A kind of Ginkgo biloba extract containing alcohol chromatography liquid 1 H NMR spectrum analysis method

Abstract

The present invention relates to the technical field of Chinese medicine component analysis, in particular to a chromatography solution containing alcohol for Ginkgo biloba extract. 1 H NMR spectrum analysis method. The method includes the following steps: (1) preparing the test sample; (2) collecting 1 H NMR spectrum; (3) 1 H NMR spectrum signal attribution; (4) quantitative analysis. The quality control method of the present invention is reasonable, has the characteristics of simple sample preparation, rapid analysis, good reproducibility, etc., and is a major breakthrough in the quality control method of the pharmaceutical process of traditional Chinese medicine preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of Chinese medicine component analysis, in particular to a chromatography solution containing alcohol for Ginkgo biloba extract. 1 HNMR spectrum analysis method. Background Art

[0002] The main active ingredients of ginkgo extract include flavonoids, terpenoid lactones, and polyphenols. Among them, flavonoids such as quercetin, kaempferol, and isorhamnetin have significant antioxidant and anti-inflammatory effects, effectively protecting cells from free radical damage. Terpenoid lactone components, including ginkgolides and bilobalide, have multiple biological activities, including improving blood circulation, anti-thrombotic effects, vasodilation, and neuroprotection. In addition, polyphenols also exhibit excellent antioxidant properties. The synergistic effect of these components makes ginkgo extract have significant pharmacological value in preventing cardiovascular disease, improving cognitive function, and anti-aging. In actual production, the extraction of active ingredients is mainly achieved through column chromatography. As a commonly used separation and purification technology, column chromatography utilizes the differences in the partition coefficients of different compounds between the stationary phase and the mobile phase to separate the active ingredients in a mixture, thereby achieving selective adsorption and purification of the active ingredients or effective parts in the traditional Chinese medicine extract. The chromatography process for producing Ginkgo biloba extract typically involves the following steps: First, the Ginkgo biloba leaves are crushed and dried, then extracted with an ethanol-water solution to obtain a crude extract. The crude extract is then subjected to rotary evaporation and filtration to remove most impurities, resulting in a concentrated filtrate. The filtrate is then separated and purified by column chromatography. During the chromatographic separation process, a fixed concentration of ethanol-water is used as the mobile phase, and a macroporous resin is used as the stationary phase. After the filtrate is loaded onto the chromatography column, the mobile phase continuously passes the ethanol-water solution through the column bed at a constant flow rate. Different components are eluted from the column in sequence due to their different affinities with the stationary phase.

[0003] Currently, the content of total flavonoids and terpenoid lactones, the main active ingredients in ginkgo extract, is primarily determined using high-performance liquid chromatography (HPLC). Flavonoids, due to their strong UV absorption, are typically detected using an ultraviolet (UV) detector, typically set at a wavelength of 360 nm. Regarding chromatographic conditions, most HPLC analyses utilize octadecylsilane-bonded silica gel (C18) as the column packing, with a mobile phase typically consisting of methanol or acetonitrile combined with an aqueous solution containing an appropriate amount of acid to optimize the separation of flavonoids. To determine the total flavonoid content in ginkgo extract, a specific proportion of hydrochloric acid solution is typically used as the solvent to hydrolyze the flavonol glycosides. The contents of quercetin, kaempferol, and isorhamnetin are then determined, and the total flavonoid glycoside content is calculated using a correction factor. Terpenoid lactones, due to their weak UV absorption, are typically detected using an evaporative light scattering detector (ELSD) or electrospray ionization detector (CAD). Sample pretreatment is required for the analysis of terpenoid lactones due to the poor selectivity of the detector. In most cases, the sample is pretreated using a complex ethyl acetate extraction method. In terms of chromatographic conditions, when using a CAD detector, the mobile phase is usually a methanol-water solution; when using an ELSD detector, the mobile phase is often a combination of n-propanol / acetonitrile-tetrahydrofuran-water to ensure effective separation and accurate detection of the components. Although the HPLC method has shown high sensitivity and accuracy in the analysis of the active ingredients of ginkgo extract, its sample pretreatment process is cumbersome and requires a lot of time and effort, especially in the sample hydrolysis and purification steps, which significantly increases the complexity and time cost of the operation. This greatly limits its application efficiency for large-scale production or rapid detection needs.

