CN113237913A - Salvia miltiorrhiza injection and process intermediate thereof1H NMR one-test-multiple-evaluation method - Google Patents

Salvia miltiorrhiza injection and process intermediate thereof1H NMR one-test-multiple-evaluation method Download PDF

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CN113237913A
CN113237913A CN202110353874.5A CN202110353874A CN113237913A CN 113237913 A CN113237913 A CN 113237913A CN 202110353874 A CN202110353874 A CN 202110353874A CN 113237913 A CN113237913 A CN 113237913A
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salvia miltiorrhiza
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CN113237913B (en
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应旭辉
瞿海斌
李文竹
刘雳
赵芳
徐绍静
潘坚扬
张琪
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CHIATAI QINGCHUNBAO PHARMACEUTICAL CO LTD
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Abstract

The invention relates to the field of quality evaluation and control of traditional Chinese medicines, in particular to a salvia miltiorrhiza bunge injection and a process intermediate thereof1HNMR one-test and multiple-evaluation method. The method comprises the following steps: (1) preparing external standard samples and test samples; (2)1h NMR spectrum determination; (3)1h NMR fingerprint spectrum 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 is1After H NMR spectrum pretreatment, 40 chemical components are assigned, including 12 amino acids, 7 small molecular organic acids, 8 saccharides and degradation products thereof, 7 salvianolic acid compounds and 6 nucleoside compounds; compared with the traditional quality detection and evaluation method, the method replaces various methods with one method, replaces multiple detections with single detection, simplifies the sample preparation process and obviously shortens the analysis time.

Description

Salvia miltiorrhiza injection and process intermediate thereof1H NMR one-test-multiple-evaluation method
Technical Field
The invention relates to the field of quality evaluation and control of traditional Chinese medicines, in particular to a method for quickly constructing a salvia miltiorrhiza injection and a related process intermediate thereof1H NMR fingerprint spectrum method for simultaneously quantifying all chemical components in the salvia miltiorrhiza bunge injection and application of the method in quality evaluation and control of salvia miltiorrhiza bunge injection and process intermediates thereof.
Background
The red sage root injection is prepared with red sage root and through water extraction, alcohol precipitation, purification, packing and disinfection, and is mainly used for treating cardiac and cerebral vascular diseases. Clinical practice shows that the Chinese medicinal preparation has quick response and obvious curative effect, and is widely applied to treatment of acute and severe patients. Modern researches show that the salvia miltiorrhiza bunge injection has various pharmacological activities of resisting lipid peroxidation, eliminating free radicals, inhibiting platelet adhesion and aggregation, improving microcirculation, improving hemorheology, regulating blood fat, resisting atherosclerosis and the like. Due to the wide clinical application of the salvia miltiorrhiza injection, the quality evaluation and control level of the salvia miltiorrhiza injection is more and more concerned.
The raw material medicine of the Salvia miltiorrhiza injection is the dried root and rhizome of Salvia miltiorrhiza bge of labiatae, namely Salvia milithiorrhiza bge, and is one of the earliest and most widely applied medicines in the field of Chinese medicine. The salvia miltiorrhiza contains complex chemical components, wherein the components with pharmacological activity reported mainly comprise fat-soluble tanshinone and water-soluble salvianolic acid. The Saviae Miltiorrhizae radix injection contains salvianolic acids as main effective components, and contains primary metabolites such as amino acids, small molecular organic acids, nucleosides, and carbohydrates, and Na+、K+、Cl-、NO3-And the like. The quality evaluation and control of the salvia miltiorrhiza injection with various and complex chemical components increase the difficulty. Most of the existing quality evaluation and control means only relate to quantitative analysis and fingerprint spectrum evaluation of salvianolic acid substances, however, the salvianolic acid substances only account for about 10 percent of the total solid of the salvia miltiorrhiza injection, and only the salvianolic acid substances are controlledThe quality content can not comprehensively and accurately evaluate the quality consistency of the salvia miltiorrhiza injection. The development of a comprehensive chemical component detection method has important significance for improving the quality evaluation and control level of the salvia miltiorrhiza bunge injection.
In the standard preparation method of Danshen injection provided in the twentieth volume of the Standard issued by the ministry of Chinese medicine, the pretreatment process of Danshen injection mainly comprises: 1) decocting Saviae Miltiorrhizae radix for three times, and concentrating; 2) precipitating with ethanol twice, and filtering; 3) recovering ethanol, and concentrating; 4) precipitating with water, adding water, refrigerating, standing, and filtering; 5) adjusting pH, boiling, and filtering. The whole process is long in time consumption and complex in operation, and the quality control of the process intermediate is worthy of being focused and researched.
Patent document with application publication number CN 104749308A discloses a quality control method of salvia miltiorrhiza injection, which adopts HPLC or HPLC-MS method to respectively determine fingerprint spectra of salvia miltiorrhiza injection reference product and salvia miltiorrhiza injection product to be tested, compares the two, and considers the quality of the product to be tested to be qualified when the similarity between the two is greater than 0.900. However, the fingerprint constructed by the patent only relates to salvianolic acid compounds.
Patent document CN 103245687 a discloses a quality control detection method based on a component structure of a salvia miltiorrhiza injection, which measures phenolic acids and carbohydrates in the salvia miltiorrhiza injection by HPLC and NMR methods, respectively, and determines quality by using phenolic acid components and carbohydrates characterizing the overall properties in the salvia miltiorrhiza injection. However, the method uses two analysis techniques, the operation is complicated and other components in the salvia miltiorrhiza injection are not considered.
