and further development and utilization of *Gnaphalium affine*. Attached Figure Description
[0018] Figure 1 Extraction ion chromatograms of 18 components in a mixed reference solution (1: p-hydroxybenzaldehyde; 2: 4-methoxysalicylaldehyde; 3: isovanthan; 4: p-coumaric acid; 5: p-hydroxycinnamic acid; 6: methyl ferulic acid; 7: genistein; 8: apigenin; 9: naringenin; 10: luteolin; 11: chlorogenic acid; 12: apigenin-7-O-β-D-glucoside; 13: isovitexin; 14: luteolin; 15: shampooside; 16: isovitexin; 17: kaempferol-3-O-rutin; 18: vitexin glucoside). Figure 2 Extraction ion chromatograms of 18 components in the test solution (1: p-hydroxybenzaldehyde; 2: 4-methoxysalicylaldehyde; 3: isovanillin; 4: p-coumaric acid; 5: p-hydroxycinnamic acid; 6: methyl ferulic acid; 7: genistein; 8: apigenin; 9: naringenin; 10: luteolin; 11: chlorogenic acid; 12: apigenin-7-O-β-D-glucoside; 13: isovitexin; 14: luteolin; 15: shampodoside; 16: isovitexin; 17: kaempferol-3-O-rutin; 18: vitexin glucoside). Detailed Implementation
[0019] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.
[0020] Example 1: Chromatographic and Mass Spectrometric Conditions Chromatographic conditions: Column: The chromatographic column specifications were 2.1 mm × 100 mm, 1.7 µm; the guard column was a Waters Van Guard BEH C18 (2.1 mm × 5 mm, 1.7 µm); the column temperature was 40 °C; the injection volume was 5 µL; mobile phase A was 0.1% formic acid acetonitrile, and mobile phase D was 0.1% formic acid water; the gradient elution program was as follows: ;
[0021] Mass spectrometry conditions: Heated electrospray ionization source (H-ESI) was used in positive mode. Ion spray voltage: 3500 V; Sheath Gas: 20 Arb; Aux Gas: 5 Arb; Sweep Gas: 10 Arb; Ion Transfer Tube Temp: 325°C; Vaporizer Temp: 275°C; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CIDGas: 2 mTorr; Selected reaction monitoring (SRM) was used for scanning; Xcalibur workstation was used for mass spectrometry data acquisition and processing. The mass spectrometry conditions for the 18 components are as follows:
[0022] Example 2: Detection of multiple components in *Gnaphalium affine* (1) Preparation of standard stock solutions: Accurately weigh p-hydroxybenzaldehyde standard, 4-methoxysalicylaldehyde standard, isovanillin standard, p-coumaric acid standard, p-hydroxycinnamic acid standard, methyl ferulic acid standard, genistein standard, apigenin standard, naringenin standard, luteolin standard, chlorogenic acid standard, apigenin-7-O-β-D-glucoside standard, isovitexin standard, luteolin standard, shamfotaside standard, isovitexin standard, kaempferol-3-O-rutin glycoside standard and vitexin glucoside standard, dissolve them in methanol and make up to volume to obtain 18 standard stock solutions with a mass concentration of 1.0 mg / mL; (2) Preparation of mixed standard solutions: Accurately pipette the 18 standard stock solutions from step (1) and dilute them to 50 μg / mL intermediate stock solutions respectively. Then, accurately pipette 2400 μL of p-hydroxybenzaldehyde intermediate stock solution, 200 μL of 4-methoxysalicylaldehyde intermediate stock solution, 800 μL of isovanillin intermediate stock solution, 1000 μL of p-coumaric acid intermediate stock solution, 1000 μL of p-hydroxycinnamic acid intermediate stock solution, 20 μL of methyl ferulic acid intermediate stock solution, 200 μL of genistein intermediate stock solution, 200 μL of apigenin intermediate stock solution, and naringenin intermediate stock solution. 1780 μL of intermediate stock solution, 40 μL of luteolin intermediate stock solution, 100 μL of chlorogenic acid intermediate stock solution, 80 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 80 μL of isovitexin intermediate stock solution, 80 μL of luteolin intermediate stock solution, 800 μL of succinyl benzoate intermediate stock solution, 40 μL of isovitexin intermediate stock solution, 3 μL of kaempferol-3-O-rutin intermediate stock solution, and 3 μL of vitexin glucoside intermediate stock solution were transferred into a 10 mL volumetric flask and diluted to volume with 50% methanol to obtain the mixed standard solution. (3) Preparation of test solution: Accurately weigh 0.5g of *Gnaphalium affine* and add 50mL of 50% methanol solution. After weighing, reflux extract at 80℃ for 1.5h, cool to room temperature, add weight, mix well, centrifuge at 14000r / min at 4℃ for 10min, and take the supernatant to obtain the test solution. (4) Standard curve plotting: Take the mixed standard solution and inject it according to the chromatographic and mass spectrometric conditions in Example 1. Plot the standard curve with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of each component. (5) Content detection: Take the test solution and inject it according to the chromatographic and mass spectrometric conditions in Example 1. Calculate the content of 18 components in the test solution according to the regression equation of the standard curve in step (4).
