CN113237913B - Red sage root injection and its technological intermediate 1 H NMR one-measurement-multiple-evaluation method - Google Patents
Red sage root injection and its technological intermediate 1 H NMR one-measurement-multiple-evaluation method Download PDFInfo
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Abstract
The invention relates to the field of quality evaluation and control of traditional Chinese medicines, in particular to a red sage root injection and a process intermediate thereof 1 HNMR one-measurement-multiple-evaluation method. The method comprises the following steps: (1) preparation of external standard samples and test samples; (2) 1 H NMR spectrum measurement; (3) 1 H NMR fingerprint construction and analysis; and (4) content measurement and comprehensive quality evaluation. The method of the invention has simple and quick operation and is used for testing the sample solution 1 The pretreatment of the H NMR spectrum is followed by attributing 40 chemical components, including 12 amino acids, 7 small molecule 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 a plurality of methods with one method, replaces a plurality of times of detection with a single measurement, simplifies the sample preparation process and obviously shortens the analysis time.
Description
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 red sage root injection and a related process intermediate thereof 1 Method for simultaneously quantifying comprehensive chemical components in H NMR fingerprint spectrum and application thereof in quality evaluation and control of Saviae Miltiorrhizae radix injection and its process intermediate.
Background
The red sage root injection is prepared with red sage medicine material and through water extraction, alcohol precipitation, purification, encapsulation and disinfection, and is mainly applied to cardiac and cerebral vascular diseases. Clinical practice shows that it has quick and obvious curative effect and wide application in treating acute and severe patients. Modern researches have shown that the red sage root injection has various pharmacological activities of resisting lipid peroxidation, scavenging free radicals, inhibiting platelet adhesion and aggregation, improving microcirculation, improving hemorheology, regulating blood lipid and resisting atherosclerosis. Because of the wide clinical application of the red sage root injection, the quality evaluation and control level of the red sage root injection is more and more concerned.
The raw material of the red sage root injection is the dried root and rhizome of red sage Salvia miltiorrhiza Bge, which is one of the earliest and most widely used medicines in the field of Chinese medicine. The red sage root contains complex chemical components, and the components reported in pharmacological activity are mainly liposoluble tanshinone and water-soluble salvianolic acid. The red sage root injection is a preparation with salvianolic acid as main effective component, and its chemical components include amino acid, small molecular organic acid, nucleoside, carbohydrate and other primary metabolites and Na + 、K + 、Cl - 、NO 3- And inorganic salt ions. The quality evaluation and control of the red sage root injection with various and complex chemical components are difficult. The existing quality evaluation and control means only relate to quantitative analysis and fingerprint evaluation of salvianolic acid substances, however, the salvianolic acid substances only account for about 10% of total solid of the salvianolic acid injection, and the quality consistency of the salvianolic acid injection cannot be comprehensively and accurately evaluated by only controlling the content of the salvianolic acid substances. The development of a comprehensive chemical component detection method has important significance for improving the quality evaluation and control level of the salvia miltiorrhiza injection.
In the standard preparation method of the salvia miltiorrhiza injection provided by the twenty-first book of the standards issued by the Chinese medicine ministry of the sciences, the pretreatment process of the salvia miltiorrhiza injection is mainly divided into: 1) Extracting Saviae Miltiorrhizae radix for three times, and concentrating; 2) Alcohol precipitation twice and filtering; 3) Recovering ethanol and concentrating; 4) Precipitating with water, adding water, cooling, standing, and filtering; 5) Adjusting pH, boiling, and filtering. The whole process is time-consuming and complex to operate, and the quality control of the process intermediate is worth being focused and studied.
Patent document with application publication number CN 104749308A discloses a quality control method of radix salviae miltiorrhizae injection, which adopts HPLC or HPLC-MS method to respectively measure fingerprint patterns of radix salviae miltiorrhizae injection reference substance and radix salviae miltiorrhizae injection product to be detected, compares the fingerprint patterns, and considers the quality of the product to be detected to be qualified when the similarity of the fingerprint patterns and the fingerprint patterns is more than 0.900. However, the fingerprint constructed by the patent only relates to salvianolic acid compounds.
