Bubble extraction method suitable for component analysis of complex biological matrix sample and application

Abstract

The invention discloses a bubble extraction method suitable for component analysis of a complex biological matrix sample and application, and belongs to the technical field of chemical analysis. According to the method, ammonia gas is creatively used as a bubble breaking carrier gas, so that effective formation of aerosol in complex matrix bubble extraction is realized, and direct mass spectrometry of complex matrixes such as mulberry juice and serum is further realized. In addition, a bubble extraction technology taking ammonia gas as carrier gas also has excellent matrix purification capability. By means of the good aerosol forming ability of the ammonia gas, direct bubble extraction mass spectrometry of samples such as fruit juice and serum is achieved, and quantitative analysis of specific components such as mulberry acid can also be achieved. In conclusion, the rapid, efficient, non-toxic and accurate sample pretreatment method is provided for analysis of complex matrix samples such as food, urine and serum, and the method is expected to be popularized into a universal sample pretreatment technology in the research fields of food, biochemistry and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis, and particularly relates to a bubble extraction method applicable to the component analysis of complex biological matrix samples and its application. Background Art

[0002] Mass spectrometry (MS) has the advantages of fast analysis speed, high sensitivity, wide range of detection objects, and high efficiency, and has been widely used in many fields such as food analysis, drug analysis, and environmental analysis. Complex matrices can enhance or suppress ion intensity by interfering with the ionization process of target analytes, resulting in an increase or decrease in the detection signal, causing deviations in the detection results, insufficient sensitivity of the lower limit of quantification, low accuracy of high-concentration samples, and sometimes precipitation, which can clog the chromatographic column, contaminate the instrument, and shorten the service life of the instrument. This phenomenon will have an adverse effect on the ionization efficiency of analytes, and further affect important method performance parameters. Therefore, the main challenge faced when using mass spectrometry technology to analyze complex matrices (such as biological and food samples) is its susceptibility to matrix effects.

[0003] Biological samples and food samples are the most complex matrices, containing many substances that may interfere with target molecules, so complex pretreatment steps are required. These interfering substances include high-concentration salts, fats, etc., and they still exist even after sample pretreatment. Therefore, appropriate sample pretreatment is always required to remove these interferences.

[0004] Conventional pretreatment methods for eliminating matrix effects, such as protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE), sample dilution (or reduction of injection volume), etc., have problems such as numerous operation steps, long extraction time, and poor reproducibility. In addition, the current sample preparation process or subsequent sample treatment often involves the use of a large amount of organic solvents. For example, buffer salts, ion-pair reagents, or anticoagulants, anti-adsorbents (urine, cerebrospinal fluid), etc. will be added to the sample collection or storage containers. For samples such as food, urine, serum, and plasma, their matrix types are numerous, the components are complex, and the content of analytes to be measured is low. Therefore, for different matrix samples, specific sample pretreatment methods are usually required, which cover cumbersome processes such as extraction, purification, and separation. In addition, during the processing, there is also a risk of sample loss in the multi-step and complex sample preparation and purification processes for eliminating matrix effects (MEs). Therefore, there is an urgent need to establish a fast, accurate, green, and efficient sample pretreatment method to process complex matrix samples.

[0005] Bubble bursting technology (BB) has efficient gas / liquid interfacial properties, as well as preconcentration and ionization effects. When combined with an analytical detector (such as MS), it can improve the sensitivity of target analytes and achieve efficient qualitative and quantitative analysis of organic substances, thus effectively overcoming the problems brought by the above-mentioned conventional sample treatment methods. In terms of preconcentration, prior art has disclosed a bubble extraction system based on the greenhouse gas CO2. In this system, in addition to observing the preconcentration of organic substances, metal ions and inorganic anions, the bubble extraction (BE) triggered by bubble bursting can also effectively reduce the content of metal organic compounds in the sample. Therefore, this technology can be used to analyze and identify volatile components in the sample.

[0006] Bubble extraction technology theoretically has high enrichment ability and high greenness, so it has application potential in the component analysis of complex matrix samples. However, so far, this technology is generally only used for the analysis of samples with simple matrices such as natural water bodies, and rarely shows good application in the field of complex matrices, far from meeting the processing requirements of complex matrices. The main reason is that there are a large number of surface active substances in complex matrices, and these active substances boil out together with the bubbles and cannot form effective aerosols. This limits the application and development of bubble extraction technology in the component analysis of complex matrices.

