Method for identifying volatile component substance difference before and after sand blanching processing of fructus xanthii based on GC-IMS

Abstract

The invention discloses a GC-IMS-based method for identifying volatile component substance differences before and after sand blanching processing of fructus xanthii, and belongs to the technical field of traditional Chinese medicine processing and quality control. The preparation method comprises the following steps: taking cocklebur fruit, removing impurities to obtain a crude product, putting river sand which is 8 times of the amount of the medicinal material into a pot, heating to a smooth state by medium fire, when the sand temperature is stabilized to 160 DEG C, putting the cocklebur fruit, frying for 7 minutes to obtain a sand hot product, and crushing by a crusher for later use. Setting parameters of a gas chromatography-ion mobility spectrometry instrument, feeding a sample, analyzing through software of the instrument, and screening out the specific volatile components of the crude siberian cocklour fruit product and the sand-burnt siberian cocklour fruit product. According to the processing method, the limitations of non-uniform heating and inconsistent processing degrees of a traditional processing method of a clear frying method are improved, and the limitation can be effectively improved by adopting a sand heating method, so that the purposes of toxicity reduction and effect storage of processing are achieved. The established gas chromatography-ion mobility spectrometry fingerprint spectrum can be used for quickly distinguishing crude siberian cocklour fruit products and sand-burnt siberian cocklour fruit products.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine processing and quality control, and particularly relates to a method for identifying the differences in volatile components of Xanthium strumarium before and after sand roasting based on GC-IMS. Background Art

[0002] Xanthium strumarium L. is the dried ripe fruit with involucre of the plant Xanthium strumarium in the Compositae family. It is pungent, bitter, and warm; and is poisonous. It belongs to the lung meridian and has the effects of dispersing wind-cold, dredging nasal orifices, and dispelling wind-dampness. It is mainly used for wind-cold headache, nasal congestion and runny nose, allergic rhinitis, nasal sinusitis, etc. However, Xanthium strumarium contains a certain amount of toxic components such as atractyloside and carboxyatractyloside. Direct use of the raw product may cause poisoning reactions. Therefore, it needs to be processed before clinical application to reduce toxicity and enhance efficacy.

[0003] According to the Chinese Pharmacopoeia 2020 Edition, the processing method of Xanthium strumarium is the stir-frying method. Through research, it is found that the sand roasting method is superior to the stir-frying method. By heating Xanthium strumarium together with river sand, the Xanthium strumarium can be heated evenly to achieve the processing purpose. The processing method adopted in the present invention is the sand roasting method. Compared with the stir-frying method, the sand-roasted Xanthium strumarium is easier to control the firing temperature, and the heating area is more uniform, and it is superior in terms of internal quality, appearance color, and the effect of reducing toxicity during processing. On the basis of retaining the original effects of dispersing wind-cold, dredging nasal orifices, and dispelling wind-dampness, the sand-roasted Xanthium strumarium can reduce toxic components and relieve irritation. As the degree of processing deepens, although the toxic components in Xanthium strumarium decrease, the active ingredients also decrease, and it is difficult to ensure the medicinal effect during clinical application. Therefore, appropriate processing is crucial. Traditional methods for quality control of traditional Chinese medicine mainly rely on empirical identification and some simple chemical analysis methods. Moreover, the color of Xanthium strumarium in the same batch is uneven, and the traditional visual inspection and smelling by nose are difficult to comprehensively and accurately reflect the complex changes of volatile components in the processed Xanthium strumarium, and it requires those with long-term work experience, and the results are accidental, which cannot meet the requirements of modern traditional Chinese medicine quality control for precision and scientificity.

[0004] The working principle of Gas Chromatography-Ion Mobility Spectrometry (GC-IMS) technology is to combine two techniques, Gas Chromatography (GC) and Ion Mobility Spectrometry (IMS), to achieve efficient separation and detection of volatile organic compounds (VOCs) in complex samples. Regarding the Gas Chromatography (GC) part, Sample injection: The sample is first injected into the gas chromatography system, usually introduced into the system through an injection port. Separation process: The sample enters the chromatographic column driven by the carrier gas (such as nitrogen, hydrogen or helium). The chromatographic column is usually coated with a stationary phase. Each component in the sample moves at different rates in the chromatographic column according to the difference in its interaction intensity with the stationary phase, thus achieving separation. The residence time of each component in the chromatographic column is unique, and this characteristic can be used to determine its identity. Detection: The separated components leave the chromatographic column in turn and enter the next detection stage.