1The H NMR technology has great advantages in traditional Chinese medicine analysis, has the capability of quantification and qualitative, and has the advantages of broad spectrum, rapidness, good reproducibility and the like. Can realize one-time detection to obtain qualitative and quantitative information of the full-scale chemical substances of the salvia miltiorrhiza bunge injection. Compared with the traditional detection means which needs different methods for different classes of substances,1the H NMR method greatly shortens the analysis time, simplifies the operation, and realizes that one method replaces a plurality of methods, and a single analysis replaces a plurality of analyses.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a novel device based on1The quality-test-multiple evaluation method and application of the salvia miltiorrhiza injection and the process intermediate thereof by the H NMR technology comprise the steps of quickly constructing the salvia miltiorrhiza injection and the related process intermediate thereof1H NMR fingerprint spectrum quantifies the whole chemical components simultaneously, and the method is applied to the quality evaluation and control of the salvia miltiorrhiza injection and the process intermediate thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
based on1A method for single-test multiple-evaluation of H NMR, comprising the steps of:
(1) preparation of external standard sample and test sample: weighing calcium formate, and dissolving with 10% deuterated water to prepare a calcium formate external standard sample solution; preparing a salvia miltiorrhiza sample to be detected into a sample solution by using deuterated water containing TSP and deionized water;
(2)1h NMR spectrum determination: determination of test sample solution and external standard sample solution by pressing water peak with presaturated water peak pressing pulse sequence1H NMR spectrum;
(3)1h NMR fingerprint spectrum construction and analysis: will be original1Preprocessing an H NMR spectrum, performing signal attribution, constructing a fingerprint spectrum by using the attributed chemical component characteristic peaks, and analyzing the fingerprint spectrum; wherein, the sample solution1After H NMR spectrum pretreatment, 40 chemical components are assigned, including 12 amino acids, 7 small molecular organic acids, 8 saccharides and degradation products thereof, 7 salvianolic acid compounds and 6 nucleoside compounds;
(4) content determination and comprehensive quality evaluation: and integrating the characteristic peaks corresponding to the chemical components exceeding the quantitative limit, absolutely quantifying by using an external standard method, and comprehensively evaluating the quality of the test sample by combining a fingerprint.
Preferably, the salvia miltiorrhiza sample to be detected in the step (1) is salvia miltiorrhiza injection or a process intermediate thereof;
preferably, when the sample to be tested is the salvia miltiorrhiza injection, the preparation method of the sample to be tested comprises the following steps: the volume ratio of the salvia miltiorrhiza injection to the deuterated water containing TSP is 9: 1, and the total volume of the mixed solution is 600-1000 mu l;
preferably, when the sample is a salvia miltiorrhiza process intermediate, centrifugally concentrating the salvia miltiorrhiza process intermediate, volatilizing the solvent, and adding a solvent with a volume ratio of 9: 1, redissolving the deionized water and the deuterated water containing the TSP, centrifuging, and taking supernatant to obtain the product; the total volume of the mixed solution is 600-;
preferably, the concentration of the calcium formate external standard sample prepared in the step (1) is 0.8-1.2 mmol/L.
Preferably, the conditions of the determination in step (2) are: the temperature of the probe is 288 and 300K; pulse sequence NOESYGPPR 1D; the spectral width is 9.9974-12.9836 ppm; center frequency 4.696 ppm; the relaxation delay time is 14.0-30.0 s; the collection time is 2.27 s; the mixing time is 50-100 ms; the collection times are 32 or 64; the gain value is 32-57; the detected data points are 32K or 64K;
preferably, the temperature of the probe is 290-; pulse sequence NOESYGPPR 1D; with 90% H as solvent2O+10％D2O, field locking; the spectral width is 11.4912-12.4967 ppm; center frequency 4.696 ppm; the relaxation delay time is 14.5-15.5 s; the collection time is 2.27 s; the mixing time is 50-100 ms; the collection times are 32 times; the gain value is 40.3-45.2; the detected data point was 32K;
preferably, in step (2)1The H NMR data acquisition requires tuning, probe matching and shimming, and the corresponding 90 ° pulse width is determined.
Preferably, the atlas preprocessing in step (3) includes fourier transform, baseline correction, phase correction and atlas alignment of the original FID signal;
preferably, prior to Fourier transformation, the spectra are processed with an exponential function at 0.30Hz as a window function; preferably, the baseline correction, phase correction and atlas alignment are all performed automatically in the MestReNova software.
Preferably, the 40 chemical components assigned in step (3) are respectively: leucine, isoleucine, valine, threonine, alanine, proline, glutamic acid, glutamine, pyroglutamic acid, aspartic acid, asparagine, tryptophan, malonic acid, gamma-aminobutyric acid, lactic acid, acetic acid, formic acid, malic acid, succinic acid, glucose, galactose, fructose, sucrose, raffinose, mannotriose, stachyose, 5-hydroxymethylfurfural, salvianolic acid B, salvianolic acid A, rosmarinic acid, lithospermic acid, protocatechualdehyde, protocatechuic acid, tanshinol, uridine, 2' -deoxyadenosine, adenine, adenosine, guanosine, and cytidine;
preferably, 1-2 peaks of each chemical component are selected as characteristic peaks for constructing a fingerprint.
Preferably, the chemical component characteristic peaks for constructing the fingerprint in step (3) are required to be independent signal peaks or independent signal peaks separated by simple deconvolution, and the 40 chemical components and their corresponding characteristic peaks are shown in table 1;
TABLE 1 names of chemical components and characteristic peak information of Salvia miltiorrhiza Bunge injecta and its process intermediate species attribution
Figure BDA0003002564420000031
Figure BDA0003002564420000041
Preferably, the fingerprint analysis in step (3) requires that the fingerprint is derived by a "binning" method;
preferably, the "binning" methods include, but are not limited to, "sum", "average", "fractional integral", "median"; preferably, the method of analysis comprises a similarity evaluation method and a multivariate statistical method; preferably, the similarity evaluation method comprises a Pearson correlation coefficient, an included angle cosine value, an Euclidean distance and a standardized Euclidean distance; the multivariate statistical method comprises principal component analysis, hierarchical clustering analysis, partial least square discriminant analysis and orthogonal partial least square analysis.
