Online extraction sequential ionization device and application thereof in high-similarity traditional Chinese medicine identification

Abstract

The invention provides a novel on-line extraction and sequential ionization mass spectrometry device (S-oEESI-MS). The device comprises a multi-solvent pump parallel structure, a sample extraction bin and an electrospray ion source. The device can be used for sequential online extraction and sequential ionization characterization of substances with different polarities in a complex sample. Compared with the traditional LC-MS (liquid chromatography-mass spectrometry) and oEESI-MS (oEESI-MS), the device has more accurate distinguishing and identifying capability. The invention also provides the application of the on-line extraction sequential ionization mass spectrometry device (S-oEESI-MS) in the production area analysis and / or detection of the traditional Chinese medicine bighead atractylodes rhizome. Mass spectrum information obtained by the device is subjected to multivariate statistical analysis, so that highly similar samples from different producing areas can be completely distinguished. The method has wide applicability and popularization value for quality control and standardized development of traditional Chinese medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to an online extraction sequential ionization mass spectrometry analysis device and an application thereof. Background Art

[0002] Accurately distinguishing and identifying different samples is one of the key research topics in traditional Chinese medicine, which is of great significance to producers, consumers or regulatory agencies. Traditional Chinese medicines are mostly derived from animal and plant tissues and organs, and their quality and price are greatly affected by differences in variety and origin. Highly similar samples, such as traditional Chinese medicines produced in adjacent origins, usually have similar appearances and contain basically the same major secondary metabolites. They are the main source of confusion and adulteration in the market and are difficult to accurately distinguish through traditional identification methods.

[0003] With the continuous development of modern analytical chemistry and instrument manufacturing technology, the rapid detection technology of traditional Chinese medicine has also made continuous progress. Spectroscopic technology, chromatography technology and molecular biology technology are the main means of identifying traditional Chinese medicine at present, and these technologies each have their own characteristics and limitations. Specifically, near-infrared spectroscopy technology and hyperspectral imaging have the advantages of strong versatility, rapidity, and non-destructiveness to samples, but their accuracy is relatively low, and the spectral signal is easily interfered by non-target components, which can easily lead to false positive misjudgments. Chromatographic technology, especially chromatography-mass spectrometry technology, can accurately analyze secondary metabolites in traditional Chinese medicine for quality evaluation and identification. Its accuracy is relatively high, but it requires a complex and lengthy sample pretreatment process, which may lead to the loss or loss of key substances in the sample. In addition, the interference and ion suppression of high-content main components on key trace components are also difficult to avoid. Molecular biological methods (such as PCR, etc.) are effective tools for identifying the source of animal and plant raw materials, but the quality of traditional Chinese medicine is not only determined by genes, but also affected by quality differences of the same species caused by environmental factors. Such quality differences caused by environmental factors cannot be identified by molecular biological tools. Therefore, there is still a lack of reliable identification technology for traditional Chinese medicine, especially when faced with highly similar samples, which cannot meet the identification requirements of being fast, accurate, simple pre-processing and low-cost.

[0004] In recent years, direct mass spectrometry has attracted more and more attention due to its advantages of rapid and high-throughput mass spectrometry analysis of complex matrix samples without the need for sample pretreatment. Since mass spectrometry has extremely strong molecular qualitative and quantitative capabilities, it can achieve in situ rapid detection and qualitative identification of different traditional Chinese medicine samples to a certain extent. However, due to the lack of pretreatment operations such as extraction and pre-separation, the ability of direct mass spectrometry to simultaneously characterize complex multi-components is relatively insufficient. In mass spectra, the phenomenon of trace components being suppressed by high-content components is relatively common, while the main components of highly similar samples are often consistent, but the differences are reflected in trace or even ultra-trace substances. Therefore, improving the ability of direct mass spectrometry to simultaneously analyze complex multi-components is of great significance for the accurate identification of traditional Chinese medicine. Summary of the invention

[0005] In order to improve the above technical problems, the present invention provides an online extraction sequential ionization mass spectrometry analysis device and its application.

[0006] First aspect

[0007] The present invention provides an online extraction sequential ionization mass spectrometry analysis device (S-oEESI-MS), which comprises a multi-solvent pump parallel structure, a sample extraction chamber (6), and an electrospray ion source (7).

[0008] According to an embodiment of the present invention, the multi-solvent pump parallel structure includes at least 2 solvent pump structures, such as 2, 3, 4, etc.

[0009] According to an embodiment of the present invention, the solvent pump structure includes a solvent storage bottle (1) and a precision peristaltic pump (5); the solvent storage bottle contains an extraction solvent; the solvent storage bottle (1) is connected to the precision peristaltic pump (5) through a solvent delivery pipeline (4); the precision peristaltic pump (5) pumps the extraction solvent to a sample extraction chamber (6).

[0010] According to an embodiment of the present invention, the extraction solvent is selected from organic or inorganic solvents of different polarities, such as water, methanol, ethanol, acetonitrile, dichloromethane, ethyl acetate, and the like.

[0011] According to an embodiment of the present invention, each solvent storage bottle independently contains the same or different extraction solvents; preferably contains different extraction solvents, such as a single extraction solvent or a mixed extraction solvent of different polarities.

[0012] According to an embodiment of the present invention, the multi-solvent pump parallel structure includes two solvent pump structures.

[0013] According to an embodiment of the present invention, the solvent storage bottle comprises a solvent storage bottle I and a solvent storage bottle II; preferably, the solvent storage bottle I and the solvent storage bottle II contain extraction solvents of different polarities.

[0014] According to an embodiment of the present invention, by setting a solvent gradient, extraction solvents with different gradients and / or polarities are pumped into the sample extraction chamber (6) via a precision peristaltic pump (5).