[0023] Example 3: Detection of multiple components of *Gnaphalium affine* (1) Preparation of standard stock solutions: Accurately weigh p-hydroxybenzaldehyde standard, 4-methoxysalicylaldehyde standard, isovanillin standard, p-coumaric acid standard, p-hydroxycinnamic acid standard, methyl ferulic acid standard, genistein standard, apigenin standard, naringenin standard, luteolin standard, chlorogenic acid standard, apigenin-7-O-β-D-glucoside standard, isovitexin standard, luteolin standard, shamfotaside standard, isovitexin standard, kaempferol-3-O-rutin glycoside standard and vitexin glucoside standard, dissolve them in methanol and make up to volume to obtain 18 standard stock solutions with a mass concentration of 0.5 mg / mL; (2) Preparation of mixed standard solutions: Accurately pipette the 18 standard stock solutions from step (1) and dilute them to 40 μg / mL intermediate stock solutions. Then, accurately pipette 2300 μL of p-hydroxybenzaldehyde intermediate stock solution, 190 μL of 4-methoxysalicylaldehyde intermediate stock solution, 90 μL of isovanillin intermediate stock solution, 900 μL of p-coumaric acid intermediate stock solution, 1100 μL of p-hydroxycinnamic acid intermediate stock solution, 19 μL of methyl ferulic acid intermediate stock solution, 190 μL of genistein intermediate stock solution, 210 μL of apigenin intermediate stock solution, and 10 μL of naringenin intermediate stock solution. 1700 μL of intermediate stock solution, 35 μL of luteolin intermediate stock solution, 110 μL of chlorogenic acid intermediate stock solution, 90 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 70 μL of isovitexin intermediate stock solution, 70 μL of luteolin intermediate stock solution, 810 μL of succinyl benzoate intermediate stock solution, 45 μL of isovitexin intermediate stock solution, 4 μL of kaempferol-3-O-rutin intermediate stock solution, and 4 μL of vitexin glucoside intermediate stock solution were transferred into a 10 mL volumetric flask and diluted to volume with 50% methanol to obtain the mixed standard solution. (3) Preparation of test solution: Accurately weigh 0.7g of *Gnaphalium affine* and add 60mL of 50% methanol solution. After weighing, reflux extract at 70℃ for 2h, cool to room temperature, add weight, mix well, centrifuge at 16000r / min for 5min at 5℃, and take the supernatant to obtain the test solution. (5) Standard curve plotting: Take the mixed standard solution and inject it according to the chromatographic and mass spectrometric conditions in Example 1. Plot the standard curve with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of each component. (6) Content detection: Take the test solution and inject it according to the chromatographic and mass spectrometric conditions in Example 1. Calculate the content of 18 components in the test solution according to the regression equation of the standard curve in step (4).