Preferably, the content determination method in the step (4) is a PULCON method by taking calcium formate as an external standard, and the calculation is carried out according to the formula (1);
Figure BDA0003002564420000042
wherein, CxAnd CRThe mass concentrations of the chemical component to be detected and the external standard are respectively; sxAnd SRRespectively is the characteristic peak area of the chemical component to be detected and the external standard; t isxAnd TRRespectively acquiring the experiment temperature of a sample to be tested and the experiment temperature of an external standard sample;
Figure BDA0003002564420000043
and
Figure BDA0003002564420000044
respectively acquiring 90-degree pulse widths of a sample to be tested and an external standard sample; n isxAnd nRRespectively is the proton number of the characteristic peak of the chemical component to be detected and the external standard; mxAnd MRRelative molecular weights of the chemical component to be detected and the external standard are respectively; f. ofTIn order to correct the factor, in the present invention, f is the same as the collection and treatment parameters of the sample to be tested and the external standard sampleTCan be considered to be 1.
Preferably, the integration method in step (4) includes, but is not limited to, a linear fitting method in MestReNova software and a GSD method;
preferably, the salvia miltiorrhiza injection sample has 20 chemical components exceeding the quantitative limit, namely valine, threonine, alanine, gamma-aminobutyric acid, acetic acid, proline, pyroglutamic acid, malonic acid, glucose, fructose, galactose, sucrose, raffinose, uridine, alkannic acid, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechualdehyde;
preferably, the salvia miltiorrhiza process intermediate sample has 22 chemical components exceeding the limit of quantitation, which are respectively: valine, threonine, alanine, gamma-aminobutyric acid, proline, pyroglutamic acid, succinic acid, malic acid, malonic acid, glucose, fructose, galactose, sucrose, mannotriose, stachyose, uridine, salvianolic acid A, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechualdehyde.
Preferably, the total content of the 20 chemical components accounts for 75-80% of the total solid content and accounts for 90-95% of the organic matter content.
Another objective of the present invention is to provide an application of the one-test-multiple-evaluation method described in any one of the above items in analyzing and evaluating the quality of a product containing salvia miltiorrhiza medicinal material;
preferably, the product is a salvia miltiorrhiza injection or a process intermediate thereof.
The invention has the following beneficial effects:
(1) simple and convenient to operate, and the invention provides1The method of the H NMR technology is simple and rapid to operate, simple in pretreatment, free of damage to the sample and recyclable after detection.
(2) The detection speed is high, only 15 minutes are needed for one-time measurement, and compared with the traditional measurement methods such as high performance liquid chromatography, the analysis time is greatly shortened.
(3) Compared with the traditional detection means which needs different methods for different classes of compounds, the method provided by the invention replaces multiple methods with one method, replaces multiple detections with single detection, and realizes one-detection and multiple-evaluation.
(4) The quantification can be carried out without a reference substance of each compound, drawing a standard curve and only one external standard.
(5) The invention provides1The method of the H NMR technique has good reproducibility.
(6) The total content of chemical components quantified by NMR of the invention accounts for 74.69-80.84% of the total solid content in the salvia miltiorrhiza injection and accounts for 89.13-95.50% of the total organic component content, so compared with the prior art, the invention has more complete and precise quality control and more comprehensive and effective quality evaluation on the salvia miltiorrhiza injection.
Drawings
FIG. 1 shows the typical example of Danshen injection1H NMR fingerprint spectrum and signal peak attribution thereof.
FIG. 2 shows the preparation of a Saviae Miltiorrhizae radix process intermediate1H NMR fingerprint spectrum and signal peak attribution thereof.
Fig. 3 is a schematic diagram of automatic integration using the linear fitting method in MestReNova software.
FIG. 4 shows the injection of 10 batches of radix Salviae Miltiorrhizae1H NMR fingerprint spectrum.
FIG. 5A shows the preparation of 10 batches of the red sage root injection1PCA analysis of H NMR fingerprint.
FIG. 5B shows the injection of 10 batches of radix Salviae Miltiorrhizae1HCA analysis of H NMR fingerprints.
FIG. 6-1 is a histogram of valine content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIG. 6-2 is a histogram of threonine content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIG. 6-3 is a histogram of alanine content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIG. 6-4 is a histogram of the gamma-aminobutyric acid content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIG. 6-5 is a histogram of acetic acid content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIG. 6-6 is a histogram of proline content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIG. 6-7 is a histogram of pyroglutamic acid content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIG. 6-8 is a histogram of malonic acid content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIGS. 6-9 are histograms of glucose content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIG. 6-10 is a histogram of fructose content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIGS. 6-11 are histograms of galactose content in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIGS. 6-12 are histograms of sucrose content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIGS. 6-13 are histograms of the raffinose content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIGS. 6-14 are histograms of uridine content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIG. 6-15 is a histogram of the lithospermic acid content in 10 batches of the Salvia miltiorrhiza Bunge injecta;
FIG. 6-16 are histograms of salvianolic acid B content in 10 batches of the Saviae Miltiorrhizae radix injection;
FIGS. 6-17 are histograms of the content of tanshinol in 10 batches of the Salvia miltiorrhiza Bunge injection;
FIG. 6-18 is a bar chart of rosmarinic acid content in 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-19 are histograms of formic acid content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIGS. 6-20 are histograms of protocatechuic aldehyde content in 10 batches of Salvia miltiorrhiza Bunge injecta;
FIG. 7 is a flow chart of quality evaluation of Salvia miltiorrhiza Bunge injection and intermediates.
Detailed Description
The following is a further description with reference to the drawings and examples.