[0007] Therefore, developing a bubble extraction method suitable for the component analysis of complex biological matrix samples has important application value and significance. Summary of the Invention

[0008] Based on the problem that the existing bubble extraction technology cannot effectively form aerosols when dealing with complex biological matrices, the present invention proposes the following solutions.

[0009] The present invention provides the application of ammonia in the bubble extraction of biological matrices.

[0010] In the above application, the ammonia is used as the carrier gas for bubble extraction.

[0011] In the above application, the biological matrix is selected from simple biological matrices or complex biological matrices; the simple biological matrix is selected from one of clarified food, drug and cosmetic systems (such as clarified drinks, oral liquids, aqueous skin care products, etc.); the complex biological matrix is selected from one of food systems such as fruit juices, emulsions, fermentation broths, and biological samples such as urine, serum, plasma, lymph fluid, interstitial fluid, cerebrospinal fluid, gastric juice, intestinal juice, bile, etc.

[0012] The present invention provides a method for bubble extraction of a biological matrix, wherein the method uses ammonia as a carrier gas to perform bubble extraction; the parameters of the bubble extraction can be conventionally selected or adjusted according to the actual application scenario or extraction device; the present invention also provides a bubble extraction parameter selected from the following conditions: a sand plate porosity of 20-35 μm, a sand plate diameter of 15-30 mm, a bubble extraction volume of 10-20 mL, a flow rate of 1-3 mL / min, and a bubble extraction time of 1-20 min.

[0013] In the present invention, the bubble extraction adopts a conventional bubble extraction device; for example, a sand core funnel (Chongqing Xinweier Glass Instrument Co., Ltd.).

[0014] The present invention provides application of ammonia in quantitative analysis of mulberry acid or phospholipid in mulberry juice.

[0015] The present invention provides application of ammonia in quantitative analysis of amino acids, phospholipids or peptides in serum.

[0016] In the above quantitative analysis application, the quantitative analysis is performed by means of a bubble extraction method using ammonia as a carrier gas and conventional mass spectrometry analysis; specifically, the aerosol collected by the bubble extraction is subjected to mass spectrometry analysis to quantitatively analyze the corresponding components.

[0017] The beneficial effects of the present invention are: The present invention pioneered the use of ammonia as a bubble burst carrier gas, which has very good aerosol-forming ability, thereby achieving effective aerosol formation in complex matrix bubble extraction, and further enabling direct mass spectrometry analysis of complex matrices such as mulberry juice and serum. The aerosol formed under ammonia conditions can be directly used for mass spectrometry analysis without clogging the chromatographic column with a particle size of 1.7 μm. In addition, the bubble extraction technology using ammonia as a carrier gas also has excellent matrix purification capabilities. With the good aerosol-forming ability of ammonia, not only direct bubble extraction mass spectrometry analysis of samples such as juice and serum is achieved, but also the quantitative analysis of specific components such as moric acid can be achieved. In summary, the present invention provides a rapid, efficient, non-toxic, and accurate sample pretreatment method for the analysis of complex matrix samples such as food, urine, and serum, and is expected to be promoted as a universal sample pretreatment technology in research fields such as food and biochemistry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A process for analyzing the composition of biological matrix samples, including bubble extraction and mass spectrometry analysis; Figure 2 Schematic diagram of bubbles in the bubble extraction process; Figure 3 The extraction effects of different carrier gases on anthocyanins, moric acid and phospholipids; Figure 4Aerosols collected for bubble extraction; wherein, A is H2O; B is the original mulberry juice; C is the CO2 bubble extraction aerosol; D is the NH3 bubble extraction aerosol; Figure 5 Particle size test results of the original mulberry juice Figure 6 Particle size test results of aerosols; wherein, A is the aerosol extracted by CO2 bubbles, and B is the aerosol extracted by NH3 bubbles; Figure 7 Comparison of mass spectrometry diagrams of serum samples of donkey, sheep, rabbit, horse, chicken, dog, guinea pig, and pig in positive ion mode Figure 8 Comparison of mass spectrometry diagrams of serum samples of donkey, sheep, rabbit, horse, chicken, dog, guinea pig, and pig in negative ion mode Figure 9 Heat map comparison of serum samples of donkey, sheep, rabbit, horse, chicken, dog, guinea pig, and pig in positive ion mode (A) and negative ion mode (B); wherein, amino acids (Mw: 50 - 200 Da), phospholipids (Mw: 600 - 900 Da), and peptides (Mw > 1000 Da). Detailed implementation mode

[0019] In the present invention, nitrogen (≥99.9%), air (≥99.999%), carbon dioxide (≥99.9%), helium (≥99.999%), argon (≥99.999%), oxygen (≥99.999%), and ammonia (≥99.999%) are used as bubbling gases for bubble extraction.