[0005] For the Ion Mobility Spectrometry (IMS) part, Ionization: After the separated compounds enter the IMS system, they are first ionized into ions in the ion source. The ionization methods can be chemical ionization, electrospray ionization, etc. Migration process: The ions enter the drift region under the action of an electric field. The drift region is filled with an inert drift gas. The ions obtain energy in the electric field for directional drift and at the same time collide with the drift gas molecules and lose energy. Due to the different masses, charges, collision cross-sections and spatial configurations of different ions, their migration rates in the electric field are also different. Detection and analysis: The ions reach the detector according to their respective migration rates, and the detector records the time when the ions arrive (i.e., the migration time). By measuring the migration time of the ions, the ions can be separated and qualitatively analyzed. IMS has the characteristics of high sensitivity and fast response, and can detect compounds at trace levels.

[0006] Advantages of GC-IMS coupling, High resolution and low detection limit: GC provides good separation ability, while IMS has high sensitivity and low detection limit. The combination of the two can simultaneously achieve efficient separation and high-sensitivity detection of volatile organic compounds in complex samples. No need for vacuum: Different from techniques such as mass spectrometry that require vacuum conditions, GC-IMS can be detected at atmospheric pressure, reducing the complexity and operating cost of the equipment. Fast analysis: The measurement speed of IMS is very fast, and it can continuously record the signal peaks of analytes in a short time, suitable for fast analysis. In summary, GC-IMS technology provides an efficient and accurate method for the analysis of volatile organic compounds through the separation ability of gas chromatography and the high-sensitivity detection ability of ion mobility spectrometry, and is widely used in environmental monitoring, food safety, drug analysis and other fields.

[0007] During the processing, the volatile components of Xanthium sibiricum Patr. will change significantly. These components not only affect the medicinal efficacy of Xanthium sibiricum Patr., but are also closely related to its toxicity. Traditional quality evaluation methods for processing mainly rely on sensory judgment and quantitative analysis of some chemical components, and it is difficult to comprehensively and accurately reflect the differences in volatile components before and after the processing of Xanthium sibiricum Patr. Gas chromatography-ion mobility spectrometry (GC-IMS) is a newly emerging analytical technique that can detect volatile components in traditional Chinese medicine. It is simple and fast to operate, the sample pretreatment is simple and convenient, the amount of consumables in the treatment process is small, the method is environmentally friendly, and it has the advantages of high resolution, high sensitivity and no labeling. It can simultaneously detect and identify multiple volatile components in complex samples. This technique has been widely applied in the fields of food, environment, materials, etc., but its application in the field of traditional Chinese medicine processing and quality control is still in its infancy. This method uses gas chromatography-ion mobility spectrometry (GC-IMS) technology to identify volatile components with different processing degrees and monitor the changes in components after the processing of Xanthium sibiricum Patr., providing a new idea for the discrimination of volatile components before and after the processing of Xanthium sibiricum Patr. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for identifying the differences in volatile components of Xanthium sibiricum Patr. before and after sand roasting based on GC-IMS, so as to realize the rapid and accurate discrimination of the differences in volatile components between the raw product and the sand-roasted product of Xanthium sibiricum Patr., and provide a scientific basis for the optimization of the processing technology and quality control of Xanthium sibiricum Patr.