[0004] 1 H NMR technology has both qualitative and quantitative functions, and has the advantages of being fast and reproducible. At present, some traditional Chinese medicine preparations have used this method as a means of quality control, such as Chinese patent CN113237913A discloses a Danshen injection and its process intermediates. 1 H NMR single-measurement multiple-evaluation method. This method includes the following steps: (1) preparation of external standard samples and test samples; (2) 1 H NMR spectrum determination; (3) 1 H NMR fingerprint construction and analysis; (4) content determination and comprehensive quality evaluation. The method of the invention is simple and fast to operate, and the test solution 1 After H NMR spectral preprocessing, a total of 40 chemical components were identified, including 12 amino acids, 7 small-molecule organic acids, 8 sugars and their degradation products, 7 salvianolic acid compounds, and 6 nucleoside compounds. Compared with traditional quality inspection and evaluation methods, this method replaced multiple methods with one method and multiple tests with a single determination, simplifying the sample preparation process and significantly shortening the analysis time.

[0005] For example, Chinese patent CN113466281B discloses a 1 A method for determining the content of total ginsenosides in Shenmai injection using H qNMR technology. The method comprises the following steps: (1) preparation of the test sample; (2) 1 HNMR analysis; (3) 1 H NMR spectrum pretreatment; (4) calculation of ginsenoside content. The invented method is simple to operate, has a short analysis time, strong specificity, does not rely on a standard curve, requires a small amount of sample and does not damage the sample, thus providing a reliable means for quality control of Shenmai injection.

[0006] But the existing 1 H NMR analysis mainly uses the presaturation method to analyze aqueous solutions such as traditional Chinese medicine injections. When the ethanol content in the sample is high, the ethanol signal will seriously affect the 1 H NMR qualitative and quantitative analysis is generally performed after evaporating the ethanol, which increases the workload and practice of pretreatment. Currently, there is no direct analysis of alcohol-containing Chinese medicine intermediates in the pharmaceutical process. 1 Therefore, it is urgent to study a method for the analysis of alcohol-containing chromatography fluid in the column chromatography process of Ginkgo biloba extract. 1 A method for rapid acquisition of H NMR spectra and quantitative component analysis. Summary of the Invention

[0007] Based on the shortcomings of the prior art, the present invention aims to provide a 1 A method for quantitative analysis of Ginkgo biloba extract by H NMR spectroscopy.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A kind of Ginkgo biloba extract containing alcohol chromatography liquid 1 The H NMR spectrum analysis method comprises the following steps:

[0010] 1) Prepare the test sample: weigh the internal standard component and dissolve it in deuterated water to prepare a deuterated water sample solution containing the internal standard; take the alcohol-containing chromatography fluid sample to be tested and prepare the test sample solution with the deuterated water sample solution containing the internal standard;

[0011] (2) Collection 1 H NMR spectrum: Use a multi-solvent peak suppression pulse sequence to suppress the water peak and ethanol peak to complete the test solution 1 Determination of H NMR spectra;

[0012] (3) 1 H NMR spectrum signal attribution: 1The H NMR spectrum was pre-processed, and the characteristic peaks of terpenoid lactone components and flavonol glycoside components were identified;

[0013] (4) Quantitative analysis: Determine the quantitative signals of quantifiable terpenoid lactone components and flavonol glycoside components, integrate the characteristic peaks corresponding to chemical components exceeding the quantitative limit, and perform absolute quantification using the internal standard method.

[0014] Preferably, the internal standard component in step (1) is 3-(trimethylsilyl) sodium deuterated propionate (TSP);

[0015] Preferably, the concentration of TSP in the deuterated water sample solution containing the internal standard prepared in step (1) is 0.30-0.70 mg / mL;

[0016] Preferably, the volume ratio of the alcohol-containing chromatographic fluid to the deuterated water sample solution containing TSP in the test sample solution described in step (1) is 9:1.