Example 1
Based on1The method for evaluating the quality of the salvia miltiorrhiza injection by H NMR technology comprises the following steps:
(1) preparing a salvia miltiorrhiza injection test sample: accurately transferring 540 mul of the salvia miltiorrhiza bunge injection and 60 mul of deuterated water containing 0.05 percent of TSP into a 2ml centrifuge tube, and uniformly mixing to prepare a salvia miltiorrhiza bunge injection sample.
(2) Preparing an external standard sample: accurately weighing 13.12mg of calcium formate in a 10ml volumetric flask, adding deuterated water to a constant volume to scale, fully dissolving and uniformly mixing, accurately transferring 1ml of the deuterated water containing calcium formate in the 10ml volumetric flask, using deionized water to a constant volume to scale, and uniformly mixing to obtain a calcium formate external standard sample with the concentration of 1.01 mmol/L.
(3)1And (3) acquisition of an H NMR spectrum: respectively placing the sample to be tested and the external standard sample in a nuclear magnetic resonance spectrometer for testing, and pressing the residual water peak by utilizing a presaturation water peak pressing pulse sequence to obtain the samples of the sample to be tested and the external standard sample1H NMR spectrum.1The H NMR acquisition instrument was a Bruker Advanced III 600 nuclear magnetic resonance spectrometer (Bruker, Germany, with 24-bit autosampler and 5mm BBO probe, Topspin workstation).1The H NMR data acquisition requires tuning, probe matching and shimming, and the corresponding 90 ° pulse width is determined.
1H NMR acquisition parameters were: the temperature of the probe is 288K; pulse sequence NOESYGPPR 1D; with 90% H as solvent2O+10％D2O, field locking; the spectral width is 9.9974 ppm; center frequency of 4696 ppm; the relaxation delay time is 15.0 s; the collection time is 2.27 s; the mixing time is 50 ms; the collection times are 32 times; the gain value is 40.3; the test data point was 64K.
(4)1Preprocessing HNMR raw data: the acquired original atlas is processed by using an exponential function of 0.30Hz as a window function, Fourier transform of an FID signal is carried out, then the atlas is led into MestReNova software to be calibrated by using TSP (0.0) as a chemical shift reference, and baseline and phase correction are automatically carried out.
(5) Red sage root injection1Attribution of H NMR spectrum and construction of fingerprint spectrum: red sage root injection1The structural assignment of H NMR spectrum is shown in figure 1, and the compounds designated by each number in figure 1 are shown in table 1 in detail.
The characteristic peaks include: leucine (δ ═ 0.96 ± 0.02, t), isoleucine (δ ═ 1.01 ± 0.02, d, J ═ 7.0 Hz; δ ═ 0.94 ± 0.02, t), valine (δ ═ 1.04 ± 0.02, d, J ═ 6.9 Hz; δ ═ 0.99 ± 0.02, d, J ═ 6.9Hz), threonine (δ ═ 1.33 ± 0.02, d, J ═ 6.6Hz), alanine (δ ═ 1.48 ± 0.02, d, J ═ 7.3Hz), proline (δ ═ 2.28 ═ 2.38, m), glutamic acid (δ ═ 2.44 ═ 2.52, m), glutamine (δ ═ 2.11 ═ 2.18, m), pyroglutamic acid (δ ═ 2.43, 39.39, m ═ 2.02, 8 ═ 4.02, 3, 15, 8, 3, 15, 8, 3, 15, 3, 8, 15, 5, 8, 15, 3, 8, 3, 8, d ═ d, 3, 8, 3, 8, d, j-8.2 Hz), acetic acid (δ -1.97 ± 0.02, s), formic acid (δ -8.45 ± 0.02, s), glucose (δ -5.24 ± 0.02, d, J-3.8 Hz; δ ═ 3.25 ± 0.02, dd, J ═ 9.6,8.0Hz), galactose (δ ═ 5.27 ± 0.02, d, J ═ 3.8Hz), fructose (δ ═ 4.11 to 4.16, m), sucrose (δ ═ 5.41 ± 0.02, d, J ═ 3.8Hz), raffinose (δ ═ 5.43 ± 0.02, d, J ═ 3.8Hz), salvianolic acid B (δ ═ 6.39 ± 0.02, d, J ═ 8.1 Hz; δ 6.21 ± 0.02, d, J2.1 Hz), salvianolic acid a (δ 7.05 ± 0.02, d, J1.9 Hz; δ 6.73 ± 0.02, d, J2.0 Hz), rosmarinic acid (δ 7.45 ± 0.02, d, J16.2 Hz), lithospermic acid (δ 7.14 ± 0.02, d, J8.6 Hz), protocatechualdehyde (δ 9.62 ± 0.02, s), protocatechuic acid (δ 6.94 ± 0.02, d, J8.3 Hz), tanshinol (δ 6.78 ± 0.02, d, J2.1 Hz; δ 2.78 ± 0.02, d, J14.1, 7.7Hz), uridine (δ 7.86 ± 0.02, d, J8.0 Hz), 2' -deoxyadenosine (δ 8.20 ± 0.02, s; δ 8.30 ± 0.02, s), adenine (δ 8.21 ± 0.02, s; δ -8.24 ± 0.02, s), adenosine (δ -8.26 ± 0.02, s; δ -8.36 ± 0.02, s), guanosine (δ -8.00 ± 0.02, s), cytidine (δ -5.87 ± 0.02, d, J-3.2 Hz).
(6) Integral of characteristic peak and quantification of chemical composition; the salvia miltiorrhiza injection contains 20 chemical components which are higher than the limit of quantification and are respectively: valine, threonine, alanine, gamma-aminobutyric acid, acetic acid, proline, pyroglutamic acid, malonic acid, glucose, fructose, galactose, sucrose, raffinose, uridine, lithospermic acid, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechuic aldehyde. The characteristic peaks of the 20 chemical components were integrated using the linear fit method in the MestReNova software, and the formate peak in the external standard samples was integrated, and the integration scheme is shown in fig. 3. Then, the concentrations of the above-mentioned 20 chemical components were calculated according to the formula (1).