[0024] Example 4: Detection of multiple components in *Gnaphalium affine* (1) Preparation of standard stock solutions: Accurately weigh p-hydroxybenzaldehyde standard, 4-methoxysalicylaldehyde standard, isovanillin standard, p-coumaric acid standard, p-hydroxycinnamic acid standard, methyl ferulic acid standard, genistein standard, apigenin standard, naringenin standard, luteolin standard, chlorogenic acid standard, apigenin-7-O-β-D-glucoside standard, isovitexin standard, luteolin standard, shamfotaside standard, isovitexin standard, kaempferol-3-O-rutin glycoside standard and vitexin glucoside standard, dissolve them in methanol and make up to volume to obtain 18 standard stock solutions with a mass concentration of 1.5 mg / mL; (2) Preparation of mixed standard solutions: Accurately pipette the 18 standard stock solutions from step (1) and dilute them to 60 μg / mL intermediate stock solutions respectively. Then accurately pipette 2500 μL of p-hydroxybenzaldehyde intermediate stock solution, 210 μL of 4-methoxysalicylaldehyde intermediate stock solution, 70 μL of isovanillin intermediate stock solution, 1100 μL of p-coumaric acid intermediate stock solution, 900 μL of p-hydroxycinnamic acid intermediate stock solution, 21 μL of methyl ferulic acid intermediate stock solution, 210 μL of genistein intermediate stock solution, 190 μL of apigenin intermediate stock solution, and 100 μL of naringenin intermediate stock solution. 1800 μL of intermediate stock solution, 45 μL of luteolin intermediate stock solution, 90 μL of chlorogenic acid intermediate stock solution, 70 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 90 μL of isovitexin intermediate stock solution, 90 μL of luteolin intermediate stock solution, 790 μL of succinyl benzoate intermediate stock solution, 45 μL of isovitexin intermediate stock solution, 2 μL of kaempferol-3-O-rutin glycoside intermediate stock solution, and 2 μL of vitexin glucoside intermediate stock solution were transferred into a 10 mL volumetric flask and diluted to volume with 50% methanol to obtain the mixed standard solution. (3) Preparation of test solution: Accurately weigh 0.3g of *Gnaphalium affine* and add 40mL of 50% methanol solution. After weighing, reflux extract for 1h in a 90℃ water bath. Cool to room temperature, add weight, mix well, centrifuge at 12000r / min for 15min at 3℃, and take the supernatant to obtain the test solution. (4) Standard curve plotting: Take the mixed standard solution and inject it according to the chromatographic and mass spectrometric conditions in Example 1. Plot the standard curve with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of each component. (5) Content detection: Take the test solution and inject it according to the chromatographic and mass spectrometric conditions in Example 1. Calculate the content of 18 components in the test solution according to the regression equation of the standard curve in step (4).
[0025] To obtain the solution and verify the technical effects of this invention, the inventors conducted extensive experimental research, some of which are recorded below: 1. Materials 1.1 Instruments and Equipment Ultimate 3000 ultra-high performance liquid chromatograph, TSQ ENDURA triple quadrupole mass spectrometer (Thermo Fisher Scientific); Allegra 64R low-temperature high-speed centrifuge (Beckman Coulter, USA); VX-III multi-tube vortex mixer (Tengjin (Beijing) Pharmaceutical Technology Co., Ltd.); EL204 0.001 g electronic balance (Mettler-Toledo Instruments Ltd.); 0.001 g balance (AE-240, Mettler-Toledo Instruments Ltd.); micropipette (Eppendorf AG, Germany).
[0026] 1.2 Medicinal Materials and Reagents 1.2.1 Medicinal Material: Sorghum vlgare Pers. is the root and rhizome of the grass plant Sorghum vlgare Pers. 13 batches of Sorghum vlgare Pers. are numbered S1 to S13. They were identified as genuine products by Researcher Liu Chunhua of Guizhou Provincial Key Laboratory of Pharmaceutical Preparations. Detailed information is shown in Table 1. 1.2.2 Reagents: p-Hydroxybenzaldehyde, isovanillin, 4-methoxysalicylaldehyde, methyl ferulic acid, genistein, isoxavorin, p-coumaric acid, p-hydroxycinnamic acid, and luteolin were purchased from Chengdu Efa Biotechnology Co., Ltd. (batch numbers: AFCC0754, AFEE1454, AFDF0551, AFCE0852, AFDA0354, AFEC0661, AFDE2451, AFCA3052, AFBL0755, all with a purity ≥98%); naringenin, isovitexin, xavorin, and vitexin glucose were also used. Glucosides, apigenin, chlorogenic acid, apigenin-7-O-β-D-glucoside, luteolin, and kaempferol-3-O-rutin were purchased from Sichuan Weikeqi Biotechnology Co., Ltd. (batch numbers: WP24111204, WP25011303, WP24092309, WP2111107, WP24052109, WP24012303, WP24060306, WP24093002, WP24080702, all with a purity ≥98%); mass spectrometry grade methanol and acetonitrile were purchased from Merck GmbH, Germany.