[0017] Preferably, step (2) 1 The H NMR spectra were collected using a Bruker Avance 500 nuclear magnetic resonance spectrometer (Bruker, Germany).

[0018] Preferably, the conditions for the measurement in step (2) are: probe temperature of 290-316K; pulse sequence LC1PNGPPS; locked with solvent 90% H2O + 10% D2O; spectral width of 12.000-20.000ppm; suppression frequency of 0.800-1.300ppm (ethanol methyl signal), 3.200-3.700ppm (ethanol methylene signal), 4.600-4.900ppm (water peak); relaxation delay time of 10.0-25.0s; mixing time of 50-100ms; number of empty scans of 0-8 times; number of acquisitions of 32 or 64 times; detection data points of 32K or 64K;

[0019] Further preferably, the probe temperature is 295-300K; the pulse sequence is LC1PNGPPS; the solvent is 90% H2O + 10% D2O for locking; the spectral width is 13.000-16.000ppm; the suppression frequency is 0.900-1.100ppm, 3.300-3.600ppm, 4.650-4.800ppm, the relaxation delay time is 15-20s; the mixing time is 50-100ms; the number of empty scans is 4 times; the number of acquisitions is 32 times; the detection data point is 32K;

[0020] Further preferably, the probe temperature is 298K; the pulse sequence is LC1PNGPPS; the solvent is 90% H2O + 10% D2O; the spectral width is 14.2756ppm; the suppression frequencies are 4.714, 3.468 and 1.012ppm; the relaxation delay time is 15s; the mixing time is 100ms; the number of scans is 4 times; the number of acquisitions is 32 times; and the detection data points are 32K.

[0021] Preferably, the suppression frequency in the determination conditions described in step (2) is obtained by conventional hydrogen spectrum acquisition, and the conditions for conventional hydrogen spectrum acquisition are: pulse sequence zg30, probe temperature 295-300K, solvent 90% H2O + 10% D2O lock field, spectral width 13.000-16.000ppm, relaxation delay time 1s, number of empty scans 0 times, number of acquisitions 4 times, and detection data points 32K.

[0022] Preferably, in step (2) 1 During H NMR data acquisition, the multi-solvent peak suppression power needs to be adjusted so that the gain value is greater than 50 to ensure the suppression of the ethanol and water solvent peaks.

[0023] Preferably, in step (2) 1 H NMR data acquisition requires tuning, probe matching, and field shimming, and the corresponding 90° pulse width is measured;

[0024] Preferably, the spectrum preprocessing in step (3) includes Fourier transform, baseline correction, phase correction and chemical shift correction of the original FID signal;

[0025] Preferably, before Fourier transformation, the spectrum is processed using a 1.00 Hz exponential function as a window function;

[0026] Preferably, the baseline correction, phase correction and chemical shift correction are all completed manually in the NMR data processing software / NMR data processing website;

[0027] Preferably, the characteristic peak information of the five terpenoid lactones and total flavonol glycosides assigned in step (3) is shown in Table 1;

[0028] Table 1 Chemical components and characteristic peak information of alcohol-containing chromatographic fluid (500M NMR)

[0029]

[0030] Preferably, the internal standard method described in step (4) is calculated according to formula (1);

[0031]

[0032] Among them, Cx and C TSP are the mass concentrations of the chemical components to be tested and the internal standard TSP; A x and A TSP are the characteristic peak areas of the chemical components to be tested and the internal standard TSP; N x and N TSP are the characteristic peak proton numbers of the chemical component to be tested and the internal standard TSP; M x and M TSP are the relative molecular weights of the chemical components to be tested and the internal standard TSP, respectively.