[0015] According to an embodiment of the present invention, the solvent gradient is set as: 0-10s 100% water, 10-50s 100%-0% water, 50-60s 100% methanol.

[0016] The parallel structure of multiple solvent pumps can further significantly expand the polarity coverage of the extraction solvent and realize the sequential extraction and ionization of high, medium and low polarity components.

[0017] According to an embodiment of the present invention, additives such as formic acid, acetic acid, ammonia water, trifluoroacetic acid, etc. may be optionally added to the extraction solvent.

[0018] According to an embodiment of the present invention, the sample extraction chamber (6) comprises a filter, such as a filter membrane. The filter or filter membrane can prevent solid powder or particles from entering the nebulizer.

[0019] According to an embodiment of the present invention, the filter membrane is a microporous filter membrane (0.22 μm-0.45 μm).

[0020] According to the embodiment of the present invention, the main body of the sample extraction chamber (6) is made of inert materials such as alloy, inert silica gel or PP plastic, etc. A microporous filter membrane (0.22-0.45 μm) made of materials such as PES is placed at the bottom.

[0021] According to embodiments of the present invention, the sample does not need to undergo any other pre-treatment or pre-separation.

[0022] According to an embodiment of the present invention, the solvent delivery line (4) is a quartz capillary or a Peek tube.

[0023] According to an embodiment of the present invention, the solvent delivery pipeline (4) is connected to the sample chamber via a section of metal pipe, and the metal pipe is connected to a high voltage power supply HV (9).

[0024] According to an embodiment of the present invention, the quartz capillary is fixed on each component through a peek joint.

[0025] According to an embodiment of the present invention, a high voltage electric field HV (9) is applied to the extraction solvent before the extraction solvent enters the sample extraction chamber (6).

[0026] According to an embodiment of the present invention, after the sample is extracted by the extraction solvent, it passes through the electrospray ion source (7), under the action of the nebulizer gas (10), an electrospray is formed and the sample is ionized, and then enters the mass spectrometer (8) through the ion transfer tube.

[0027] According to an embodiment of the present invention, the atomizing gas is selected from inert gases such as nitrogen and helium, for example nitrogen.

[0028] According to the embodiments of the present invention, the device can be coupled with various mass spectrometers (such as QTOF / TQ / Orbitrap-MS, etc.).

[0029] Second aspect

[0030] The present invention provides application of the above device in sample analysis and / or detection.

[0031] According to an embodiment of the present invention, the sample is selected from medicines (such as traditional Chinese medicine samples), foods, cosmetics, health products, and the like.

[0032] The present invention also provides the use of the above device in combination with a chemometric multivariate statistical analysis method in the analysis and / or detection of samples.

[0033] According to an embodiment of the present invention, the sample is selected from medicines (such as traditional Chinese medicine samples), foods, cosmetics, health products, and the like.

[0034] According to an embodiment of the present invention, the chemometric multivariate statistical analysis method includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA).

[0035] The third aspect

[0036] The present invention provides application of the above device in origin analysis and / or detection of a sample of the traditional Chinese medicine Atractylodes macrocephala.

[0037] According to an embodiment of the present invention, the solvent gradient is set as: 0-10s 100% water, 10-50s 100%-0% water, 50-60s 100% methanol.

[0038] According to an embodiment of the present invention, the solvent flow rate is 100 μL / min.

[0039] According to an embodiment of the present invention, the extraction solvent in the solvent storage bottle 1 is water.

[0040] According to an embodiment of the present invention, the extraction solvent in the solvent storage bottle II is methanol.

[0041] According to an embodiment of the present invention, 0.01%-10% acetic acid is added to the extraction solvent, for example, 1% acetic acid.

[0042] According to an embodiment of the present invention, the Atractylodes macrocephala sample is in powder form.

[0043] According to an embodiment of the present invention, the amount of sample added to the sample extraction chamber (6) is 0.05 mg-5.0 mg, 0.1 mg-2.0 mg, for example 0.5 mg.

[0044] According to embodiments of the present invention, the sample does not need to undergo any other pre-treatment or pre-separation.

[0045] According to an embodiment of the present invention, mass spectrometry is performed in positive ion mode.

[0046] According to an embodiment of the present invention, the mass range of the mass spectrometry scan is m / z 100-1000.

[0047] According to an embodiment of the present invention, the flow rate of the atomizing gas is 3 L / min to 15 L / min, preferably 5 L / min to 11 L / min, for example 9 L / min.

[0048] According to an embodiment of the present invention, the fragmentation voltage is 50V-250V, preferably 50V-150V, such as 100V.

[0049] According to an embodiment of the present invention, the capillary voltage is 2 kV to 4.5 kV, preferably 3 kV to 4 kV, for example 3.5 kV.

[0050] According to an embodiment of the present invention, the atomizing gas temperature is 100°C-350°C, preferably 100°C-200°C, such as 150°C.

[0051] According to an embodiment of the present invention, the Chinese medicine Atractylodes macrocephala samples are Atractylodes macrocephala samples from different origins, for example, highly similar Atractylodes macrocephala samples from different origins; preferably, Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang; further preferably, Atractylodes macrocephala samples from Anhui, Atractylodes macrocephala samples from Henan, Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang.

[0052] According to an embodiment of the present invention, after the mass spectrum information is obtained using the device, the characteristic markers of the sample are screened.

[0053] According to an embodiment of the present invention, the characteristic markers are compounds corresponding to the following m / z values: 318, 158, 489, 133, 533, 577, 149, 445, 319, 579, 349, 413, 377, 302, 365, 175, 317, 116, 301, 429, 279, 117.