[0020] In the present invention, the mass spectrometry parameters were set as follows (Waters Xevo G2-XS UPLC-QTOF high-resolution liquid chromatography-mass spectrometry system): Cone voltage: 40 V, Voltage offset: 80 V, Ion source temperature: 120 °C, Desolvation temperature: 400 °C, Cone gas flow rate: 50 L / h, Desolvation gas flowrate: 800 L / h, Ion signal acquisition time for single massspectrum recording: 0.2 s. Argon (Ar) was used as the Collision gas. Collision-induced dissociation (CID) was performed on the selected parent ions using different collision energies to obtain tandem mass spectra (MS / MS). Mass spectrometry data were acquired in Profile mode.

[0021] In the present invention, the compound identification and chromatographic conditions were as follows: The extracted compounds were identified using a Waters Xevo G2-XS QTOF mass spectrometer. The UPLC system was equipped with a photodiode array detector (PDA) and an electrospray ionization source (ESI). Chromatographic column: Waters BEH C18 column (2.1 mm × 100 mm, 1.7 μm; Waters Corporation, Milford, Massachusetts, USA). Chromatographic analysis conditions: Mobile phase A: aqueous solution containing 0.1% formic acid; Mobile phase B: acetonitrile; Gradient elution program: 0 - 5 min (95% A - 5% A / 5% B - 95% B), 5 - 8 min (90% A - 10% A / 10% B - 90% B), 8 - 14 min (60% A - 40% A / 40% B - 60% B), 14 - 18 min (20% A - 80% A / 80% B - 20% B), 18 - 20 min (0% A - 100% A / 100% B - 0% B), returning to the initial conditions within 3 min. Injection volume: 1 μL, Flow rate: 0.3 mL / min.

[0022] In the present invention, the mass spectrometry analysis conditions are as follows: The compounds are analyzed in the positive ionization mode. Detection conditions: Capillary voltage: 3.0 kV, Desolvation gas flow: 800 L / h, Cone gas flow: 50 L / h, Desolvation temperature: 400 °C, Ion source temperature: 120 °C, m / z scanning range: 50–1200 Da.

[0023] In the present invention, the aerosol is characterized as follows: The collected aerosol is analyzed for particle size and potential using a Nanoparticle size and Zeta potential analyzer (ZEN5600), and each sample is measured three times. Zeta potential parameters: Refractive index (RI, Material): 1.590, Absorbance: 0.010, Dispersant: Water, Temperature: 25 °C, Viscosity: 0.8872 centipoise (cp), Refractive index (RI, Dispersant): 1.330, Dielectric constant: 78.5, Equilibrium time: 120 seconds (s), Measurement angle: 173°, Backscattering: Optical non-invasive backscattering (ONIBS), Number of runs: 11 times, Run time: 10 seconds (s).

[0024] In the present invention, the bubble rupture with weak acidic and weak basic gases (such as CO2 and NH3) will change the pH value of the solution. Therefore, the pH of the aerosol formed with carbon dioxide and ammonia as the carrier gas is detected (PHS-320).

[0025] In the present invention, the chromaticity determination method is as follows: Use a Minolta CR400 (Japan) color difference meter to measure the color of mulberries after different treatments. Each sample is measured 3 times repeatedly, and the average value is taken to ensure accurate L*, a*, and b* values. The L* value represents the lightness coefficient of the sample, ranging from 0 to 100; when the L* value is 0, the sample color is black (no reflection); when the L* value is 100, the sample color is white (fully reflected). The a* value represents the red-green chromaticity coefficient, indicating the range from red (positive value) to green (negative value). The b* value represents the yellow-blue chromaticity coefficient, indicating the range from yellow (positive value) to blue (negative value). If both a* and b* values are 0, it means the sample color is gray. Data analysis method: The obtained results are expressed as mean ± standard deviation. Use SPSS software (version 21.0, SPSS Inc., Chicago, IL, USA) for analysis of variance (ANOVA). Multiple comparisons are performed according to the Duncan test at a 5% significance level (p < 0.05).