[0033] Preferably, the quantitative signal information in step (4) is as shown in Table 2;

[0034] Table 2 Chemical component names and characteristic peak information of alcohol-containing chromatography fluid

[0035] serial number chemical composition Characteristic peak chemical shift and its multiplet information 1 Ginkgolide A 6.06(s) 2 Ginkgolide B 6.14(s) 3 Ginkgolide C 6.16(s) 4 Ginkgolide J 6.08(s) 5 bilobalide 6.41(s) 6 Total flavonol glycosides 6.39-6.27(m)

[0036] Preferably, the integration method described in step (4) includes but is not limited to the linear fitting method and GSD method in the nuclear magnetic data processing software;

[0037] In the present invention, the volume fraction of ethanol in the alcohol-containing chromatography liquid is 40%-70%.

[0038] Compared with existing analysis methods, the advantages of the present invention are as follows:

[0039] (1) No pretreatment such as hydrolysis and solvent evaporation is required. Analysis can be performed after simple sample preparation. It has rapid detection capabilities, and a single test takes less than 20 minutes, greatly shortening the analysis time.

[0040] (2) Suppressing the solvent signals of ethanol and water greatly improves the signal-to-noise ratio of the signal of the component to be measured, enabling direct analysis of alcohol-containing samples;

[0041] (3) The analytical method is highly reproducible, non-invasive, and non-destructive, and the sample can be recovered after the test is completed;

[0042] (4) The total flavonol glycosides in the chromatographic fluid can be quantified without hydrolysis. No reference substance for each compound is required for quantification, and no standard curve needs to be drawn. Only one internal standard is required for quantification.

[0043] The present invention provides 1 The H NMR analysis method has the ability to simultaneously obtain qualitative and quantitative information on the chemical components in the alcohol-containing chromatographic fluid in a single detection. Therefore, compared with the existing quality control methods that only focus on indicator components, the quality evaluation of the alcohol-containing chromatographic fluid in the present invention is more scientific and complete. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The chromatogram of the alcohol-containing chromatographic solution before and after solvent peak suppression and the signal peak attribution of Example 1;

[0045] Figure 2 The full spectrum and the amplified spectrum of the main secondary metabolite signal region obtained under the test conditions of Example 2;

[0046] Figure 3 The full spectrum and the amplified spectrum of the main secondary metabolite signal region obtained under the test conditions of Example 3;

[0047] Figure 4 The full spectrum and the amplified spectrum of the main secondary metabolite signal area obtained under the test conditions of Comparative Example 1;

[0048] Figure 5 The full spectrum and the amplified spectrum of the main secondary metabolite signal area obtained under the test conditions of Comparative Example 2;

[0049] Figure 6 The spectra obtained under the test conditions of Examples 1-3 and Comparative Examples 1-2 (the upper figure is the full spectrum, and the lower figure is the enlarged spectrum of the 5.6-9.2 ppm main secondary metabolite signal area). DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with specific examples, which are intended only to illustrate the present invention and do not limit the scope of protection of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements fall within the scope of protection of the present invention.

[0051] Example 1 A method based on 1 A method for quantitative analysis of Ginkgo biloba extract using H NMR spectroscopy

[0052] Specifically:

[0053] (1) Preparation of test sample: Accurately weigh 5.26 mg of TSP into a 10 mL volumetric flask, add deuterated water to the mark, dissolve thoroughly, and mix well to obtain a deuterated aqueous solution containing TSP with a concentration of 0.526 mg / mL; Accurately pipette 450 μL of alcohol-containing chromatography fluid and 50 μL of deuterated water containing TSP into a 2 mL centrifuge tube and mix well to prepare the alcohol-containing chromatography fluid test sample;

[0054] (2) Collection 1H NMR spectrum: The sample was placed in a nuclear magnetic resonance instrument for testing under the following conditions: probe temperature of 298K; pulse sequence LC1PNGPPS; lock field with solvent 90% H2O + 10% D2O, spectral width of 14.2756ppm, suppression frequencies of 4.714, 3.468 and 1.012ppm, relaxation delay time of 15s, mixing time of 100ms; number of empty scans 4 times, number of acquisitions 32 times, detection data points at 32K. The water peak and ethanol peak were suppressed by using a multi-solvent peak suppression pulse sequence to obtain the sample's peak. 1 HNMR spectrum; 1 H NMR data acquisition requires tuning, probe matching, and field shimming, as well as measuring the corresponding 90° pulse width.