Example 2
Based on1The quality one-test-multiple-evaluation method of the salvia miltiorrhiza process intermediate by the H NMR technology comprises the following steps:
(1) preparing a salvia miltiorrhiza process intermediate sample: accurately transferring 200 μ l of Saviae Miltiorrhizae radix ethanol precipitation solution into 2ml centrifuge tube, centrifuging and concentrating to completely volatilize solvent, adding 540 μ l of deionized water and 60 μ l of deuterated water containing 0.05% TSP, mixing well, centrifuging at 10000r/min for 10min, collecting supernatant, and making into Saviae Miltiorrhizae radix process intermediate sample.
(2) Preparing an external standard sample: accurately weighing 13.12mg of calcium formate in a 10ml volumetric flask, adding deuterated water to a constant volume to scale, fully dissolving and uniformly mixing, accurately transferring 1ml of the deuterated water containing calcium formate in the 10ml volumetric flask, using deionized water to a constant volume to scale, and uniformly mixing to obtain a calcium formate external standard sample with the concentration of 1.01 mmol/L.
(3) Red sage root process intermediate1And (3) acquisition of an H NMR spectrum: respectively placing the sample to be tested and the external standard sample in a nuclear magnetic resonance spectrometer for testing, and pressing the residual water peak by utilizing a presaturation water peak pressing pulse sequence to obtain the samples of the sample to be tested and the external standard sample1H NMR spectrum.1The H NMR acquisition instrument was a Bruker Advanced III 600 nuclear magnetic resonance spectrometer (Bruker, Germany, with 24-bit autosampler and 5mm BBO probe, Topspin workstation).1The H NMR data acquisition requires tuning, probe matching and shimming, and the corresponding 90 ° pulse width is determined.1H NMR acquisition parameters were: the temperature of the probe is 300K; pulse sequence NOESYGPPR 1D; with 90% H as solvent2O+10％D2O, field locking; the spectral width is 12.9836 ppm; center frequency 4.696 ppm; the relaxation delay time is 30.0 s; the collection time is 2.27 s; the mixing time is 100 ms; the collection times are 64 times; the gain value is 57; the check data point was 32K.
(4)1H NMR raw data preprocessing: the acquired original atlas is processed by using an exponential function of 0.30Hz as a window function, Fourier transform of an FID signal is carried out, then the atlas is led into MestReNova software to be calibrated by using TSP (0.0) as a chemical shift reference, and baseline and phase correction are automatically carried out.
(5) Red sage root process intermediate1Attribution of H NMR spectrum and construction of fingerprint spectrum: red sage root alcohol precipitation liquid1The structural assignment of H NMR spectrum is shown in figure 2, and the compounds designated by each number in figure 2 are shown in table 1 in detail. The characteristic peaks include: leucine (δ ═ 0.96 ± 0.02, t), isoleucine (δ ═ 1.01 ± 0.02, d, J ═ 7.0 Hz; δ 0 ═ 0.94 ± 0.02, t), valine (δ 1 ═ 1.04 ± 0.02, d, J ═ 6.9 Hz; δ 2 ═ 0.02, d, J ═ 6.9Hz), threonine (δ 3 ═ 1.33 ± 0.02, d, J ═ 6.6Hz), alanine (δ 4 ═ 1.48 ± 0.02, d, J ═ 7.3Hz), proline (δ 5 ═ 2.28 to 2.38, m), glutamic acid (δ 6 ═ 2.44 to 2.52, m), glutamine (δ ═ 2.11 to 2.18, m ═ 2.8, 8 ═ 2.43 to 2.38, m), aspartic acid (δ ═ 6 ═ 2.44 to 2.52, m), glutamine (δ ═ 2.02, δ ═ 2.8, 8 ═ 2.9.9, 9.9, 9 ═ 9.9, 9, 8, 9, 8, d, 1, d, 8, d, c, m), lactic acid (δ 3 ═ 1.40 ± 0.02, d, J ═ 8.2Hz), formic acid (δ 4 ═ 8.45 ± 0.02, s), malic acid (δ ═ 2.63 ± 0.02, dd, J ═ 19.2,5.0Hz), succinic acid (δ ═ 2.59 ± 0.02, s), glucose (δ ═ 5.24 ± 0.02, d, J ═ 3.8 Hz; δ being 3.25 ± 0.02, dd, J being 9.6,8.0Hz), galactose (δ being 5.27 ± 0.02, d, J being 3.8Hz), fructose (δ being 4.11-4.16, m), sucrose (δ being 5.41 ± 0.02, d, J ═ 3.8Hz), mannotriose (δ ═ 5.24 ± 0.02, d, J ═ 3.8Hz), stachyose (δ ═ 5.00 ± 0.02, m), 5-hydroxymethylfurfural (δ 0 ═ 9.44 ± 0.02, s), salvianolic acid B (δ 1 ═ 6.39 ± 0.02, d, J ═ 8.1 Hz; δ 2 ═ 6.21 ± 0.02, d, J ═ 2.1Hz), salvianolic acid a (δ 3 ═ 7.05 ± 0.02, d, J ═ 1.9 Hz; δ 4 ═ 6.73 ± 0.02, d, J ═ 2.0Hz), rosmarinic acid (δ 5 ═ 7.45 ± 0.02, d, J ═ 16.2Hz), lithospermic acid (δ 6 ═ 7.14 ± 0.02, d, J ═ 8.6Hz), protocatechualdehyde (δ 7 ═ 9.62 ± 0.02, s), protocatechuic acid (δ 8 ═ 6.94 ± 0.02, d, J ═ 8.3Hz), tanshinol (δ 9 ═ 6.78 ± 0.02, d, J ═ 2.1 Hz; δ 2.78 ± 0.02, d, J14.1, 7.7Hz), uridine (δ 0 ═ 7.86 ± 0.02, d, J ═ 8.0Hz), 2' -deoxyadenosine (δ 1 ═ 8.20 ± 0.02, s; δ 8.30 ± 0.02, s), adenine (δ 8.21 ± 0.02, s; δ -8.24 ± 0.02, s), adenosine (δ -8.26 ± 0.02, s; δ -8.36 ± 0.02, s), guanosine (δ -8.00 ± 0.02, s), cytidine (δ -5.87 ± 0.02, d, J-3.2 Hz).