[0026] In the present invention, ultrapure water was purchased from Wahaha Company (Hangzhou, China). Methanol (MeOH), acetonitrile (ACN), ethanol (EtOH), acetone (Ace), n-hexane (n-Hex), dichloromethane (DCM), ethyl acetate (EtOAc), and formic acid were all high-performance liquid chromatography (HPLC)-grade reagents, purchased from Thermo Fisher Scientific (Shanghai, China). Hydrochloric acid was purchased from Kelong Chemical Co., Ltd. (Chengdu, China). Cyanidin-3-O-rutinoside (≥ 98%) and pelargonidin-3-O-rutinoside (≥ 90%) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Cyanidin-3-O-glucoside (≥ 98%) was purchased from Guangzhou Qiyun Biotechnology Co., Ltd. (Guangzhou, China). Pelargonidin-3-O-glucoside chloride (≥ 98%), pelargonidin chloride (≥ 97%), delphinidin chloride (≥ 97%), and cyanidin chloride (≥ 95%) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). cis-4-Hydroxy-L-proline (98%) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Donkey serum, sheep serum, rabbit serum, horse serum, chicken serum, dog serum, guinea pig serum (10%), and pig serum (negative) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The pH buffers potassium hydrogen phthalate and sodium tetraborate were purchased from Tianjin Aoran Fine Chemical Research Institute.

[0027] In the present invention, due to the instrument range, the particle size of the mulberry original juice exceeds the particle size measurement range of the nano particle size and Zeta potential analyzer. Therefore, a Laser particle size analyzer (HELOS-OSIS) is used for detection. The particle size measurement parameters are set as follows: Sample state: Wet sample, Measurement range: R6, 0.5~1750 μm, Trigger condition: 10 seconds (SUCELL), Dispersion system: SUCELL 800 s-8 clearn, Pump speed: 60%, Stirrer speed: 80%, Ultrasound: No ultrasound, Display trigger condition: HELOS / RF, Blank measurement duration: 10 seconds (s), Time base: 100.0 milliseconds (ms).

[0028] In the present invention, the analysis process and experimental device of the biological matrix sample components are as Figure 1 shown, including bubble extraction (sample pretreatment) and mass spectrometry analysis process.

[0029] Other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way.

[0030] I. Mulberry juice 1. Preparation of mulberry juice Select mulberry fruits with similar shapes, weights, and colors, place them in a foam box for refrigeration and quickly transport them to the laboratory, and then select fruits with uniform diameters and colors for the experiment. Mix 100 g of fresh mulberry fruits with distilled water (1:1, weight / volume ratio) and mash them to obtain turbid mulberry juice (crude extract). To remove the fruit residues, filter the crude extract through 5 layers of gauze. Transfer the filtered crude extract into a 1.5 L bottle and store it at -4°C for analysis.

[0031] 2. Sample pretreatment (1) Bubble extraction The schematic diagram of the bubble extraction experimental device is as Figure 1 shown.

[0032] Add 5 mL of pure water to 5 mL of the sample (mulberry juice). Then add the internal standard. For anthocyanin compounds, cyanidin chloride is used as the internal standard, and for moronic acid and phospholipids, cis-4-hydroxy-L-proline is used as the internal standard. According to the concentration of the target components to be measured at the μg / mL level, prepare an internal standard of 1 mg / mL. Take 100 μL of the internal standard and add it to the above 10 mL of the sample to make its concentration in the sample 10 μg / mL. Then mix well and perform bubble extraction. The aerosol after bubble extraction is collected with a quartz sample plate (sand plate) and directly measured.

[0033] The bubble extraction parameters are as follows: The porosity of the sand plate is 20 - 35 μm, the diameter of the sand plate is 20 mm, the bubble extraction volume is 15 mL, the flow rate is 2 mL / min, and the bubble extraction time is 5 min; The carrier gases are respectively CO2, Air, N2, He, Ar, O2, NH3 to investigate the influence of different carrier gases on the bubble extraction effect (i.e., the extraction effect on anthocyanins, moronic acid, and phospholipid compounds in mulberry juice).

[0034] Collect the aerosol after the bubble rupture of the sample using the above method and parameters. The present invention provides a schematic diagram of the aerosol, as Figure 2 shown.