[0009] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] A method for identifying the differences in volatile components of Xanthium sibiricum Patr. before and after sand roasting based on GC-IMS includes the following steps:

[0011] (1) Sample treatment: Take Xanthium sibiricum Patr., remove impurities, which is the raw product; put 8 times the amount of river sand of the medicinal material into the pot, heat it over medium heat until it is in a smooth state, take the temperature at 1-1.2 cm from the bottom of the pot as the sand temperature, and when it is stable at 160 °C, put in Xanthium sibiricum Patr. and stir-fry for 7 minutes respectively to obtain the sand-roasted product, and crush it with a pulverizer;

[0012] (2) Sample detection: Incubate in a powder headspace vial and inject by automatic headspace injection; perform gas chromatography-ion mobility spectrometry (GC-IMS) detection on the Xanthium sibiricum Patr. sample to obtain detection data, specifically:

[0013] ① Instrument setting: Turn on the GC-IMS instrument, set parameters such as the injection port temperature, column oven temperature program, carrier gas flow rate of gas chromatography, and the drift tube temperature and drift gas flow rate parameters of the ion mobility spectrometry to ensure the stable operation of the instrument;

[0014] ② Sample injection: Inject the processed sample filtrate into the GC-IMS instrument through an automatic injector for analysis;

[0015] ③Data collection: The instrument automatically collects the gas chromatography-ion mobility spectrometry data of the samples, and records the chromatographic peaks and ion mobility spectrometry peaks of the volatile components in each sample;

[0016] (3) Data processing and discrimination, specifically:

[0017] ①Use the software supporting the instrument to classify the collected GC-IMS sample data to obtain the volatile components among different samples;

[0018] ②Perform PCA dimensionality reduction on the peak heights of all detected VOC signals to study the differences in volatile components;

[0019] ③Conduct qualitative analysis on the chromatograms of volatile components in the samples.

[0020] As a preferred technical solution of the present invention, in the sample detection step of step (2):

[0021] The incubation conditions are: temperature: 80°C; time: 15 min; injection volume: 100 μL; splitless injection; rotation speed: 500 r / min; injection needle temperature: 110°C;

[0022] The GC detection conditions are: column temperature: 60°C; carrier gas: high-purity nitrogen; programmed pressure increase: initial flow rate 2.0 mL / min for 2 min, linearly increased to 10.0 mL / min in 8 min, linearly increased to 100.0 mL / min in 10 min, and then maintained for 30 min to end, chromatographic run time: 50 min; injection port temperature: 80°C;

[0023] The IMS conditions are: ionization source: tritium source (3H); migration tube length: 53 mm; electric field strength: 500 V / cm; migration tube temperature: 45°C; drift gas: high-purity nitrogen (purity ≥ 99.999%); flow rate: 150.0 mL / min; positive and negative ion modes: positive ion; opening time: 100 μs, closing voltage: 90 dgt.

[0024] As a preferred technical solution of the present invention, in step (3), use the GC-IMS software to process the obtained chromatographic-mobility spectrometry diagrams, extract characteristic ion peaks, and establish the fingerprint chromatograms of the volatile components of raw Xanthium sibiricum and different processed products.

[0025] Compared with the prior art, the beneficial effects of the present invention are shown in:

[0026] 1. Fast and accurate: The GC-IMS technology has high resolution and high sensitivity, can simultaneously detect and identify multiple volatile components in Xanthium sibiricum, without complex sample pretreatment and labeling processes, greatly shortening the analysis time, and improving the discrimination efficiency and accuracy.

[0027] 2. Comprehensive analysis: Compared with traditional analysis methods, GC-IMS can more comprehensively reflect the changes in volatile components before and after the processing of Xanthii Fructus, providing richer data support for the optimization of the processing technology and quality control of Xanthii Fructus.

[0028] 3. Scientific basis: Through the method of the present invention, the change law of volatile components during the sand roasting process of Xanthii Fructus can be clarified, providing a scientific basis for optimizing the processing parameters (such as temperature, time, etc.) and formulating quality standards, which helps to improve the medicinal efficacy and safety of Xanthii Fructus. Description of the Drawings

[0029] Figure 1 Appearance of 6 batches of samples of Xanthii Fructus before and after sand roasting.

[0030] Figure 2 Three-dimensional GC-IMS spectrum of volatile components in the sample.

[0031] Figure 3 Two-dimensional GC-IMS spectrum of volatile components in the sample.

[0032] Figure 4 Fingerprint spectrum of volatile components in the sample.