(6) Integration of characteristic peaks and quantification of chemical composition. The salvia miltiorrhiza alcohol precipitation solution contains 22 chemical components which are higher than the limit of quantitation: valine, threonine, alanine, gamma-aminobutyric acid, proline, pyroglutamic acid, succinic acid, malic acid, malonic acid, glucose, fructose, galactose, sucrose, mannotriose, stachyose, uridine, salvianolic acid A, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechuic aldehyde. The characteristic peaks of the 22 chemical components were integrated by linear fitting in MestReNova software, and the integration scheme is shown in FIG. 3. Then, the concentrations of the above 22 chemical components were calculated according to formula (1).
Example 3
Red sage root injection and process intermediate of red sage root1H NMR quantitative methodology investigation:
(1) the linear relationship is: adding deionized water and deuterated water containing TSP in a ratio of 9: 1, preparing the deuterated solvent. ② precisely weighing a certain amount of reference substances of valine, threonine, alanine, gamma-aminobutyric acid, acetic acid, proline, pyroglutamic acid, succinic acid, malic acid, malonic acid, glucose, fructose, galactose, sucrose, raffinose, stachyose, uridine, lithospermic acid, salvianolic acid A, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechualdehyde, and diluting the volume with a deuterated solvent to obtain the reference substancesAnd (4) mother liquor. ③ diluting the mother liquor of the reference substance by using the deuterated solvent in a multiplying ratio to obtain the diluted solutions of the reference substance with the concentrations of 1/2, 1/4, 1/8 and 1/16 of the original mother liquor. (iv) collecting all control stock solutions and control dilutions by the method described in step 3 of examples 1 and 21H NMR spectra and integrated the characteristic peaks for each control using the method described in step 6 of examples 1 and 2. Using characteristic peak area of reference substance as ordinate and mass concentration as abscissa to perform linear regression to obtain coefficient r of regression equation2All are more than 0.999, which proves that the linearity relationship of the NMR instrument is good.
(2) The specificity is as follows: taking the salvia miltiorrhiza injection and the salvia miltiorrhiza alcohol precipitation solution to prepare a sample of the test solution according to the method in the step 1 in the examples 1 and 2, verifying the specificity of the quantitative peak by an HSQC method, and indicating that the quantitative peak has no impurity peak interference, the method has good specificity.
(3) Authenticity: the method includes the steps of calculating the concentration of characteristic peaks of each reference substance by using the method described in the step 6 in the examples 1 and 2, calculating the variation coefficient by using the calculated value and the weighing value of each reference substance concentration, and obtaining the variation coefficient of each reference substance NMR measurement within 2.48% by using the calculation formula (2), so that the method is proved to have good authenticity.
Figure BDA0003002564420000101
Wherein c isvIs the coefficient of variation, CmFor the quantification of the measured values obtained by said method, CwIs the actual value of weighing.
(4) Precision of the instrument: taking the salvia miltiorrhiza bunge injection to prepare a sample of the test solution according to the method in the step 1 in the embodiment 1, and continuously collecting the sample for six times by the method in the step 31And (4) H NMR spectrum, and integrating each characteristic peak by using the method in the step 6, and calculating peak area ratios and RSD values of each chemical component and an external standard, wherein the result shows that the RSD value of each characteristic peak is less than 2.07%, and the method is proved to have good instrument precision.
(5) And (3) sample preparation repeatability: taking the salvia miltiorrhiza injection and the salvia miltiorrhiza alcohol precipitation solution to prepare 6 parts of the salvia miltiorrhiza injection and the salvia miltiorrhiza alcohol precipitation solution in parallel according to the method of the step 1 in the examples 1 and 2 respectivelySamples of test solutions, six replicates of each of which were collected by the method described in step 3 of examples 1 and 21And H NMR spectra are obtained, and the method described in step 6 of examples 1 and 2 is used for integrating all characteristic peaks and calculating peak area ratios and RSD values of all chemical components and external standards, and the results show that the RSD values of all the characteristic peaks are less than 2.84%, so that the sample preparation method is proved to have good repeatability.
(6)24h stability: taking the red sage root injection and the red sage root ethanol precipitation solution to prepare test solution samples according to the method of the step 1 in the examples 1 and 2, and collecting the samples at the 0h, 2h, 4h, 6h, 9h, 12h and 24h by the method of the step 3 in the examples 1 and 21And H NMR spectrum, and integrating all characteristic peaks by using the method in step 6 of examples 1 and 2, and calculating peak area ratios and RSD values of all chemical components and external standards, wherein the results show that no new characteristic peak appears within 24H, and the RSD value of each characteristic peak is less than 2.47%, which proves that the sample of the test solution has good stability within 24H.
(7) Sample adding and recovering experiment: taking salvia miltiorrhiza injection with known content, adding quantitative reference substances into groups with high, medium and low concentrations, preparing a test solution sample by using the salvia miltiorrhiza injection with the reference substances according to the method in the step 1 in the example 1, performing content measurement by using the method in the steps 3-6 in the example 1, and calculating the recovery rate of each chemical component. The result shows that the recovery rate of each component is between 97.79% and 102.34%, which indicates that the method has good quantitative accuracy on the salvia miltiorrhiza injection.