[0035] (2) Conventional treatment Measure 5 mL of mulberry juice and then add 5 mL of pure water. Using cyanidin chloride as the internal standard, prepare an internal standard of 1 mg / mL, and then take 100 μL of the internal standard and add it to the above 10 mL of the sample to make its concentration in the sample 10 μg / mL. Then place the beaker in an ultrasonic bath at room temperature for 5 min and filter it through a 0.22 μm organic filter membrane into a vial for UPLC-MS analysis.

[0036] 3. Mass spectrometry analysis of bubble extraction Perform mass spectrometry analysis on the aerosol collected by bubble extraction using the mass spectrometry instrument and parameters disclosed above. At the same time, perform pH detection on the aerosols formed with CO2 and NH3 as the carrier gases. And measure the chromaticity of the aerosol.

[0037] The mass spectrometry analysis results are as Figure 3 shown: Among them, in Figure 3Among them, 1 is chlorinated pelargonidin; 2 is chlorinated delphinidin; 3 is chlorinated cyanidin-3-O-glucoside; 4 is chlorinated cyanidin-3-O-rutinoside; 5 is chlorinated pelargonidin-3-O-glucoside; 6 is chlorinated delphinidin-3-O-rutinoside; 7 is Morμsimic acid C; 8 is Morμsimic acid A; 9 is Morμsimic acid B; 10 is Morμsimic acid D or Morμsimic acid F; 11 is hexadecasphinganine; 12 is dehydrophytosphingosine; 13 is Lμteolin-8-C-glμcoside; 14 is lysophosphatidylcholine 14:3; 15 is C 25 H 39 N3O6; 16 is Lysophosphatidylcholine 18:2; 17 is 1-palmitoyl-sn-glycero-3-phosphoethano-lamine; 18 is Lysophosphatidylcholine 16:0; 19 is Lysophosphatidylcholine 18:1; 20 is Lysophosphatidylcholine 18:0lamine.

[0038] Depend on Figure 3 As can be seen, bubble extraction using air, N2, He, Ar, and O2 as carrier gases is very poor. This is because complex matrix samples contain a large amount of surfactant, which makes continuous bubbles more likely to emulsify and form continuous foam, resulting in poor aerosol formation and, consequently, inability to effectively collect and analyze the aerosol composition.

[0039] When CO2 is introduced, more and stronger peaks can be obtained compared to other carrier gases (except ammonia). When CO2 comes into contact with water, the solvent weakly acidifies the aqueous solution, and the interaction between CO2 molecules and analytes is stronger, resulting in higher enrichment.

[0040] Notably, this invention utilizes ammonia as a bubble-bursting carrier gas for the first time. It has been shown to have a strong bubble-bursting aerosol collection capability (more pronounced than CO2) in complex matrices like mulberry juice. The collection of moric acid and phospholipids is particularly efficient. This may be due to ammonia's strong affinity for water, which allows for better aerosol formation.

[0041] The pH test results are shown in Table 1: Table 1 Effect of different carrier gases on aerosol pH

[0042] In bubble extraction, CO2 can enhance the acidity of the aqueous solution. However, in the mulberry juice system, it does not make the acidity stronger. The pH value of the original mulberry juice is 3.1617, and the pH value of the aerosol extracted by CO2 is 3.3953. Instead, the pH value increases. The possible reason is the buffering effect between the bicarbonate formed by CO2 and water and the proton or anthocyanin cation.

[0043] Introducing ammonia makes the aerosol highly alkaline, with a pH value of 10.4610, greater than 10. Since ammonia bubbles are alkaline, the negatively charged hydroxyl groups in ammonia water have electrostatic interactions with the positively charged phospholipid compounds. As the hydroxyl groups are consumed, the concentration of NH 4+ increases and will have electrostatic interactions with the negatively charged mulberry acid. This electrostatic interaction enables the good enrichment of both mulberry acid and phospholipids.

[0044] The aerosol collected by bubble extraction is as Figure 4 shown; the results of its chromaticity test are shown in Table 2: Table 2 Effects of different treatment methods on the color parameters of mulberry juice

[0045] In Table 2, L* is the lightness coefficient; a* is the redness coefficient; b* is the yellowness coefficient. Each value is expressed as the mean ± standard deviation (n = 3). The means with different letters in the same column are significantly different (p < 0.05).