[0054] According to an embodiment of the present invention, after the characteristic marker information is obtained, it is further analyzed and identified by a chemometric multivariate statistical analysis method.

[0055] According to an embodiment of the present invention, the chemometric multivariate statistical analysis method includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA).

[0056] The present invention also provides an application of the above device in combination with a chemometric multivariate statistical analysis method in origin analysis and / or detection of a Chinese medicine Atractylodes macrocephala sample.

[0057] According to an embodiment of the present invention, the chemometric multivariate statistical analysis method includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA).

[0058] According to an embodiment of the present invention, the solvent gradient is set as: 0-10s 100% water, 10-50s 100%-0% water, 50-60s 100% methanol.

[0059] According to an embodiment of the present invention, the solvent flow rate is 100 μL / min.

[0060] According to an embodiment of the present invention, the extraction solvent in the solvent storage bottle 1 is water.

[0061] According to an embodiment of the present invention, the extraction solvent in the solvent storage bottle II is methanol.

[0062] According to an embodiment of the present invention, 0.01%-10% acetic acid is added to the extraction solvent, for example, 1% acetic acid.

[0063] According to an embodiment of the present invention, the Atractylodes macrocephala sample is in powder form.

[0064] According to an embodiment of the present invention, the amount of sample added to the sample extraction chamber (6) is 0.05 mg-5.0 mg, 0.1 mg-2.0 mg, for example 0.5 mg.

[0065] According to embodiments of the present invention, the sample does not need to undergo any other pre-treatment or pre-separation.

[0066] According to an embodiment of the present invention, mass spectrometry is performed in positive ion mode.

[0067] According to an embodiment of the present invention, the mass range of the mass spectrometry scan is m / z 100-1000.

[0068] According to an embodiment of the present invention, the flow rate of the atomizing gas is 3 L / min to 15 L / min, preferably 5 L / min to 11 L / min, for example 9 L / min.

[0069] According to an embodiment of the present invention, the fragmentation voltage is 50V-250V, preferably 50V-150V, such as 100V.

[0070] According to an embodiment of the present invention, the capillary voltage is 2 kV to 4.5 kV, preferably 3 kV to 4 kV, for example 3.5 kV.

[0071] According to an embodiment of the present invention, the atomizing gas temperature is 100°C-350°C, preferably 100°C-200°C, such as 150°C.

[0072] According to an embodiment of the present invention, the Chinese medicine Atractylodes macrocephala samples are Atractylodes macrocephala samples from different origins, for example, highly similar Atractylodes macrocephala samples from different origins; preferably, Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang; further preferably, Atractylodes macrocephala samples from Anhui, Atractylodes macrocephala samples from Henan, Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang.

[0073] According to an embodiment of the present invention, after the mass spectrum information is obtained using the device, the characteristic markers of the sample are screened.

[0074] According to an embodiment of the present invention, the characteristic markers are compounds corresponding to the following m / z values: 318, 158, 489, 133, 533, 577, 149, 445, 319, 579, 349, 413, 377, 302, 365, 175, 317, 116, 301, 429, 279, 117.

[0075] According to an embodiment of the present invention, after the characteristic marker information is obtained, it is further analyzed and identified by a chemometric multivariate statistical analysis method.

[0076] Beneficial Effects

[0077] The present invention provides a novel online extraction sequential ionization mass spectrometry (S-oEESI-MS), which comprises a multi-solvent pump parallel structure, a sample extraction chamber, and an electrospray ion source. The device can be used for sequential online extraction and sequential ionization characterization of substances of different polarities in complex samples. Compared with traditional LC-MS and oEESI-MS, the device has more accurate differentiation and identification capabilities.

[0078] The present invention also provides the use of the online extraction sequential ionization mass spectrometry device (S-oEESI-MS) in the origin analysis and / or detection of the Chinese medicine Atractylodes macrocephala. The mass spectrum information obtained by the device can achieve complete distinction of highly similar samples from different origins through multivariate statistical analysis. It has wide applicability and promotion value for the quality control and standardization development of Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 Schematic diagram of the structure of the sequential online extraction electrospray ionization device.

[0080] Figure 2 This is a graph showing the results of S-oEESI-MS condition optimization.

[0081] Figure 3 These are typical mass spectral fingerprints obtained by S-oEESI-MS from four different origins; A is from Anhui, B is from Henan, C is from Hunan, and D is from Zhejiang.

[0082] Figure 4 The results of chemometric differentiation of highly similar Atractylodes macrocephala from different origins based on S-oEESI-MS technology.

[0083] Figure 5 PCA differentiation contribution diagram based on S-oEESI-MS technology.

[0084] Figure 6 This is the validation diagram of the PLS-DA model based on S-oEESI-MS technology.

[0085] Figure 7 Bar graph of PLS-DA VIP values ​​based on S-oEESI-MS technology.

[0086] Figure 8 The bar graph shows the OPLS-DA VIP values ​​based on S-oEESI-MS technology.

[0087] Fig. 9 The results of chemometric differentiation of highly similar Atractylodes macrocephala from different origins based on traditional LC-MS and oEESI-MS techniques.

[0088] Fig.10 Figure 2 is the validation diagram of the PLS-DA model based on LC-MS data.

[0089] Fig.11 Figure 2 is the validation diagram of the PLS-DA model based on oEESI-MS data. DETAILED DESCRIPTION

[0090] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0091] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0092] Example 1

[0093] 1. Experimental part

[0094] 1.1 Materials and reagents

[0095] Eight batches of Atractylodes macrocephala samples were collected from Henan, Hunan, Anhui, and Zhejiang provinces, for a total of 32 batches. After identification, all the herbs were stored in the National Center for Chinese Medicine Resources, China Academy of Chinese Medical Sciences. Methanol and acetic acid (MS grade) were purchased from Thermo Fisher Scientific (China). Deionized water (18.2 MΩ / cm) was prepared by a Mill-Q water purification system (Billerica, MA, USA).