[0033] Figure 5 PCA score plot of volatile components in the sample. Detailed Description of the Invention

[0034] The present invention will be further described in detail below in conjunction with the embodiments and the drawings.

[0035] Example 1

[0036] This example presents a sand roasting processing technology for Xanthii Fructus, including the following steps:

[0037] (1) Sample preparation

[0038] Raw Xanthii Fructus: Select 6 batches of Xanthii Fructus from different origins that meet the standards of the Chinese Pharmacopoeia ( Figure 1 as shown), and remove impurities.

[0039] Sand-roasted Xanthii Fructus: Put 8 times the amount of river sand of the medicinal material into the pot, heat it over medium heat until it is in a smooth state, take the temperature at about 1 cm from the bottom of the pot as the sand temperature, when it stabilizes at 160 °C, put in Xanthii Fructus and stir-fry for 7 minutes respectively, stir-fry until the surface is slightly yellow, take out and let it cool for later use.

[0040] (2) Sample pretreatment

[0041] Take the raw product and sand-roasted product of Xanthii Fructus. After drying, crush them into fine powder, and pass through a No. 3 sieve (50 mesh) after crushing with a pulverizer for later use.

[0042] Example 2

[0043] This example presents a method for identifying the differences in volatile components of Xanthii Fructus before and after stir-frying with sand based on GC-IMS, including the following steps:

[0044] (1) Use GC-IMS to detect the volatile components of raw Xanthii Fructus and Xanthii Fructus stir-fried with sand, and obtain the three-dimensional, two-dimensional, and differential spectral data of the GC-IMS samples, as shown in Figures 2 - 3 , specifically as follows:

[0045] ① Sample preparation

[0046] Accurately weigh 0.5 g of Xanthii Fructus sample into a 20 mL headspace vial, add 2-octanol as the internal standard, incubate at 80 °C for 15 min, and then inject the sample. The samples are divided into two categories, namely 6 batches of raw Xanthii Fructus from different origins (XF01-XF06) and the corresponding Xanthii Fructus stir-fried with sand (PXF07-PXF12), as shown in Table 1 below.

[0047] Table 1 Sample information table

[0048]

[0049] ② Headspace injection conditions

[0050] Incubation temperature: 80 °C; incubation time: 15 min; injection volume: 100 μL; splitless injection; incubation rotation speed: 500 r / min; injection needle temperature: 110 °C.

[0051] ③ GC conditions:

[0052] Column temperature: 60 °C; carrier gas: high-purity nitrogen (purity ≥ 99.999%); programmed pressure increase: initial flow rate 2.0 mL / min for 2 min, linearly increased to 10.0 mL / min in 8 min, linearly increased to 100.0 mL / min in 10 min, and then maintained for 30 min to end. Chromatographic running time: 50 min; injection port temperature: 80 °C; drift gas flow rate: 150.0 mL / min.

[0053] ④ IMS conditions

[0054] Ionization source: tritium source (3H); migration tube length: 53 mm; electric field strength: 500 V / cm; migration tube temperature: 45 °C; drift gas: high-purity nitrogen (purity ≥ 99.999%), flow rate: 150.0 mL / min; positive and negative ion modes: positive ion. Door opening time: 100 μs, door closing voltage: 90 dgt.

[0055] ⑤ Data processing

[0056] The obtained chromatogram-migration spectrogram was processed using GC-IMS software to extract characteristic ion peaks, and a fingerprint of the volatile components of raw and processed Xanthii Fructus was established. Statistical methods such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for analysis to achieve the discrimination between raw and processed Xanthii Fructus.

[0057] A mixed standard of 6 ketones was detected to establish a calibration curve for retention time and retention index. Subsequently, the retention index of the substance was calculated from the retention time of the target compound, and the target compound was qualitatively analyzed by retrieving and comparing with the built-in GC retention index database (NIST 2020) and IMS migration time database in VOCal software.

[0058] The Reporter, Gallery Plot, and Dynamic PCA plugins in VOCal data processing software were used to generate three-dimensional spectrograms, two-dimensional spectrograms, difference spectrograms, fingerprint spectrograms, and PCA plots of volatile components respectively for the comparison of volatile organic compounds between samples.