[0027] Table 1. Origin and Batch Number of 13 Batches of Groundhog Guinea Pig
[0028] 2. Methods and Results 2.1 Preparation of mixed standard solutions Accurately weigh the following standards: p-hydroxybenzaldehyde, 4-methoxysalicylaldehyde, isovanillin, p-coumaric acid, p-hydroxycinnamic acid, methyl ferulic acid, genistein, apigenin, naringenin, luteolin, chlorogenic acid, apigenin-7-O-β-D-glucoside, isovitexin, luteolin, shampooside, isoshampooside, and kaempferol. Phenol-3-O-rutin glycoside and vitexin glucoside standards were used to prepare stock solutions of p-hydroxybenzaldehyde (1.054 mg / mL), 4-methoxysalicylaldehyde (1.089 mg / mL), isenhanyl aldehyde (1.042 mg / mL), p-coumaric acid (0.931 mg / mL), and p-hydroxycinnamic acid (1.121 mg / mL), respectively. 0.022 mg / mL methyl ferulic acid standard stock solution, 1.015 mg / mL genistein standard stock solution, 0.662 mg / mL apigenin standard stock solution, 1.004 mg / mL naringenin standard stock solution, 1.054 mg / mL luteolin standard stock solution, 1.022 mg / mL chlorogenic acid standard stock solution, 0.835 mg / mL apigenin-7-O-β-D-glucan Stock solutions of glucosinolate standards, 0.967 mg / mL of isovitexin standard stock solution, 1.031 mg / mL of luteolin standard stock solution, 1.005 mg / mL of shampodoside standard stock solution, 0.952 mg / mL of isovitexin standard stock solution, 0.975 mg / mL of kaempferol-3-O-rutin standard stock solution and 0.965 mg / mL of vitexin glucoside standard stock solution; Accurately pipette the above-mentioned standard stock solutions and dilute them to an intermediate stock solution of approximately 50 μg / mL. Then, accurately pipette each intermediate stock solution (2400 μL of p-hydroxybenzaldehyde intermediate stock solution, 200 μL of 4-methoxysalicylaldehyde intermediate stock solution, 80 μL of isovanillin intermediate stock solution, 1000 μL of p-coumaric acid intermediate stock solution, 1000 μL of p-hydroxycinnamic acid intermediate stock solution, 20 μL of methyl ferulic acid intermediate stock solution, 203 μL of genistein intermediate stock solution, 200 μL of apigenin intermediate stock solution, and 1781 μL of naringenin intermediate stock solution). The following intermediate stock solutions were transferred into a 10 mL volumetric flask: 40 μL of luteolin intermediate stock solution, 102 μL of chlorogenic acid intermediate stock solution, 80 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 80 μL of isovitexin intermediate stock solution, 82 μL of luteolin intermediate stock solution, 799 μL of succinyl benzoate intermediate stock solution, 40 μL of isovitexin intermediate stock solution, 3 μL of kaempferol-3-O-rutin intermediate stock solution, and 3 μL of vitexin glucoside intermediate stock solution. The solutions were then diluted to volume with 50% methanol to obtain a mixed standard solution for linearity studies.
[0029] 2.2 Preparation of the test solution Accurately weigh 0.5g of *Gnaphalium affine* into a 100mL round-bottom flask, add 50mL of 50% methanol (v / v), weigh the sample, reflux extract at 80℃ for 1.5h, cool to room temperature, add weight, mix well, centrifuge at 14000r / min for 10min at 4℃, and inject the supernatant for analysis.
[0030] 2.3 Chromatographic and Mass Spectrometry Conditions Chromatographic conditions: Column: (2.1 mm × 100 mm, 1.7 µm) post; Protective post: Waters Van Guard BEH C 18 (2.1 mm × 5 mm, 1.7 µm); column temperature 40°C; injection volume: 5 µL; mobile phase: 0.1% formic acid acetonitrile (A) - 0.1% formic acid water (D); gradient elution (see Table 2).