Taking salvia miltiorrhiza alcohol precipitation liquid with known content, adding quantitative reference substances into groups with high, medium and low concentrations, preparing a test solution sample by using the salvia miltiorrhiza alcohol precipitation liquid with the reference substances according to the method in the step 1 in the embodiment 2, performing content measurement by using the method in the steps 3-6 in the embodiment 2, and calculating the recovery rate of each chemical component. The result shows that the recovery rate of each component is between 97.66% and 101.94%, which indicates that the method has good quantitative accuracy on the salvia miltiorrhiza process intermediate.
Example 4
Based on1The application of the salvia miltiorrhiza injection quality one-test-multiple evaluation by the H NMR technology comprises the following steps:
1. taking 10 batches of pillsThe salvia miltiorrhiza injection is obtained by the method of the steps 1 to 6 in the example 11H NMR fingerprint spectrum, and quantifying 20 chemical components in the H NMR fingerprint spectrum.
2. Fingerprint analysis of the salvia miltiorrhiza injection: fig. 4 shows the fingerprint of 10 batches of salvia miltiorrhiza injection collected, and the fingerprint of the 10 batches of salvia miltiorrhiza injection is analyzed by the Pearson correlation coefficient method in the similarity evaluation method and the PCA and HCA methods in multivariate analysis in the embodiment, but the analysis and evaluation methods of the fingerprint are not limited to the above, and the specific steps are as follows:
(1) the fingerprint of 10 batches of salvia miltiorrhiza injection is exported in a box separation mode of 'sectional integration' with the width of 0.02ppm, a water peak inhibition area and signal-free areas at two ends are deleted, and a matrix X (10 multiplied by 415) containing 10 observed values and 415 variables is obtained.
(2) Taking the average chromatogram of the fingerprint of 10 batches of salvia miltiorrhiza injection as reference, selecting a data point where a fingerprint characteristic peak is located, and calculating the Pearson correlation coefficient of 10 batches of samples as a similarity evaluation index. The lot number and similarity of each batch of salvia miltiorrhiza injection are shown in table 2, and the similarity evaluation results show that the similarity of the other batches is greater than 0.99 except the batch S3, and the similarity of the batch S3 is slightly lower than that of other samples but is also greater than 0.98, so that all samples have higher similarity, which indicates that the consistency of 10 batches of salvia miltiorrhiza injection is good. In addition, the similarity of the six batches S5-S10 is greater than 0.995, which shows that the six batches have higher similarity.
TABLE 210 batch number and similarity of Salvia miltiorrhiza Bunge injecta batches
Figure BDA0003002564420000111
(2) The matrix X was introduced into SIMCA software for PCA analysis, and the PCA score chart is shown in fig. 5(a), from which it can be seen that lot S4 differs significantly from other lots.
(3) On the basis, HCA analysis is carried out by SIMCA software, an HCA dendrogram is shown in fig. 5(B), and the fingerprint spectrum consistency of batches S5-S10 of salvia miltiorrhiza injection is higher, the batches S1-S3 are less, the relative distance between the batch S4 and other batches is the largest, which shows that the chemical components of the injection are the largest in difference with the injection of other batches, and the conclusion is consistent with the similarity evaluation result.
3. Quantification of 20 chemical components in 10 batches of salvia miltiorrhiza injection: applications of1The results of measuring the contents of valine, threonine, alanine, γ -aminobutyric acid, acetic acid, proline, pyroglutamic acid, malonic acid, glucose, fructose, galactose, sucrose, raffinose, uridine, lithospermic acid, salvianolic acid B, tanshinol, rosmarinic acid, formic acid, and protocatechualdehyde in 10 batches of injections by H NMR are shown in table 3. The visualized bar charts are shown in fig. 6-1 to 6-20.
Table 3 is based on1H NMR quantitative result (unit: mg/ml) of chemical components of 10 batches of salvia miltiorrhiza injection
Figure BDA0003002564420000112
Figure BDA0003002564420000121
4. Determining total solid content of 10 batches of Saviae Miltiorrhizae radix injection by dry constant weight method, determining cation content by atomic absorption spectrometry, determining anion content by anion chromatography, and calculating1The ratio of the total solids content to the total chemical component content that can be quantified by H NMR (ratio 1) and1the H NMR can quantify the proportion of all chemical components to the solid content except inorganic ions (ratio 2), and the ratio 2 can also be regarded as1H NMR can quantitatively determine the proportion of all chemical components in the total organic components in the salvia miltiorrhiza injection. The results of the measurement of the total solid content and the inorganic ion content and the calculation results of the ratios 1 and 2 are shown in table 4, and it can be seen from the results that the total chemical components in the NMR quantification account for 74.69 to 80.84% of the total solid content in the salvia miltiorrhiza injection and 89.13 to 95.50% of the total organic component content.
TABLE 410 Total solid content, inorganic ion content determination results and NMR quantitative component content ratios of Saviae Miltiorrhizae radix injection batches
Figure BDA0003002564420000122
The quality evaluation flow chart of the salvia miltiorrhiza injection and the intermediate is shown in figure 7.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.

Claims (10)

1. Based on1The one-test-multiple-evaluation method of the salvia miltiorrhiza injection or the process intermediate thereof by H NMR is characterized by comprising the following steps of:
(1) preparation of external standard sample and test sample: weighing calcium formate, and dissolving with 10% deuterated water to prepare a calcium formate external standard sample solution; preparing a salvia miltiorrhiza sample to be detected into a sample solution by using deuterated water containing TSP and deionized water;
(2)1h NMR spectrum determination: determination of test sample solution and external standard sample solution by pressing water peak with presaturated water peak pressing pulse sequence1H NMR spectrum;
(3)1h NMR fingerprint spectrum construction and analysis: will be original1Preprocessing an H NMR spectrum, performing signal attribution, constructing a fingerprint spectrum by using the attributed chemical component characteristic peaks, and analyzing the fingerprint spectrum; wherein, the sample solution1After H NMR spectrum pretreatment, 40 chemical components are assigned, including 12 amino acids, 7 small molecular organic acids, 8 saccharides and degradation products thereof, 7 salvianolic acid compounds and 6 nucleoside compounds;
(4) content determination and comprehensive quality evaluation: and integrating the characteristic peaks corresponding to the chemical components exceeding the quantitative limit, absolutely quantifying by using an external standard method, and comprehensively evaluating the quality of the test sample by combining a fingerprint.