[0096] 1.2 Mass spectrometry parameter optimization

[0097] All experiments were performed on an Agilent 6470B mass spectrometer (Agilent Technologies (China) Co., Ltd.). Mass spectrometry parameters are key factors in determining the signal response of target compounds. Therefore, the signal response intensity of atractylodes lactone (m / z203), an important component in Atractylodes macrocephala, was first used as an indicator to optimize a series of mass spectrometry parameters, including fragmentation voltage, capillary voltage, gas flow rate, and gas temperature. The fragmentation voltage was optimized in the range of 50 to 300 V, the gas flow rate was optimized in the range of 3 to 15 L / min, the capillary voltage was optimized in the range of 2 to 4.5 kV, and the gas temperature was optimized in the range of 100 to 350 °C.

[0098] 1.3 Direct analysis of Atractylodes macrocephala samples based on S-oEESI-MS

[0099] The S-oEESI-MS experiment used a new improved S-oEESI source installed on a triple quadrupole mass spectrometer (Agilent 6470B). The schematic diagram of the setup for sample extraction and ionization is shown in Figure 1 . Among them, the sample extraction chamber refers to the literature [Qiu, ZD Huang, LQ et al. (2020). Limitation standard of toxic aconitines in Aconitum proprietary Chinese medicines using on-line extraction electrosprayionization mass spectrometry. Acta Pharm Sin B, 10 (8), 1511-1520.].

[0100] Figure 1In the figure, 1 is a solvent storage bottle, 2 is an extraction solvent I, 3 is an extraction solvent II, 4 is a solvent delivery pipeline, 5 is a precision peristaltic pump, 6 is a sample extraction chamber, 7 is an electrospray ion source, 8 is a mass spectrometer, 9 is a high voltage power supply HV, and 10 is a nebulizer gas. The solvent storage bottle (1) is connected to the precision peristaltic pump (5) through the solvent delivery pipeline (4); the precision peristaltic pump (5) pumps the extraction solvent to the sample extraction chamber (6). After the sample is extracted by the extraction solvent, it passes through the electrospray ion source (7), and under the action of the nebulizer gas (10), an electrospray is formed and the sample is ionized, and then enters the mass spectrometer (8) through the ion transfer tube.

[0101] The experiment used water and methanol as extraction solvents, and 1% acetic acid was added to both phases of solvent. The two phases of solvent were placed in two solvent bottles respectively, and were extracted in order according to the established procedure using a precision peristaltic pump. The gradient elution process was completed within 1 min, and the solvent ratio was 0-10s 100% water, 10-50s 100%-0% water, and 50-60s 100% methanol. The flow rate was 100μL / min. An air filter membrane was installed at the bottom of the sample chamber to prevent solid powder or particles from entering the nebulizer. The nebulization auxiliary gas was nitrogen. Other mass spectrometry parameters used the above optimized parameters or instrument default values ​​in positive ion scanning mode. In order to improve the reproducibility and stability of the sample, the Atractylodes macrocephala sample used in the experiment was powdered (0.5 mg) without any other pretreatment or pre-separation. The mass spectrometer adopted the positive ion mode with a mass range of m / z 100-1000.

[0102] 1.4 Differentiation strategy of Atractylodes macrocephala production areas based on S-oEESI-MS fingerprint and index component groups

[0103] The full scan mass spectrum fingerprint of each Atractylodes macrocephala sample obtained by S-oEESI-MS was exported to Microsoft Excel to obtain the m / z and corresponding intensity data of each mass spectrum peak. The samples were grouped according to different origins, and each sample was sorted according to the intensity of each peak. The top 100 peaks of each sample signal intensity and the peaks common to the samples from the same origin were selected to construct the candidate characteristic marker group of the origin; the candidate characteristic marker groups from different origins were merged, and repeated peaks, isotope peaks, impurity peaks, etc. were removed to finally obtain the characteristic markers of Atractylodes macrocephala. PCA, PLS-DA, and OPLS-DA techniques were used to further model and distinguish Atractylodes macrocephala from different origins using the m / z signal intensity of the characteristic marker as an indicator.

[0104] 1.5 LC-MS and oEESI-MS analysis

[0105] 1.5.1 LC-MS analysis

[0106] The traditional LC-MS method was used to analyze 32 batches of samples from 4 production areas to obtain their LC-MS fingerprints, which were further distinguished by combining chemometric models (PCA / PLS-DA / OPLS-DA).

[0107] LC-MS sample preparation: 10 mg of each sample powder was accurately weighed, 1.0 mL of methanol was added for ultrasound-assisted extraction for 15 min, and then the extract was passed through a 0.22 μm microporous membrane.

[0108] LC-MS conditions: The compounds were separated on a chromatographic column (ZORBAX Eclipse Plus C18 2.1x100 mm, 3.5 μm), mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, column temperature was 25°C, flow rate was 0.2 mL / min.

[0109] The mobile phase B gradient elution was as follows: 0–4 min, 28% B to 60% B; 4–6 min, 60% B to 90% B; 6–15 min, 90% B to 100% B; 15–20 min, 100% B; 20–22 min, 100–28% B; 22–24 min 28–28% B. The injection volume was 3.0 μL. The mass spectrometer was an Agilent 6470B (Agilent Technologies (China) Co., Ltd.), which was coupled to an ESI ion source. The parameters of the mass spectrometer were the same as those of the S-oEESI-MS device.