[0031] Table 2 Gradient elution conditions
[0032] Mass spectrometry conditions: Heated electrospray ionization source (H-ESI) was used, positive mode scanning, ion spray voltage: 3500 V, Sheath Gas: 20 Arb; Aux Gas: 5 Arb; Sweep Gas: 10 Arb; Ion Transfer Tube Temp: 325°C; Vaporizer Temp: 275°C; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CID Gas: 2 mTorr; The scanning method was selected reaction monitoring (SRM), and the mass spectrometry data acquisition and processing software was the Xcalibur workstation. The monitoring ions used for the quantitative analysis of 18 components, including p-hydroxybenzaldehyde, are shown in Table 3.
[0033] Table 3 Mass spectrometry conditions for 18 components including p-hydroxybenzaldehyde
[0034] 2.4 Validation of Analytical Methods 2.4.1 Specificity Accurately pipette 5 μL of the mixed reference standard and the *Gnaphalium affine* sample solution, and inject them for analysis according to the chromatographic and mass spectrometric conditions described in section "2.3". Record the chromatograms. Results are shown below. Figure 1 and Figure 2 The chromatograms of the test solution and the mixed reference standard showed the same chromatographic peaks at the corresponding positions, and the separation of each component was good, indicating that the method has good specificity and meets the requirements of quantitative analysis.
[0035] 2.4.2 Linearity Precisely pipette the series of mixed reference solutions at the concentrations specified in section "2.1", and analyze them according to the chromatographic and mass spectrometric conditions specified in section "2.3". Plot a standard curve with concentration (x) on the x-axis and peak area (y) on the y-axis to obtain the regression equations for each component. The results are shown in Table 4. The 18 components, including p-hydroxybenzaldehyde, showed good linearity within their respective concentration ranges, with R... 2 ≥0.993.
[0036] Table 4. Results of linearity studies for 18 components including p-hydroxybenzaldehyde
[0037] 2.4.3 Precision Test Take *Gnaphalium affine* (S1), prepare the test solution according to the method in section "2.2", and continuously inject and determine it 6 times under the chromatographic and mass spectrometric conditions in section "2.3". Record the peak areas of 18 components, including p-hydroxybenzaldehyde, and calculate the RSD value of the peak area of each component to evaluate the precision of the instrument. The results show (see Table 5) that the RSD of the peak areas of p-hydroxybenzaldehyde, 4-methoxysalicylaldehyde, etc., is < 3%, indicating that the instrument precision is good.
[0038] Table 5 Precision test results (n = 6)
[0039] 2.4.4 Repeatability Test Six aliquots of the same *Gnaphalium affine* sample were taken, and test solutions were prepared according to the method described in section "2.2". The samples were then analyzed under the chromatographic and mass spectrometric conditions described in section "2.3". The peak areas of 18 components, including p-hydroxybenzaldehyde, were recorded. The content and RSD values of each component were calculated based on the regression equation obtained from the linear relationship study to evaluate the repeatability of the method. The results (see Table 6) show that the RSDs of p-hydroxybenzaldehyde and other components are <3%, indicating good repeatability of this method.
[0040] Table 6 Results of repeatability testing (n = 6)
[0041] 2.4.5 Stability Test The test solution under section "2.4.4" was tested at 0, 4, 6, 12, and 24 hours. The peak areas of 18 components, including p-hydroxybenzaldehyde, were recorded, and the RSD values of the peak areas of each component were calculated to evaluate the stability of the test solution. The results showed (see Table 7) that the RSD of the peak areas of the 18 components, including p-hydroxybenzaldehyde, was <3%, indicating that the test solution had good stability within 24 hours.
[0042] Table 7. Stability test results (n = 5)
[0043] 2.4.6 Recovery rate Six portions of *Gnaphalium affine* sample (S1) with known content were accurately weighed, each approximately 0.25 g. A certain amount of each reference solution was added to each portion, and samples were prepared according to the method described in section "2.2". The samples were then analyzed under the chromatographic and mass spectrometric conditions described in section "2.3". The peak areas of 18 components, including p-hydroxybenzaldehyde, were recorded. The recovery rate and RSD of each component were calculated based on the regression equation obtained from the linear relationship study to evaluate the recovery rate of the method. The results (see Table 8) showed that the average recovery rate of the 18 components, including p-hydroxybenzaldehyde, was 96.25%–102.29%, with an RSD <3%, indicating that the recovery rate of each component using this method was good.