[0055] (3) 1 H NMR spectrum signal attribution: The collected original spectrum was processed with a 1.0 Hz exponential function as the window function, and the FID signal was Fourier transformed. The spectrum was then imported into the NMR data processing software and calibrated with TSP (0.0 ppm) as the chemical shift reference, and baseline and phase correction were performed manually; the alcohol-containing chromatography solution 1 The signal identification of terpenoid lactones and flavonol glycosides in the H NMR spectrum was shown in Figure 2. Figure 1 , Figure 1 The chemical components indicated by the numbers are shown in Table 1.

[0056] (4) Quantitative analysis: The characteristic peaks of terpenoid lactones and flavonol glycosides and the TSP internal standard peak were integrated according to Table 2, and then the concentrations of the five terpenoid lactones and total flavonol glycosides were calculated according to formula (1). The results are shown in Table 3.

[0057] Table 3

[0058]

[0059] Example 2 A method based on 1 The specific method for quantitative analysis of Ginkgo biloba extract by H NMR spectroscopy is as follows:

[0060] The only difference from Example 1 is that in step (2) 1 The conditions for H NMR spectrum measurement are as follows: the probe temperature is 298K, the pulse sequence is LC1PNGPPS, the solvent is 90% H2O + 10% D2O, the spectral width is 12.0160ppm, the suppression frequencies are 4.707ppm, 3.462ppm and 0.989ppm, the relaxation delay time is 10s, the mixing time is 50ms; the number of empty scans is 0, the number of acquisitions is 32, and the detection data points are 32K. The test sample is placed in the nuclear magnetic resonance instrument for testing. The spectrum obtained is as follows Figure 2 .

[0061] Example 3 A method based on 1 The specific method for quantitative analysis of Ginkgo biloba extract by H NMR spectroscopy is as follows:

[0062] The only difference from Example 1 is that in step (2) 1 The H NMR spectrum was measured under the following conditions: a probe temperature of 298 K, a pulse sequence of LC1PNGPPS, a lock field of 90% H2O + 10% D2O solvent, a spectral width of 19.9947 ppm, suppression frequencies of 4.709 ppm, 3.480 ppm, and 1.007 ppm, a relaxation delay time of 25 s, a mixing time of 100 ms, 8 empty scans, 32 acquisitions, and a detection data point of 32 K. The sample was placed in a nuclear magnetic resonance instrument for testing. The spectrum obtained is shown below. Figure 3 shown.

[0063] Comparative Example 1 is based on the comparative document CN113237913A Example 1 1 The quantitative analysis of Ginkgo biloba extract was performed using H NMR spectroscopic analysis conditions as follows:

[0064] The only difference from Example 1 is that in step (2) 1 The H NMR spectrum was measured under the following conditions: probe temperature of 298 K, pulse sequence of NOESYGPPR1D, lock field of H2O+10% D2O solvent, spectral width of 12.0160 ppm, center frequency of 4.696 ppm, relaxation delay time of 15.0 s, acquisition time of 2.27 s, mixing time of 50 ms, acquisition times of 32, gain of 40.3, and detection data points at 32 K. The spectrum obtained is shown in the figure below. Figure 4 shown.

[0065] Comparative Example 2: A method based on 1 The specific method for quantitative analysis of Ginkgo biloba extract by H NMR spectroscopy is as follows:

[0066] The only difference from Example 1 is that in step (2) 1 The conditions for H NMR spectrum measurement are as follows: the sample was placed in a nuclear magnetic resonance instrument for testing at a probe temperature of 298K, a pulse sequence of LC1PNGPPS, a solvent of 90% H2O + 10% D2O for locking, a spectral width of 9.9974ppm, suppression frequencies of 4.294ppm, 3.113ppm, and 0.568ppm, a relaxation delay time of 2s, 0 empty scans, 16 acquisitions, and a detection data point of 32K. The spectrum obtained is shown in FIG. Figure 5 shown.