[0110] 1.5.2oEESI-MS analysis

[0111] oEESI-MS analysis was performed using an oEESI ion source. The oEESI ion source device was referenced to the literature [Qiu, ZD Huang, LQ et al. (2020). Limitation standard of toxic aconitines in Aconitum proprietary Chinese medicines using on-line extraction electrosprayionization mass spectrometry. Acta Pharm Sin B, 10 (8), 1511-1520.]. The extraction solvent was methanol-water (1:1, v:v). The flow rate was 100 μL / min. In order to improve the analysis efficiency and experimental stability, the direct sample load was 0.5 mg. Other mass spectrometry conditions were the same as the settings of the S-oEESI-MS device.

[0112] 2. Results and Discussion

[0113] 2.1 Creation and optimization of S-oEESI-MS device

[0114] The present invention first establishes a two-phase solvent delivery device, and selects methanol and water with 0.1% formic acid added as the two-phase extraction solvent. The specific operation steps are to extract in 100% methanol phase for 10s, then gradually transition from 100% methanol phase to 100% water phase for 40s, and finally extract with 100% water phase for 10s. The extraction time for each sample is 1.0min. Obviously, due to the gradient solvent extraction process, the S-oEESI-MS technology takes longer to analyze a single sample than the oEESI-MS technology (10-30s), but compared with the LC-MS technology (at least about 20-30min), it still has obvious advantages.

[0115] According to the signal intensity of key target components, key parameters such as nebulizer gas flow rate, ionization voltage, capillary voltage and gas temperature of the S-oEESI source were first optimized to achieve more effective ionization and determination ( Figure 2 ).from Figure 2 A It can be clearly seen that within the range of 3L / min to 15L / min of nebulizing gas flow rate, the mass spectrometry signal of the target substance first increases significantly and then gradually decreases. The optimization process of the fragmentation voltage shows that ( Figure 2 B), in the relatively low fragmentation voltage range of 50V-150V, the signal response is good, and the response is best at 100V. The optimization results of the capillary voltage reveal ( Figure 2 C), too high ionization voltage and too low ionization voltage will lead to reduced ionization effect of the analyte, while the target analyte has a relatively good ionization effect in the range of 3kV to 4kV. Finally, adjust the nebulizing gas temperature in the range of 100℃-350℃ ( Figure 2 D); It is not difficult to find that the best signal response can be obtained at 150°C, while after 200°C, the signal decays rapidly.

[0116] Therefore, the optimal gas flow rate of 9.0 L / min, the optimal fragmentor voltage of 100 V, the optimal capillary voltage of 3.5 kV, and the optimal gas temperature of 150 °C were selected in subsequent experiments.

[0117] 2.2 Screening of characteristic markers of Atractylodes macrocephala from different origins

[0118] Using the optimized mass spectrometry analysis conditions, 32 batches of Atractylodes macrocephala samples from 4 production areas were rapidly characterized by mass spectrometry. Figure 3The typical mass spectrometry fingerprints of samples from different origins obtained based on S-oEESI-MS are shown in Figure 2. Overall, although most of the main compounds in the typical fingerprints of Atractylodes macrocephala from four different origins are common peaks (such as m / z 371, 301, 279, 429, etc.), there are still large differences in the relative intensity of the signals. At the same time, there are also obvious differences in the types of compounds in substances with relatively low signals.

[0119] For example, Henan origin ( Figure 3 The peak intensities of m / z 205 and m / z 391 in B) are very high, with relative intensities of about 30-50% of that of compound m / z 317, while in Anhui ( Figure 3 A) and Zhejiang origin ( Figure 3 D), the peak intensities of these two compounds are less than 10% of the base peak m / z 317 intensity. Figure 3 C), it is even difficult to see these two compounds in the overall outline. From the perspective of absolute signal intensity, the differences in the substance content of the four origins are basically within one order of magnitude. Figure 3 D) had a relatively higher content of the main components in Atractylodes macrocephala than those in the other three origins (the signal intensity of m / z 317 was 1.2*10 6 ), suggesting that the Atractylodes macrocephala produced in Zhejiang may have better pharmacological activity. This is consistent with the traditional belief that the Atractylodes macrocephala produced in Zhejiang is an authentic medicinal material.

[0120] Although the fingerprints of the four origins of Atractylodes macrocephala obtained based on the S-oEESI-MS technology show certain differences in the overall outline, it is still difficult to intuitively and accurately distinguish different origins. Therefore, the present invention further screens the characteristic markers of each origin, in order to simply, quickly and accurately distinguish different Atractylodes macrocephala samples through a small amount of characteristic markers. The selection of characteristic markers is based on the mass spectrometry fingerprints of all 32 batches of Atractylodes macrocephala samples. All the mass spectrometry primary spectrum mass-to-charge ratios (m / z 100-1000) and signal intensities of each sample are exported to Excel (>10,000 compounds for each sample), grouped according to origin and arranged according to signal intensity for compounds with different mass-to-charge ratios. Finally, the signals with the top 100 intensities in all samples of the same origin are selected as candidate characteristic markers of Atractylodes macrocephala from the origin. Further excluding isotope peaks, summed ion peaks, blank matrix interference peaks, etc., characteristic markers of Atractylodes macrocephala from different origins can be obtained. Finally, 12, 15, 13 and 14 characteristic markers were screened out from Anhui, Henan, Hunan and Zhejiang respectively, and the results from the four origins were merged and duplicates were removed to obtain a total of 22 characteristic markers for the differentiation and identification of Atractylodes macrocephala (m / z 318, 158, 489, 133, 533, 577, 149, 445, 319, 579, 349, 413, 377, 302, 365, 175, 317, 116, 301, 429, 279 and 117). The possible structures of some compounds were further attributed based on literature search and other methods (Table 1).