[0059] (2) The sample data was classified using the software supporting the instrument to obtain the volatile components between different samples, as shown in Figures 4 - 5 as follows:

[0060] It can be very intuitively seen the differences in volatile components among several groups of samples. The contents of 2-Methyl-1-propanol, alpha-Thujene, alpha-humulene, Butanoic acid ethyl ester, 1-octanal, Borneol, Menthol, beta-Cedrene, Ethyl nonanoate, Linalool, isomenthone, (-)-alpha-Cubebene, alpha-Phellandrene, gamma-Terpinene, Decanal, Acetic acid ethyl ester, 1-pentanol-4-methyl, (+)-limonene, 2-methyl-2-hepten-6-one, alpha-Terpinene, 1-(1-methylethyl)-4-methylbenzene, ethanol, beta-Thujene, beta-Pinene, 4-Nonanone, 2-Butanone, delta 3-carene, (Z)-2-pentenal, 1-butanol, and 1-hexanol were higher in the group of raw Xanthii Fructus samples; see Table 2 below for details.

[0061] Table 2. Characteristic volatile components of crude Xanthii Fructus

[0062]

[0063] The contents of 1-hexanal, 2,3-pentadione, Acetone, 1-nonanal, 2-Propanol, Benzaldehyde, 3-Pentanone, Propanal, 1-Hydroxy-2-propanone, methylacetate, Propanoic acid, 1-Pentanol, 2-pentyl furan, (E)-2-octenal, (E)-2-hexen-1-al, 2,5-Dimethyl thiophene, Heptaldehyde, n-Pentanal, 3-Methyl butanal, Butanal, 2-Methyl propanal, 2,3-dimethyl-5-ethylpyrazine, 2-furaldehyde, 2,5-Dimethylpyrazine, 2,3,5-trimethylpyrazine, 2-hexen-1-ol, 2-Heptanone, (E)-2-Methyl-2-butenal, 2-Methylpyrazine, (E)-2-Heptenal in the stir-fried Xanthii Fructus with sand samples were relatively high. See Table 3 below for details.

[0064] Table 3. Characteristic volatile components of processed Xanthii Fructus

[0065]

[0066] (3) In order to study the differences in volatile components, PCA dimensionality reduction was performed on the peak heights of all detected VOC signals;

[0067] Principal component analysis (PCA) is a method for dimensionality reduction of multivariate variables, belonging to unsupervised learning, also known as principal component analysis. Through the PCA graph, the overall statistical differences between groups of samples and the level of parallelism between samples within a group can be roughly understood.

[0068] In order to study the differences in volatile components of Xanthii Fructus from different origins and processes, PCA dimensionality reduction was performed on the peak heights of all detected VOC signals, and the principal component score graph is as Figure 5As shown, it can be seen that the cumulative contribution rate of the first two principal components (PCs) is 68%, and the cumulative contribution rate of the first three PCs is 77.1%. Through PCA, the differences between samples can be seen, which is consistent with the intuitive observation results of the fingerprint spectra.

[0069] (4) Finally, qualitative analysis was performed on the volatile component spectra in the samples.

[0070] To further compare the differences between volatile components, fingerprint spectra of the samples were drawn. The fingerprint spectra can visually reflect the differential compounds, and chemometric analysis was performed based on the peak areas of various substances in different fingerprint spectra to obtain the characteristic markers of volatile components, that is, the characteristic flavors.

[0071] Through the analysis and detection of the spectra before and after stir-frying Xanthii Fructus with sand, it was found that a total of 65 volatile odor substances were detected, including 30 characteristic volatile odor substances in raw Xanthii Fructus and 30 characteristic volatile odor substances in stir-fried Xanthii Fructus with sand. Combining cluster analysis, the differences in odor substances between raw Xanthii Fructus and stir-fried Xanthii Fructus were analyzed, and the results were highly accurate, realizing a new idea for quickly judging Xanthii Fructus before and after processing from volatile odor substances.