[0044] Table 8 Results of the recovery rate study (n = 6)
[0045]
[0046]
[0047] 2.5 Determination of the content of multiple components Each batch of *Gnaphalium affine* samples was collected, and test solutions were prepared according to the method described in section "2.2". The samples were then analyzed under the chromatographic and mass spectrometric conditions described in section "2.3". The contents of 18 components, including p-hydroxybenzaldehyde, in the samples were calculated based on the regression equation obtained from the linear relationship of the standard curve. The results of the content determination of the 18 components in 13 batches of *Gnaphalium affine* are shown in Tables 9 and 10.
[0048] Table 9. Results of content determination of 18 components in batches S1-S7 (n=3)
[0049] Table 10. Results of content determination of 18 components in batches S8-S13 (n = 3)
[0050] 3. Discussion and Conclusion This study established an analytical method for the simultaneous determination of 18 components, including p-hydroxybenzaldehyde, chlorogenic acid, and apigenin, in *Gnaphalium affine* based on UHPLC-MS / MS technology, and the contents of 13 batches of *Gnaphalium affine* samples were determined. The chromatographic and mass spectrometric conditions were optimized. A Waters ACQUITY UPLC BEH C18 column was used, with gradient elution using 0.1% formic acid acetonitrile-0.1% formic acid water as the mobile phase. Heated electrospray ionization (H-ESI) was used for positive mode scanning, and reaction monitoring (SRM) mode was selected for detection. Method validation results showed that the 18 components exhibited good linearity within their respective linear ranges, good specificity, and RSDs for precision, repeatability, stability, and recovery were all less than 3%, indicating that the method is reliable and accurate, meeting the requirements for multi-component quantitative analysis of *Gnaphalium affine*.
[0051] The content determination results of 13 batches of *Gnaphalium affine* samples showed significant differences in the content of each component among different batches. The contents of p-hydroxybenzaldehyde, p-coumaric acid, p-hydroxycinnamic acid, and schafodal were relatively high, all exceeding 30 μg / g, with some batches showing the highest p-hydroxybenzaldehyde content reaching 1148.69 μg / g. The contents of kaempferol-3-O-rutin and vitexin glucoside were relatively low. The most significant differences were found between different batches of genistein and apigenin, with contents ranging from 1.37 to 65.45 μg / g and 1.35 to 70.47 μg / g, respectively.
[0052] Significant differences in the component content of 13 batches of *Gnaphalium affine* samples were observed, which are presumably related to the following factors: First, differences in origin and growing environment. *Gnaphalium affine* refers to the roots and rhizomes of sorghum, and environmental factors such as soil fertility, sunlight, and rainfall may affect the biosynthesis of flavonoids and benzaldehydes. Second, harvesting time and storage conditions. The accumulation of active ingredients in sorghum roots and rhizomes varies at different harvesting periods, and changes in temperature and humidity during storage may lead to the degradation of some components. Third, differences in processing techniques. Steps such as cleaning and drying during sample pretreatment may affect the retention rate of components. Among these, the differences in flavonoid components such as genistein and apigenin were the most prominent, possibly related to the high sensitivity of these components to environmental factors.
[0053] Of the 18 components determined in this study, chlorogenic acid exhibits pharmacological activities such as antioxidant and anti-inflammatory effects; flavonoids such as luteolin, apigenin, and genistein possess antibacterial, antiviral, and immunomodulatory effects; and benzaldehydes such as p-hydroxybenzaldehyde and isovanthan are also confirmed to have certain biological activities. The content determination results of these components not only reveal the pharmacological material basis of *Gynostemma pentaphyllum*, but also provide chemical evidence for its effectiveness in clinical application. Furthermore, the detection and quantification of flavonoid glycosides such as shampooside and isoshampooside enrich the research on the chemical composition of *Gynostemma pentaphyllum*, providing a reference for the formulation of quality standards and further development and utilization of *Gynostemma pentaphyllum*.