[0067] Comparison of the patterns collected under the conditions of Examples 1-3 and Comparative Examples 1-2 Figure 6 As shown, the spectra of Examples 1-3 are basically the same. Compared with Example 1, the signal suppression effect of Example 2 is slightly worse, which is because the relaxation delay time of Example 2 is shorter; the suppression effect and signal-to-noise ratio of Example 3 are comparable to those of Example 1, but due to the increase in the number of acquisitions, the time cost is significantly increased, and the acquisition time of Example 3 is twice that of Example 1; when the spectrum is acquired under the conditions of Comparative Example 1, since Comparative Example 1 only suppresses the water peak and does not process the ethanol signal, the RG is too high during spectrum acquisition, causing the final spectrum to be completely distorted; the suppression frequency of Comparative Example 2 is not within the range specified in the invention, and the solvent peaks of water and ethanol cannot be successfully suppressed. The solvent signal intensity of ethanol and water in the spectrum is very large, and the signal intensity of the components between 5.8-8.0ppm is extremely weak, and further analysis cannot be performed.

[0068] Effect experiment

[0069] 1. Ginkgo biloba extract alcohol chromatography fluid 1 Investigation of H NMR quantitative methodology:

[0070] (1) Precision: An NMR sample was prepared from the alcohol-containing chromatographic solution according to the method described in step (2) of Example 1. The NMR spectrum of the sample was collected six times continuously and the content of each component was calculated. The RSD value was calculated. The results showed that the RSD value of each characteristic peak was less than 3.72%, demonstrating that the instrument precision of this sample preparation method was good;

[0071] (2) Durability: An NMR sample was prepared from the alcohol-containing chromatographic solution according to the method described in step (2) of Example 1. The NMR spectra of the sample were collected at 296.5K, 297K, 297.5K, 298K, 298.5K, and 299K, and the content of each component was calculated. The RSD values were calculated. The results showed that the RSD values of each characteristic peak were less than 2.90%, indicating that the method has good durability.

[0072] (3) Sample preparation repeatability: Six NMR samples were prepared in parallel using the alcohol-containing chromatographic solution according to the method described in step (2) of Example 1. The NMR spectrum of each sample was collected and the content of each component was calculated. The RSD value was calculated. The results showed that the RSD value of each characteristic peak was less than 3.98%, indicating that the sample preparation repeatability of this method was good;

[0073] (4) Stability: Prepare an NMR sample from the alcohol-containing chromatographic solution according to the method described in step (2) of Example 1. Collect NMR spectra of the sample at six time points: 0, 3, 6, 12, 24, and 48 hours, calculate the content of each component, and calculate the RSD value. The results show that the RSD value of each characteristic peak is less than 3.41%. This proves that the test solution sample has good stability within 48 hours.

[0074] (5) Linear relationship: Take 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, and 800 μL of the alcohol-containing chromatographic solution, centrifuge and concentrate to evaporate the solvent, add 450 μL of 60% ethanol and 50 μL of deuterated water containing TSP prepared by the method described in step (1) of Example 1 to reconstitute, mix well to obtain linear test solution samples. Collect all samples by the method described in step (3) of Example 1. 1 H NMR spectrum, and integrate the characteristic peaks of each component using the method described in step (4) of Example 1; perform linear regression with the sampling concentration gradient of the chromatographic fluid as the horizontal axis and the concentration of the chemical component as the vertical axis to obtain the coefficient R of the regression equation 2 All of them are greater than 0.9950, which proves that the linear relationship of the NMR instrument is good;

[0075] (6) Sample recovery experiment: Take the alcohol-containing chromatographic solution with known component content, divide it into three concentration groups: high, medium and low, add a certain amount of reference solution, and then prepare the test solution sample according to the method described in Example 1, collect 1 The HNMR spectra were obtained and the content and recovery of each chemical component were calculated. The results showed that the recovery of each chemical component was between 95.00% and 105.00%, indicating that the method had good quantitative accuracy.