2. The one-time multi-evaluation method according to claim 1, wherein the salvia miltiorrhiza sample to be tested in the step (1) is salvia miltiorrhiza injection or a process intermediate thereof;
preferably, when the sample is a salvia miltiorrhiza injection, the volume ratio of the sample to the deuterated water is 9: 1 in a certain proportion; when the sample is the salvia miltiorrhiza process intermediate, centrifugally concentrating the salvia miltiorrhiza process intermediate, volatilizing the solvent, and adding a solvent with the volume ratio of 9: 1, redissolving the deionized water and the deuterated water containing the TSP, centrifuging, and taking supernatant to obtain the product; the total volume of the mixed solution is 600-;
preferably, the concentration of the calcium formate external standard sample prepared in the step (1) is 0.8-1.2 mmol/L.
3. The method according to claim 1, wherein the conditions of the measurement in the step (2) are: the temperature of the probe is 288 and 300K; pulse sequence NOESYGPPR 1D; the spectral width is 9.9974-12.9836 ppm; center frequency 4.696 ppm; the relaxation delay time is 14.0-30.0 s; the collection time is 2.27 s; the mixing time is 50-100 ms; the collection times are 32 or 64; the gain value is 32-57; the detected data points are 32K or 64K;
preferably, the temperature of the probe is 290-; pulse sequence NOESYGPPR 1D; with 90% H as solvent2O+10％D2O, field locking; the spectral width is 11.4912-12.4967 ppm; center frequency 4.696 ppm; the relaxation delay time is 14.5-15.5 s; the collection time is 2.27 s; the mixing time is 50-100 ms; the collection times are 32 times; the gain value is 40.3-45.2; the detected data point was 32K;
preferably, in step (2)1The H NMR data acquisition requires tuning, probe matching and shimming, and the corresponding 90 ° pulse width is determined.
4. The method of claim 1, wherein the atlas preprocessing in step (3) includes fourier transform, baseline correction, phase correction, and atlas alignment of the original FID signal; preferably, prior to Fourier transformation, the spectra are processed with an exponential function at 0.30Hz as a window function; preferably, the baseline correction, phase correction and atlas alignment are all performed automatically in the MestReNova software.
5. The method according to claim 1, wherein the 40 chemical components assigned in step (3) are respectively: leucine, isoleucine, valine, threonine, alanine, proline, glutamic acid, glutamine, pyroglutamic acid, aspartic acid, asparagine, tryptophan, malonic acid, gamma-aminobutyric acid, lactic acid, acetic acid, formic acid, malic acid, succinic acid, glucose, galactose, fructose, sucrose, raffinose, mannotriose, stachyose, 5-hydroxymethylfurfural, salvianolic acid B, salvianolic acid A, rosmarinic acid, lithospermic acid, protocatechualdehyde, protocatechuic acid, tanshinol, uridine, 2' -deoxyadenosine, adenine, adenosine, guanosine, and cytidine;
preferably, 1-2 peaks of each chemical component are selected as characteristic peaks for constructing a fingerprint, the characteristic peaks being required to be independent signal peaks or independent signal peaks separated by simple deconvolution.
6. The one-test-and-multiple-evaluation method according to claim 1, wherein the fingerprint analysis in step (3) requires that the fingerprint is derived by a "binning" method;
preferably, the "binning" methods include, but are not limited to, "sum", "average", "fractional integral", "median"; preferably, the method of analysis comprises a similarity evaluation method and a multivariate statistical method; preferably, the similarity evaluation method comprises a Pearson correlation coefficient, an included angle cosine value, an Euclidean distance and a standardized Euclidean distance; the multivariate statistical method comprises principal component analysis, hierarchical clustering analysis, partial least square discriminant analysis and orthogonal partial least square analysis.
7. The method according to claim 1, wherein the content determination method in step (4) is a PULCON method using calcium formate as an external standard;
preferably, the method of integrating the characteristic peaks exceeding the limit of quantitation includes a linear fitting method and a GSD method in MestReNova software.
8. The method according to claim 1, wherein 20 chemical components of the sample of the red sage root injection in step (4) exceeding the quantitation limit are valine, threonine, alanine, γ -aminobutyric acid, acetic acid, proline, pyroglutamic acid, malonic acid, glucose, fructose, galactose, sucrose, raffinose, uridine, lithospermic acid, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechualdehyde;
preferably, 22 chemical components of the salvia miltiorrhiza process intermediate sample exceeding the limit of quantitation in the step (4) are respectively: valine, threonine, alanine, gamma-aminobutyric acid, proline, pyroglutamic acid, succinic acid, malic acid, malonic acid, glucose, fructose, galactose, sucrose, mannotriose, stachyose, uridine, salvianolic acid A, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechualdehyde.
9. The one-test-multiple-evaluation method according to claim 8, wherein the total content of the 20 chemical components of the salvia miltiorrhiza bunge injection accounts for 75-80% of the total solid content and accounts for 90-95% of the organic matter content.
10. Use of the Danshen injection or the Danshen injection intermediate one-test-multiple-evaluation method according to any one of claims 1 to 9 for the analytical evaluation of the quality of a product containing Danshen; preferably, the product is a salvia miltiorrhiza injection or a salvia miltiorrhiza process intermediate.
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