Claims
1. A method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS, characterized in that: The following steps are to be followed: (1) Preparation of standard stock solutions: Accurately weigh p-hydroxybenzaldehyde standard, 4-methoxysalicylaldehyde standard, isovanillin standard, p-coumaric acid standard, p-hydroxycinnamic acid standard, methyl ferulic acid standard, genistein standard, apigenin standard, naringenin standard, luteolin standard, chlorogenic acid standard, apigenin-7-O-β-D-glucoside standard, isovitexin standard, luteolin standard, shamfotaside standard, isovitexin standard, kaempferol-3-O-rutin glycoside standard and vitexin glucoside standard, dissolve them in methanol and make up to volume to obtain 18 standard stock solutions with a mass concentration of 0.5-1.5 mg / mL; (2) Preparation of mixed standard solution: Accurately pipette the 18 standard stock solutions in step (1) and dilute them to 40-60 μg / mL intermediate stock solutions. Then accurately pipette the above 18 intermediate stock solutions and dilute them to volume with 50% methanol to obtain the mixed standard solution. (3) Preparation of test solution: Accurately weigh 0.3-0.7g of *Gnaphalium affine* and add 40-60mL of 50% methanol solution. After weighing, reflux extract at 70-90℃ for 1-2h. Cool to room temperature, add weight, mix well, and centrifuge at 12000-16000r / min at 3-5℃ for 5-15min. Take the supernatant to obtain the test solution. (4) Chromatographic and mass spectrometric conditions: Chromatographic conditions: Column: The chromatographic column specifications were 2.1 mm × 100 mm, 1.7 µm; the guard column was a Waters Van Guard BEH C18 (2.1 mm × 5 mm, 1.7 µm); the column temperature was 40 °C; the injection volume was 5 µL; mobile phase A was 0.1% formic acid acetonitrile, and mobile phase D was 0.1% formic acid water; the gradient elution program was as follows: ； Mass spectrometry conditions: Heated electrospray ionization source (H-ESI) was used in positive mode. Ion spray voltage: 3500 V; Sheath Gas: 20 Arb; Aux Gas: 5 Arb; Sweep Gas: 10 Arb; Ion Transfer Tube Temp: 325°C; Vaporizer Temp: 275°C; Auxiliary gas and sheath gas: N2; Q1 Resolution: 0.7; Q3 Resolution: 0.7; CID Gas: 2 mTorr; Selected reaction monitoring (SRM) was used for scanning; Xcalibur workstation was used for mass spectrometry data acquisition and processing. The mass spectrometry conditions for the 18 components are as follows: (5) Standard curve plotting: Take the mixed standard solution and inject it according to the chromatographic and mass spectrometric conditions in step (4). Plot the standard curve with concentration as the abscissa and peak area as the ordinate to obtain the regression equation of each component. (6) Content detection: Take the test solution and inject it according to the chromatographic and mass spectrometry conditions in step (4). Calculate the content of 18 components in the test solution according to the regression equation of the standard curve in step (5).
2. The method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS according to claim 1, characterized in that: In step (1), the preparation of the mixed standard solution involves accurately weighing the following standards: p-hydroxybenzaldehyde standard, 4-methoxysalicylaldehyde standard, isovanillin standard, p-coumaric acid standard, p-hydroxycinnamic acid standard, methyl ferulic acid standard, genistein standard, apigenin standard, naringenin standard, luteolin standard, chlorogenic acid standard, apigenin-7-O-β-D-glucoside standard, isovitexin standard, luteolin standard, and shampooside standard. Standards for isosorbide dimethylbenzyl, kaempferol-3-O-rutin, and vitexin glucoside were dissolved in methanol and diluted to volume to obtain stock solutions of p-hydroxybenzaldehyde (1.054 mg / mL), 4-methoxysalicylaldehyde (1.089 mg / mL), isovanillin (1.042 mg / mL), p-coumaric acid (0.931 mg / mL), and 1.121 mg / mL... p-Hydroxycinnamic acid standard stock solution, methyl ferulic acid standard stock solution (1.022 mg / mL), genistein standard stock solution (1.015 mg / mL), apigenin standard stock solution (0.662 mg / mL), naringenin standard stock solution (1.004 mg / mL), luteolin standard stock solution (1.054 mg / mL), chlorogenic acid standard stock solution (1.022 mg / mL), apigenin-7 standard stock solution (0.835 mg / mL). O-β-D-glucoside standard stock solution, 0.967 mg / mL isovitexin standard stock solution, 1.031 mg / mL luteolin standard stock solution, 1.005 mg / mL shampodoside standard stock solution, 0.952 mg / mL isovitexin standard stock solution, 0.975 mg / mL kaempferol-3-O-rutin standard stock solution, and 0.965 mg / mL vitexin glucoside standard stock solution.