Claims

1. A Ginkgo biloba extract containing alcohol chromatography solution 1 The H NMR spectrum analysis method is characterized by: The following steps are involved: (1) Prepare the test sample: weigh the internal standard component and dissolve it in deuterated water to prepare a deuterated water sample solution containing the internal standard; take the alcohol-containing chromatography fluid sample to be tested and use the deuterated water sample solution containing the internal standard to prepare the test sample solution; (2) Collection 1 H NMR spectrum: Use a multi-solvent peak suppression pulse sequence to suppress the water peak and ethanol peak, and perform the test solution 1 Determination of H NMR spectra; (3) 1 H NMR spectrum signal attribution: 1 The H NMR spectrum was pre-processed, and the characteristic peaks of terpenoid lactone components and flavonol glycoside components were identified; (4) Quantitative analysis: Determine the quantitative signals of terpenoid lactone components and flavonol glycoside components, integrate the characteristic peaks corresponding to the chemical components exceeding the quantitative limit, and perform absolute quantification using the internal standard method; The measurement conditions in step (2) are as follows: probe temperature of 290-316 K, pulse sequence LC1PNGPPS, locked with solvent 90% H2O+10% D2O, spectral width of 12.000-20.000 ppm, suppression frequency of 0.800-1.300 ppm, 3.200-3.700 ppm and 4.600-4.900 ppm, relaxation delay time of 10.0-25.0 s, mixing time of 50-100 ms, number of empty scans of 0-8 times, number of acquisitions of 32 or 64 times, and detection data points of 32K or 64K.

2. according to claim 1 1 The H NMR spectrum analysis method is characterized by: The internal standard component described in step (1) is sodium 3-(trimethylsilyl)deuterated propionate; the concentration of sodium 3-(trimethylsilyl)deuterated propionate in the deuterated water sample solution containing the internal standard prepared in step (1) is 0.30-0.70 mg / mL.

3. according to claim 1 1 The H NMR spectrum analysis method is characterized by: The volume ratio of the alcohol-containing chromatographic solution to the deuterated water sample solution containing sodium 3-(trimethylsilyl)deuterated propionate in the test sample solution described in step (1) is 9:

1.

4. according to claim 1 1 The H NMR spectrum analysis method is characterized by: The measurement conditions described in step (2) are as follows: probe temperature of 298 K, pulse sequence LC1PNGPPS, locked with solvents of 90% H2O and 10% D2O, spectral width of 14.2756 ppm, suppression frequencies of 4.714, 3.468, and 1.012 ppm, relaxation delay time of 15 s, mixing time of 100 ms, number of empty scans of 4 times, number of acquisitions of 32 times, and detection data points of 32 K.

5. according to claim 1 1 The H NMR spectrum analysis method is characterized by: The suppression frequency in the determination conditions described in step (2) is obtained by conventional hydrogen spectrum acquisition, and the conditions for conventional hydrogen spectrum acquisition are: pulse sequence zg30, probe temperature 295-300 K, solvent 90% H2O + 10% D2O lock field, spectral width 13.000-16.000 ppm, relaxation delay time 1 s, number of empty scans 0 times, number of acquisitions 4 times, and detection data points 32K.

6. according to claim 1 1 The H NMR spectrum analysis method is characterized by: In step (2) 1 During H NMR data acquisition, tuning, probe matching, and shimming were performed, and the corresponding 90° pulse width was measured.

7. according to claim 1 1 The H NMR spectrum analysis method is characterized by: The spectrum preprocessing in step (3) includes Fourier transform, baseline correction, phase correction and chemical shift correction of the original FID signal.

8. according to claim 1 1 The H NMR spectrum analysis method is characterized by: The integration method described in step (4) includes the linear fitting method and the GSD method in the NMR data processing software.

9. according to claim 1 1 The H NMR spectrum analysis method is characterized by: The alcohol-containing chromatography liquid in step (1) has an ethanol volume fraction of 40%-70%.

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