[0121] Table 1 22 selected characteristic markers of Atractylodes macrocephala and some possible structures

[0122] Note: a Henan, b Anhui, c Hunan, d Zhejiang.

[0123] 2.3 Multivariate statistical analysis of different Atractylodes macrocephala with high similarity based on S-oEESI-MS

[0124] Through S-oEESI-MS, the multi-component sequential ionization characterization of each medicinal material and the screening of characteristic markers of Atractylodes macrocephala from different origins were carried out, and the highly similar Atractylodes macrocephala from different origins were further distinguished and identified more accurately and the chemical differences and correlations were visualized by chemometric multivariate statistical analysis. Based on the signal intensity of 22 characteristic markers in 32 samples from different origins, three statistical analysis techniques, PCA, PLS-DA, and OPLS-DA, were used in turn for analysis ( Figure 4 ).

[0125] Figure 4 A is the PCA score graph of Atractylodes macrocephala samples from four origins, model R 2 X=0.984 is close to 1, Q 2=0.629, indicating that the model classification effect is stable and the prediction effect is high, and subsequent differential component analysis can be performed. Overall, within the 95% confidence interval, the four origin samples were relatively clearly distinguished. Only one sample from Anhui Province showed an abnormal deviation, which was relatively discrete compared with other samples in the same group. Contribution to the distinction by PCA Figure 5 It can be seen that ions such as m / z 365, 318, 317, 445, 429, 533, and 319 are the main contributing ions for distinguishing Atractylodes macrocephala samples from the four origins.

[0126] In order to better explore the metabolic differences of Atractylodes macrocephala samples from four origins, PLS-DA and OPLS-DA techniques were used to analyze the data. Figure 4 B is the PLS-DA score graph of Atractylodes macrocephala samples from four origins. 2 X=0.946,R 2 Y=0.899,Q 2 =0.69, within the 95% confidence interval, the four origin samples were clearly distinguished. The validity of the model was verified by randomly changing the order of the categorical variable y 200 times through the permutation test ( Figure 6 ), R 2 and Q 2 The values ​​are all smaller than the original values ​​on the right, and the intercept of the regression line of Q2 on the Y axis is negative, indicating that the established model is not overfitting, has good predictive ability, and can be used for difference analysis. Difference analysis usually uses VIP value to characterize the contribution of difference variables. When VIP value>1, and P<0.05 by t test, it is considered that the component is a difference chemical component with a large contribution rate to the separation between groups. The contribution diagram of VIP differentiation by PLS-DA model is as follows: Figure 7 It can be seen that m / z 133, 117, 489, 159, 377, and 175 are the main classification contribution ions, which is consistent with the main ions contributed by PCA classification. In order to further verify the effectiveness of the model, OPLS-DA was used to analyze the data. Figure 4 C is the OPLS-DA score scatter plot of Atractylodes macrocephala samples from four origins, which is very similar to the PLS-DA score scatter plot results. Figure 8 It can be seen that the ions with larger classification contribution rates are mainly m / z 133, 117, 159, 377, and 175, which are consistent with the main ions of PLS-DA, but the OPLS-DA sample is more aggregated than PLS-DA.

[0127] In summary, the full-gradient mass spectrometry fingerprint obtained based on S-oEESI-MS and the characteristic marker screening strategy, combined with the chemometric model, can achieve the differentiation and identification of highly similar samples that are difficult to distinguish and identify by conventional analytical techniques, and is expected to help promote the improvement of relevant sample quality standards and quality control levels.

[0128] 2.4 Traditional LC-MS and oEESI-MS cannot distinguish highly similar Atractylodes macrocephala samples

[0129] The present invention uses traditional LC-MS technology and oEESI-MS technology, combined with chemometric models (such as PCA, PLS-DA and OPLS-DA), to characterize and identify Atractylodes macrocephala samples from four different origins ( Fig. 9 2.4.1 LC-MS cannot distinguish highly similar Atractylodes macrocephala samples

[0130] Fig. 9 A-9C is the result obtained by distinguishing the fingerprint data obtained by LC-MS through PCA, PLS-DA and OPLS-DA models.

[0131] PCA score plot( Fig. 9 A) The results show that the samples from the four origins are distributed in a disordered manner and are difficult to distinguish. 2 The value of X is 0.215, Q 2 The value of is -0.0849. In the PCA analysis, R 2 The closer X is to 1.0, the higher the model stability is; 2 If it is greater than 0.5, it means that the model prediction rate is high. 2 X is 0.215, which is less than 0.5, and Q 2 It is a negative value, which indicates that the model classification stability is low and the prediction efficiency is not high. In addition, within the 95% confidence interval of the model, there is an abnormal sample from Hunan.

[0132] Since PCA is an unsupervised analysis method, it is easy to ignore intra-group differences and random errors when determining the variation components. Therefore, the present invention further adopts the supervised pattern recognition method PLS-DA to distinguish the data. Fig. 9 B shows the score scatter plot of the LC-MS data of four Atractylodes macrocephala samples under the PLS-DA model. 2 The value of X is 0.315, R 2 The value of Y is 0.999, Q 2 The value of is 0.701. The results show that the overall classification effect is still better than the PCA model. There is a clear distinction between Atractylodes macrocephala produced in Anhui and Henan, but the distinction between Atractylodes macrocephala produced in Zhejiang and Hunan is not obvious. In order to further verify the effectiveness of the PLS-DA model, the present invention conducted a permutation experiment. By randomly performing 200 permutation tests, the permutation order of the classification variable y was changed to obtain corresponding different random permutations. The R generated by the random permutation on the left end of the PLS-DA model permutation test 2 and Q 2When the values ​​are all smaller than the original values ​​on the right, it means that the prediction ability of the model is greater than that of the random permutation y, indicating that the model is effective and subsequent variance analysis can be performed. Fig.10 It can be seen that only the R on the right side 2 and Q 2 Some values ​​are smaller than the original values, indicating that the model is overfitting, the classification is unstable, and the prediction efficiency is low, which is consistent with the result that products produced in Zhejiang and Hunan cannot be distinguished.