3. The method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS according to claim 1, characterized in that: In step (2), the preparation of the mixed standard solution involves accurately pipetting the 18 standard stock solutions from step (1) and diluting them to 40-60 μg / mL as intermediate stock solutions. Then, accurately pipette 2300-2500 μL of p-hydroxybenzaldehyde intermediate stock solution, 190-210 μL of 4-methoxysalicylaldehyde intermediate stock solution, 70-90 μL of isovanillin intermediate stock solution, 900-1100 μL of p-coumaric acid intermediate stock solution, 900-1100 μL of p-hydroxycinnamic acid intermediate stock solution, 19-21 μL of methyl ferulic acid intermediate stock solution, 190-210 μL of genistein intermediate stock solution, and 190-210 μL of apigenin intermediate stock solution. Transfer 1700-1800 μL of naringenin intermediate stock solution, 35-45 μL of luteolin intermediate stock solution, 90-110 μL of chlorogenic acid intermediate stock solution, 70-90 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 70-90 μL of isovitexin intermediate stock solution, 70-90 μL of luteolin intermediate stock solution, 790-810 μL of succinyl benzoate intermediate stock solution, 35-45 μL of isovitexin intermediate stock solution, 2-4 μL of kaempferol-3-O-rutin intermediate stock solution, and 2-4 μL of vitexin glucoside intermediate stock solution into a 10 mL volumetric flask, dilute to volume with 50% methanol, and obtain the mixed standard solution.
4. The method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS according to claim 1, characterized in that: In step (2), the preparation of the mixed standard solution is as follows: Accurately pipette the 18 standard stock solutions from step (1) and dilute them to 50 μg / mL as intermediate stock solutions. Then, accurately pipette 2400 μL of p-hydroxybenzaldehyde intermediate stock solution, 200 μL of 4-methoxysalicylaldehyde intermediate stock solution, 80 μL of isovanillin intermediate stock solution, 1000 μL of p-coumaric acid intermediate stock solution, 1000 μL of p-hydroxycinnamic acid intermediate stock solution, 20 μL of methyl ferulic acid intermediate stock solution, 203 μL of genistein intermediate stock solution, 200 μL of apigenin intermediate stock solution, and... 1781 μL of quercetin intermediate stock solution, 40 μL of luteolin intermediate stock solution, 102 μL of chlorogenic acid intermediate stock solution, 80 μL of apigenin-7-O-β-D-glucoside intermediate stock solution, 80 μL of isovitexin intermediate stock solution, 82 μL of luteolin intermediate stock solution, 799 μL of shampodoside intermediate stock solution, 40 μL of isovitexin intermediate stock solution, 3 μL of kaempferol-3-O-rutin intermediate stock solution, and 3 μL of vitexin glucoside intermediate stock solution were transferred into a 10 mL volumetric flask and diluted to volume with 50% methanol to obtain the mixed standard solution.
5. The method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS according to claim 1, characterized in that: In step (3), the test solution is prepared as follows: accurately weigh 0.4-0.6g of *Gnaphalium affine* and add 45-55mL of 50% methanol solution. After weighing, reflux extract at 75-85℃ for 1.2-1.8h, cool to room temperature, add weight, mix well, centrifuge at 13000-15000r / min at 3-5℃ for 8-12min, and take the supernatant to obtain the test solution.
6. The method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS according to claim 5, characterized in that: In step (4), the test solution is prepared as follows: accurately weigh 0.5g of *Gnaphalium affine* and add 50mL of 50% methanol solution. After weighing, reflux extract at 80℃ for 1.5h, cool to room temperature, add weight, mix well, centrifuge at 14000r / min at 4℃ for 10min, and take the supernatant to obtain the test solution.
7. The method for determining the content of multiple components of *Gnaphalium affine* based on UHPLC-MS / MS according to claim 1, characterized in that: In step (6), the 18 components are p-hydroxybenzaldehyde, 4-methoxysalicylaldehyde, isovanillin, p-coumaric acid, p-hydroxycinnamic acid, methyl ferulic acid, genistein, apigenin, naringenin, luteolin, chlorogenic acid, apigenin-7-O-β-D-glucoside, isovitexin, luteolin, shamotexin, isovitexin, kaempferol-3-O-rutin, and vitexin glucoside.