Patent document with application publication number of CN 103245687A discloses a quality control detection method based on component structure of radix salviae miltiorrhizae injection, wherein phenolic acids and saccharides in the radix salviae miltiorrhizae injection are respectively measured by an HPLC method and an NMR method, and the quality is judged by the phenolic acid component and the saccharide component which characterize the integral property in the radix salviae miltiorrhizae injection. However, the method uses two analysis techniques, the operation is complex and other components in the red sage root injection are not considered.
1 The H NMR technique has great advantages in traditional Chinese medicine analysis, has the capacity of quantification and qualitative property, 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 comprehensive chemical substances of the salvia miltiorrhiza injection. Compared with the traditional detection means, different methods are needed for different classes of substances, 1 the H NMR method greatly shortens the analysis time, simplifies the operation, realizes one method to replace a plurality of methods, and replaces a plurality of analyses by a single analysis.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a novel base 1 A method for evaluating quality of radix Salviae Miltiorrhizae injection and its process intermediate by one test and multiple tests by H NMR technique and its application are disclosed, which comprise quickly constructing radix Salviae Miltiorrhizae injection and its related process intermediate 1 The H NMR fingerprint spectrum simultaneously quantifies the comprehensive chemical components in the red sage root injection and the quality evaluation and control of the process intermediates thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
based on 1 A method for multi-evaluation of H NMR comprising the steps of:
(1) Preparation of external standard samples and test samples: weighing calcium formate, and dissolving the calcium formate with 10% deuterated water to prepare an external standard sample solution of the calcium formate; taking a red sage root sample to be detected, and preparing a sample solution by using deuterated water containing TSP and deionized water;
(2) 1 h NMR spectrum determination: the pre-saturated water peak pressing pulse sequence is used for pressing water peaks, and the test sample solution and the external standard sample solution are determined 1 H NMR spectrum;
(3) 1 h NMR fingerprint construction and analysis: will be original 1 Preprocessing the H NMR spectrum and attaching signals, constructing a fingerprint spectrum by using attached characteristic peaks of chemical components, and analyzing the fingerprint spectrum; wherein the sample solution 1 The pretreatment of the H NMR spectrum is followed by attributing 40 chemical components, including 12 amino acids, 7 small molecule organic acids, 8 saccharides and degradation products thereof, 7 salvianolic acid compounds and 6 nucleoside compounds;
(4) Content measurement and overall quality evaluation: and (3) carrying out integral on characteristic peaks corresponding to chemical components exceeding a quantitative limit, absolute quantification by an external standard method, and comprehensively evaluating the quality of a sample by combining with a fingerprint.
Preferably, the red sage root sample to be detected in the step (1) is red sage root injection or its process intermediate;
preferably, when the sample to be tested is a red sage root injection, the preparation method of the sample for test 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 to 1000 mu l;
preferably, when the sample is the intermediate of the red sage root process, the intermediate of the red sage root process is centrifugally concentrated, the solvent is volatilized, and the volume ratio of the added solvent is 9:1, re-dissolving deionized water and deuterated water containing TSP, centrifuging, and taking supernatant to obtain; the total volume of the mixed solution is 600-1000 mu l;
preferably, the concentration of the calcium formate external standard sample prepared in the step (1) is 0.8-1.2mmol/L.
Preferably, the conditions of the determination in step (2) are: the temperature of the probe is 288-300K; a pulse sequence NOESYGPPR1D; the spectrum width is 9.9974-12.9836ppm; the center frequency was 4.696ppm; relaxation delay time is 14.0-30.0s; the acquisition time is 2.27s; the mixing time is 50-100ms; the collection times are 32 or 64 times; gain value is 32-57; the detected data point is 32K or 64K;
preferably, the probe temperature is 290-295K; a pulse sequence NOESYGPPR1D; with solvent 90% H 2 O+10％D 2 O locking a field; the spectrum width is 11.4912-12.4967ppm; the center frequency was 4.696ppm; relaxation delay time is 14.5-15.5s; the acquisition time is 2.27s; the mixing time is 50-100ms; the collection times are 32 times; gain value is 40.3-45.2; the detected data point is 32K;
preferably, in step (2) 1 The H NMR data acquisition requires tuning, probe matching and shimming, and corresponding 90 ° pulse width measurements.
Preferably, the profile pre-processing in step (3) comprises fourier transform, baseline correction, phase correction and profile alignment of the raw FID signals;
preferably, prior to fourier transformation, the map is processed with an exponential function of 0.30Hz as a window function; preferably, the baseline correction, phase correction and map alignment are automated in the MestReNova software.