[0046] From the above results, it can be seen that both treatments significantly affected the color parameters of the samples (p < 0.05). The color of the CO2 bubble aerosol is slightly lighter than that of the original mulberry juice.

[0047] Compared with fresh mulberry juice, the L* and b* values in the mulberry juice treated with NH3 increased, while the a* value decreased. This may be because the mulberry juice is alkaline under the NH3 system, and the anthocyanins are degraded, thus reducing the redness of the system.

[0048] In addition, the a* and b* values of the mulberry juice treated with CO2 increased significantly, but the increase amplitude of the latter was less than that of the mulberry juice treated with NH3. This result may be because CO2 dissolved in the mulberry juice forms H2CO3, enhancing the acidity of the environment. In a weakly acidified aqueous solution, the anthocyanins in the mulberry juice undergo a "red shift", resulting in a darker red color.

[0049] Among them, the higher L* value compared to the original mulberry juice may be that the particles in the solution blocked the light transmittance, making the original mulberry juice have a lower brightness. This also indirectly proves that the particles in the aerosol after bubble extraction are small particles, having a good matrix removal effect, and the L* value is higher than that of the original mulberry juice.

[0050] 4. Matrix purification effect The pulp particles suspended in mulberry juice (with a diameter greater than 100 µm) will precipitate quickly due to a certain density difference with the juice, and most of the fruit residues can be removed after rough filtration. There are still some complexes formed by the interaction of invisible particulate pectin substances, a small amount of fibers, proteins, carbohydrates and other macromolecular substances in the filtered mulberry juice. Their particle size is relatively large and may not be directly applicable to the analysis of chromatographic columns.

[0051] Therefore, particle size detection was carried out in the experiment. Due to the instrument range reason, the particle size detection of the original mulberry juice, the aerosol extracted by CO2 bubbles and the aerosol extracted by NH3 bubbles was not carried out with the same instrument. The nano particle size measurement range is 0.3 nm - 10 µm. After exceeding this detection range, the instrument will report an error and cannot measure, which also indirectly shows that the particle size of the original mulberry juice is relatively large.

[0052] The test results are as Figure 5 and Figure 6 shown: There are more particles with a particle size of about 10 µm in the original mulberry juice. Among the particles with a size of ≤180 µm, the particles with a size of ≤9 µm account for 51% of the total. At the same time, there are large particles with a size of more than 500 µm. The particle size of the original mulberry juice is much larger than that of the chromatographic column (1.7 µm), and the chromatographic column cannot be directly used for sample injection analysis.

[0053] The pretreatment process of complex matrix samples determines the qualitative and quantitative capabilities of the target analytes. The traditional sample pretreatment of mulberry juice usually uses filtration or centrifugation to remove the complex matrix, but the effect is not good.

[0054] Using the bubble extraction method for pretreatment, after extraction with CO2 and NH3 bubbles, the particle size of the aerosol particles is significantly reduced (equivalent to the purification of the target components from the complex matrix samples). Although the average particle size of the aerosol extracted by CO2 bubbles is close to that of the chromatographic column, most of the particle sizes are distributed at 100 nm, and no column clogging phenomenon occurred during the experiment. Most of the particle sizes of the aerosol extracted by NH3 bubbles are around 100 nm in average particle size, which is smaller than the 1.7 µm particle size of the chromatographic column.

[0055] In comparison, after bubble extraction, the particle size is significantly reduced, minimizing the interference of the matrix in the complex matrix samples as much as possible, avoiding the disadvantages of long sample loading time and easy clogging in traditional pretreatment techniques, effectively purifying the samples, and greatly reducing the analysis difficulty of complex matrix samples. It can be seen that bubble extraction has a significant purification effect on the matrix. Especially when ammonia is used as the carrier gas, the particle size can be significantly reduced, which is more conducive to subsequent mass spectrometry analysis.

[0056] 5. Semi - quantitative analysis of mulberry acid Pharmacological studies have shown that mulberry acid has significant lipid-lowering activity and can significantly regulate the expression of key target proteins (PCSK9 and LDL-R), providing a basis for the development of drugs for the treatment of obesity and hyperlipidemia.

[0057] In this experiment, semi-quantitative analysis of mulberry acid was performed based on the ratio of the peak area of the internal standard (cis-4-Hydroxy-L-proline) added to the peak area of mulberry acid.