[0133] OPLS-DA is based on PLS-DA and combines orthogonal signals to eliminate changes in the X matrix that are irrelevant to the Y matrix, maximize the relationship between the X matrix and the Y variable, and maximize the classification difference between the two, thereby more accurately characterizing the differences in the chemical components of the two groups. Fig. 9 C is the OPLS-DA score scatter plot of LC-MS data of Atractylodes macrocephala samples from four origins; R 2 X = 0.172, R 2 Y=0.966,Q 2 =0.136. Overall, the classification results are similar to those of PLS-DA, but the overall clustering of Zhejiang and Hunan samples is slightly higher than that of PLS-DA. Fig. 9 C shows that there is a clear distinction between Atractylodes macrocephala produced in Anhui and that produced in Henan, but there is no obvious distinction between Atractylodes macrocephala produced in Zhejiang and that produced in Hunan.

[0134] 2.4.2oEESI-MS cannot distinguish highly similar Atractylodes macrocephala samples

[0135] Fig. 9 D-9F is the result of PCA, PLS-DA and OPLS-DA analysis of the fingerprint data of Atractylodes macrocephala samples from four production areas, namely Anhui, Henan, Hunan and Zhejiang, obtained by oEESI-MS.

[0136] Overall, the PCA score scatter plot is still very confusing (R 2 X=0.305,Q 2 =-0.0852,R 2 Y<0.5=, there is no obvious difference among the samples from the four origins, among which two sample points from Anhui origin showed abnormalities.

[0137] Scatter plot of PLS-DA scores (R 2 X=0.288,R 2 Y=0.978,Q 2 =0.419) showed that the classification effect was better than PCA. The Atractylodes macrocephala from Anhui and Zhejiang could be clearly distinguished, but the two samples from Hunan and Henan could not be distinguished. The permutation test was used to further evaluate the effectiveness of the model ( Fig.11 ), the results show that only some R 2 and Q2 The value on the right end is smaller than the original value, indicating that the model is overfitted, the classification is unstable, and the prediction efficiency is low, so it is impossible to distinguish between samples originating from Hunan and Henan.

[0138] Fig. 9 F shows the OPLS-DA score scatter plot of four Atractylodes macrocephala samples, among which R 2 The value of X is 0.171, R 2 The value of Y is 0.835, Q 2 The value of is 0.0902. As can be seen from the figure, R 2 X and Q 2 The values ​​of are all less than 0.5. Fig. 9 It can be seen from F that within the 95% confidence interval, only Anhui and Zhejiang can be distinguished among the four types of Atractylodes samples, which is consistent with the classification results of PLS-DA.

[0139] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An online extraction sequential ionization mass spectrometry analysis device (S-oEESI-MS), the device comprising a multi-solvent pump parallel structure, a sample extraction chamber, and an electrospray ion source.

2. The device according to claim 1, characterized in that The multi-solvent pump parallel structure includes at least two solvent pump structures, such as 2, 3, 4, etc.; Preferably, the solvent pump structure comprises a solvent storage bottle and a precision peristaltic pump; the solvent storage bottle contains an extraction solvent; the solvent storage bottle is connected to the precision peristaltic pump through a solvent delivery pipeline; the precision peristaltic pump pumps the extraction solvent to the sample extraction chamber; Preferably, the extraction solvent is selected from organic or inorganic solvents of different polarities, such as water, methanol, ethanol, acetonitrile, dichloromethane, ethyl acetate, etc.; Preferably, each solvent storage bottle independently contains the same or different extraction solvents; preferably contains different extraction solvents, such as a single extraction solvent or a mixed extraction solvent of different polarity; Preferably, the solvent storage bottle comprises a solvent storage bottle I and a solvent storage bottle II; preferably, the solvent storage bottle I and the solvent storage bottle II contain extraction solvents of different polarities; Preferably, by setting a solvent gradient, extraction solvents of different gradients and / or polarities are pumped into the sample extraction chamber via a precision peristaltic pump; Preferably, the solvent gradient is set as: 0-10s 100% water, 100%-0% water, 10-50s 100% methanol, 50-60s; Preferably, additives such as formic acid, acetic acid, ammonia, trifluoroacetic acid, etc. may be optionally added to the extraction solvent; Preferably, the sample extraction chamber comprises a filter, such as a filter membrane; preferably, the filter membrane is a microporous filter membrane; Preferably, the sample extraction chamber body is made of an inert material such as alloy, inert silica gel or PP plastic; Preferably, the sample is not subjected to any other pretreatment or pre-separation; Preferably, the solvent delivery pipeline is a quartz capillary or a Peek tube; Preferably, the solvent delivery pipeline is connected to the sample chamber via a metal tube, and the metal tube is connected to a high voltage power supply HV; Preferably, the quartz capillary is fixed on each component through a peek joint; Preferably, before the extraction solvent enters the sample extraction chamber, a high voltage electric field HV is applied to the extraction solvent; Preferably, after the sample is extracted with the extraction solvent, it passes through an electrospray ion source, under the action of the atomizing gas, to form an electrospray and complete sample ionization, and then enters the mass spectrometer through an ion transfer tube; Preferably, the atomizing gas is selected from an inert gas such as nitrogen and helium, for example nitrogen; Preferably, the device can be coupled with various mass spectrometers (such as QTOF / TQ / Orbitrap-MS, etc.).