[0072] The present invention aims to solve the problem in the prior art that it is difficult to quickly and accurately distinguish between raw Xanthii Fructus and stir-fried Xanthii Fructus with sand. It is identified by analyzing the differences in volatile components before and after stir-frying with sand to optimize the processing technology and quality control of Xanthii Fructus. To sum up, the present invention has the following beneficial effects:

[0073] High accuracy: The GC-IMS technology can effectively separate and identify the complex volatile components in Xanthii Fructus, providing rich qualitative and quantitative information, and the discrimination results are accurate and reliable.

[0074] High sensitivity: This technology has a low detection limit for volatile components and can detect minute component changes, which helps to discover the minor component differences during the processing of Xanthii Fructus.

[0075] No need for labeling: Compared with traditional labeling analysis methods, GC-IMS does not require labeling treatment of samples, simplifies the analysis process, and reduces the analysis cost.

[0076] Strong applicability: The method of the present invention is not only applicable to the discrimination of processed Xanthii Fructus, but also can be extended to the quality control of the processing of other Chinese medicinal materials, and has a wide application prospect.

[0077] The above content is only an example and explanation of the concept of the present invention. Those skilled in the art of this technology make various modifications or supplements or use similar methods to replace the specific embodiments described, as long as they do not deviate from the concept of the invention or exceed the scope defined by this claim book, they should all belong to the protection scope of the present invention.

Claims

1. A method for identifying the difference in volatile components of Xanthium sibiricum before and after sand blanching based on GC-IMS, characterized in that: The steps include: (1) Sample processing: Take Xanthium sibiricum, remove impurities, and obtain the raw product; take river sand 8 times the amount of the medicinal material and put it in a pot, heat it over medium heat until it is smooth, and take the temperature 1-1.2 cm away from the bottom of the pot as the sand temperature. When it stabilizes to 160°C, add Xanthium sibiricum and stir-fry for 7 minutes to obtain the sand-scalded product, and crush it in a pulverizer; (2) Sample detection: Incubate the powder in a headspace bottle and perform automatic headspace sampling; perform gas chromatography-ion mobility spectrometry (GC-IMS) detection on the Xanthium sibiricum sample to obtain the detection data, specifically: ① Instrument settings: Turn on the GC-IMS instrument, set the gas chromatograph’s injection port temperature, column box temperature program, carrier gas flow rate and other parameters, as well as the ion mobility spectrometer’s drift tube temperature and drift gas flow rate parameters to ensure stable operation of the instrument; ② Sample injection: inject the treated sample filtrate into the GC-IMS instrument through an automatic sample injector for analysis; ③Data acquisition: The instrument automatically collects the gas chromatography-ion mobility spectrometry data of the sample, and records the chromatographic peaks and ion mobility spectrometry peaks of the volatile components in each sample; (3) Data processing and identification, specifically: ① Use the instrument software to classify the collected GC-IMS sample data and obtain the volatile components of different samples; ② Perform PCA dimensionality reduction on the peak heights of all detected VOC signals to study the differences in volatile components; ③ Qualitative analysis of the spectrum of volatile components in the sample.

2. The method according to claim 1, characterized in that In step (2) sample testing step: The incubation conditions were: temperature: 80°C; time: 15 min; injection volume: 100 μL; splitless injection; Rotation speed: 500r / min; Injection needle temperature: 110℃; GC detection conditions were: column temperature: 60°C; Carrier gas: high-purity nitrogen; programmed pressure increase: initial flow rate 2.0 mL / min, maintained for 2 min, linearly increased to 10.0 mL / min within 8 min, linearly increased to 100.0 mL / min within 10 min, and maintained for 30 min. Chromatographic running time: 50 min; injection port temperature: 80 °C; IMS conditions: ionization source: tritium source (3H); migration tube length: 53mm; electric field strength: 500V / cm; migration tube temperature: 45°C; drift gas: high-purity nitrogen (purity ≥99.999%), flow rate: 150.0mL / min; positive and negative ion mode: positive ion; door opening time: 100μs, door closing voltage: 90dgt.

3. The method according to claim 1, characterized in that Step (3) using GC-IMS software to process the obtained chromatogram-migration spectrum, extract characteristic ion peaks, and establish fingerprints of volatile components of raw Xanthium sibiricum and different processed products.

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