[0058] The test results are shown in Table 3: Table 3 Semi-quantitative analysis of mulberry acid

[0059] Comparing carbon dioxide and ammonia, the results showed that the concentration of mulberry acid obtained by ammonia bubble extraction was significantly higher than that by carbon dioxide bubble extraction, which further proved that ammonia was significantly superior to carbon dioxide in the component analysis of complex matrices.

[0060] II. Serum Based on the strong aerosol collection ability and matrix purification ability of ammonia for mulberry juice, in order to further expand and verify its effect, ammonia was used to perform bubble extraction on donkey, sheep, rabbit, horse, chicken, dog, guinea pig, and pig serum samples [dissolved in ethanol / water (1:1) solution] and mass spectrometry analysis was carried out. The bubble extraction method and mass spectrometry analysis parameters were the same as those disclosed above.

[0061] The test results are as Figure 7 、 Figure 8 and Figure 9 shown: According to Figure 7 and Figure 8 , from the total ion current chromatograms detected in both positive ion and negative ion modes, it can be seen that compared with the untreated serum that cannot be directly analyzed by mass spectrometry, the serum samples after bubble extraction can be analyzed by mass spectrometry, and the signal-to-noise ratio of the mass spectrometry spectrum and the baseline flatness are good, indicating that ammonia treatment can effectively reduce the interference of large particle substances (such as proteases, immunoglobulins, etc.) and electrolytes in serum samples on mass spectrometry analysis. Further through Figure 9 results, it can be known that in the serum aerosol after bubble extraction, amino acids (50 - 200 Da), phospholipids (600 - 900 Da), and peptides (oligopeptides 200 - 1200 Da, polypeptides 1200 - 5000 Da) components were significantly detected. Among them, the detection abundance of some components was higher in the negative ion mode, indicating that ammonia bubble extraction can effectively purify the matrix of serum samples, and can quickly and accurately separate these components from the complex blood matrix, thereby improving the analysis ability of serum sample omics and greatly promoting the research on disease biology.

[0062] In summary, by utilizing the rupture of bubbles formed by ammonia gas on the surface of the solution, the direct extraction and ionization mass spectrometry analysis of complex matrix samples such as mulberry juice and serum have been successfully achieved. In addition, ammonia also has good matrix removal ability, providing an important sample pretreatment method for complex matrix samples. Among them, as the carrier gas for bubble rupture, ammonia has excellent aerosol formation ability, and the resulting efficient enrichment and ionization performance expand the application of bubble rupture in complex matrices such as fruit juice and serum. This research has promoted the direct mass spectrometry analysis and application of bubble extraction technology in complex matrix samples, which is an important breakthrough, providing a more efficient tool and way out for future food health and drug analysis.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention in any other form. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Application of ammonia in bubble extraction of biological matrix.

2. The application according to claim 1, characterized in that The ammonia is used as the carrier gas for bubble extraction.

3. The application according to claim 1, wherein The biological matrix is selected from simple biological matrix or complex biological matrix.

4. The application according to claim 3, characterized in that The simple biological matrix is selected from one of clarified food, pharmaceutical and cosmetic systems; the complex biological matrix is selected from one of food systems such as fruit juice, emulsion, fermentation broth, etc. and biological samples such as urine, serum, plasma, lymph fluid, interstitial fluid, cerebrospinal fluid, gastric juice, intestinal juice, bile, etc.

5. A method for extracting biological matrix bubbles, characterized in that, The method uses ammonia as the carrier gas for bubble extraction.

6. The biological matrix bubble extraction method according to claim 5, wherein The bubble extraction is selected from the following parameter conditions: the porosity of the sintered glass plate is 20 - 35 µm, the diameter of the sintered glass plate is 15 - 30 mm, the volume of bubble extraction is 10 - 20 mL, the flow rate is 1 - 3 mL / min, and the bubble extraction time is 1 - 20 min.

7. The biological matrix bubble extraction method according to claim 5, wherein The bubble extraction uses a conventional bubble extraction device.

8. Application of ammonia in quantitative analysis of morolic acid or phospholipids in mulberry juice.

9. Application of ammonia in quantitative analysis of amino acids, phospholipids or peptides in serum.

10. The application according to claim 8 or 9, characterized in that, The quantitative analysis is carried out by means of bubble extraction method with ammonia as the carrier gas and conventional mass spectrometry analysis.

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