3. Use of the device according to claim 1 or 2 in the analysis and / or detection of samples; Preferably, the sample is selected from medicines (such as traditional Chinese medicine samples), foods, cosmetics, health products, etc.

4. Use of the device according to claim 1 or 2 in combination with a chemometric multivariate statistical analysis method in the analysis and / or detection of a sample; Preferably, the sample is selected from medicines (such as traditional Chinese medicine samples), food, cosmetics, health products, etc.; Preferably, the chemometric multivariate statistical analysis method includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA).

5. Use of the device according to claim 1 or 2 in origin analysis and / or detection of a sample of the Chinese herbal medicine Atractylodes macrocephala.

6. The use according to claim 5, characterized in that: The solvent gradient was set as: 0-10s 100% water, 10-50s 100%-0% water, 50-60s 100% methanol. Preferably, the solvent flow rate is 100 μL / min; Preferably, the extraction solvent in the solvent storage bottle 1 is water; Preferably, the extraction solvent in the solvent storage bottle II is methanol; Preferably, 0.01%-10% acetic acid is added to the extraction solvent, for example 1% acetic acid; Preferably, the Atractylodes macrocephala sample is in powder form; Preferably, the amount of sample added to the sample extraction chamber is 0.05 mg-5.0 mg, 0.1 mg-2.0 mg, for example 0.5 mg; Preferably, the sample is not subjected to any other pretreatment or pre-separation; Preferably, the mass spectrometer is in positive ion mode; Preferably, the mass range of the mass spectrometry scan is m / z 100 to 1000; Preferably, the flow rate of the atomizing gas is 3 L / min to 15 L / min, preferably 5 L / min to 11 L / min, for example 9 L / min; Preferably, the fragmentation voltage is 50V-250V, preferably 50V-150V, for example 100V; Preferably, the capillary voltage is 2 kV to 4.5 kV, preferably 3 kV to 4 kV, for example 3.5 kV; Preferably, the atomizing gas temperature is 100°C-350°C, preferably 100°C-200°C, for example 150°C; Preferably, the Chinese medicine Atractylodes macrocephala samples are Atractylodes macrocephala samples from different origins, such as highly similar Atractylodes macrocephala samples from different origins; preferably Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang; further preferably Atractylodes macrocephala samples from Anhui, Atractylodes macrocephala samples from Henan, Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang; Preferably, after obtaining mass spectrum information using the device, characteristic markers of the sample are screened; preferably, the characteristic markers are compounds corresponding to the following m / z values: 318, 158, 489, 133, 533, 577, 149, 445, 319, 579, 349, 413, 377, 302, 365, 175, 317, 116, 301, 429, 279, 117; Preferably, after obtaining the characteristic marker information, further analysis and identification are performed by chemometric multivariate statistical analysis methods; Preferably, the chemometric multivariate statistical analysis method includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA).

7. Use of the device according to claim 1 or 2 in combination with a chemometric multivariate statistical analysis method in the origin analysis and / or detection of a sample of the traditional Chinese medicine Atractylodes macrocephala; Preferably, the chemometric multivariate statistical analysis method includes principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA).

8. The use according to claim 7, characterized in that: The solvent gradient was set as: 0-10s 100% water, 10-50s 100%-0% water, 50-60s 100% methanol. Preferably, the solvent flow rate is 100 μL / min; Preferably, the extraction solvent in the solvent storage bottle 1 is water; Preferably, the extraction solvent in the solvent storage bottle II is methanol; Preferably, 0.01%-10% acetic acid is added to the extraction solvent, for example 1% acetic acid; Preferably, the Atractylodes macrocephala sample is in powder form; Preferably, the amount of sample added to the sample extraction chamber is 0.05 mg-5.0 mg, 0.1 mg-2.0 mg, for example 0.5 mg; Preferably, the sample is not subjected to any other pretreatment or pre-separation; Preferably, the mass spectrometer is in positive ion mode; Preferably, the mass range of the mass spectrometry scan is m / z 100 to 1000; Preferably, the flow rate of the atomizing gas is 3 L / min to 15 L / min, preferably 5 L / min to 11 L / min, for example 9 L / min; Preferably, the fragmentation voltage is 50V-250V, preferably 50V-150V, for example 100V; Preferably, the capillary voltage is 2 kV to 4.5 kV, preferably 3 kV to 4 kV, for example 3.5 kV; Preferably, the atomizing gas temperature is 100°C-350°C, preferably 100°C-200°C, for example 150°C; Preferably, the Chinese medicine Atractylodes macrocephala samples are Atractylodes macrocephala samples from different origins, such as highly similar Atractylodes macrocephala samples from different origins; preferably Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang; further preferably Atractylodes macrocephala samples from Anhui, Atractylodes macrocephala samples from Henan, Atractylodes macrocephala samples from Hunan and Atractylodes macrocephala samples from Zhejiang; Preferably, after obtaining mass spectrum information using the device, characteristic markers of the sample are screened; preferably, the characteristic markers are compounds corresponding to the following m / z values: 318, 158, 489, 133, 533, 577, 149, 445, 319, 579, 349, 413, 377, 302, 365, 175, 317, 116, 301, 429, 279, 117; Preferably, after obtaining the characteristic marker information, it is further analyzed and identified by chemometric multivariate statistical analysis methods.

Images