Preferably, the 40 chemical components belonging to the 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, protocatechuic acid, salvianic acid, 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 characteristic peaks of the chemical components used for constructing the fingerprint in the step (3) are required to be independent signal peaks or independent signal peaks separated by simple deconvolution, and 40 chemical components and corresponding characteristic peaks are shown in table 1;
TABLE 1 Red sage root injection and its technological intermediate species belonged chemical composition name and characteristic peak information
Preferably, the fingerprint analysis in the step (3) needs to be conducted by a 'binning' method on the fingerprint;
preferably, the "binning" method includes, but is not limited to, "sum", "average", "piecewise integral", "median"; preferably, the method of analysis includes 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 squares discriminant analysis and orthogonal partial least squares analysis.
Preferably, the content determination method in the step (4) is calculated according to the formula (1) by using calcium formate as an external standard and using a PULCON method;
wherein C is x And C R The mass concentrations of the chemical components to be detected and the external standard are respectively; s is S x And S is R The characteristic peak areas of the chemical component to be detected and the external standard are respectively; t (T) x And T R The experimental temperatures are respectively the experimental temperatures when the sample to be tested and the external standard sample are collected;and->90-degree pulse width when the sample to be tested and the external standard sample are collected respectively; n is n x And n R The characteristic peak proton numbers of the chemical component to be detected and the external standard are respectively; m is M x And M R The relative molecular weights of the chemical component to be measured and the external standard; f (f) T In the present invention, since the sample to be tested and the external standard sample are completely identical in terms of the parameters for the collection and processing of the correction factors, f T Can be considered as 1.
Preferably, the integration method in step (4) includes, but is not limited to, linear fitting method and GSD method in MestReNova software;
preferably, the chemical components of the red sage root injection sample exceeding the quantitative limit are 20, namely 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;
preferably, the red sage process intermediate sample has 22 chemical components exceeding the quantitative limit, which are respectively: valine, threonine, alanine, gamma-aminobutyric acid, proline, pyroglutamic acid, succinic acid, malic acid, malonic acid, glucose, fructose, galactose, sucrose, mannose, stachyose, uridine, salvianolic acid A, salvianolic acid B, salvianic acid A, rosmarinic acid, formic acid and protocatechuic aldehyde.
Preferably, the total content of the 20 chemical components accounts for 75% -80% of the total solid content, and the total content of the 20 chemical components accounts for 90% -95% of the organic content.
Another object of the present invention is to provide an application of the method for testing multiple samples according to any one of the above in analyzing and evaluating quality of a product containing red sage root;
preferably, the product is a red sage root injection or a process intermediate thereof.
The invention has the beneficial effects that:
(1) The operation is simple and convenient, and the invention provides a base 1 The method of the H NMR technology is simple and quick to operate, simple in pretreatment, free from damaging the sample, and recoverable after detection.
(2) The detection speed is high, only 15 minutes are needed for one-time determination, and compared with the traditional high performance liquid chromatography and other determination methods, the analysis time is greatly shortened.
(3) Meanwhile, the multi-class compound is measured, and compared with the traditional detection means which need different methods aiming at different classes of compounds, the method provided by the invention uses one method to replace a plurality of methods, uses single measurement to replace multiple detection, and realizes one measurement and multiple evaluation.
(4) The quantitative method does not need a reference substance of each compound, does not need to draw a standard curve, and can be used for quantitative determination only by using one external standard.
(5) The invention provides the basis 1 The method of H NMR technique has good reproducibility.
(6) The total content of chemical components of NMR quantification of the invention accounts for 74.69% -80.84% of the total solid content in the red sage root injection and accounts for 89.13% -95.50% of the total organic component content, so that compared with the prior art, the invention has more complete and precise quality control and more comprehensive and effective quality evaluation on the red sage root injection.
Drawings
FIG. 1 is a schematic representation of an injection of Salvia Miltiorrhiza 1 H NMR fingerprint and signal peak ascribed thereto.
FIG. 2 shows an intermediate of the process of Salvia Miltiorrhiza 1 H NMR fingerprint and signal peak ascribed thereto.
Fig. 3 is a schematic diagram of the automatic integration using the linear fitting method in the MestReNova software.
FIG. 4 shows 10 batches of red sage root injection 1 H NMR fingerprint.
FIG. 5A is a 10-batch salvia miltiorrhiza injection 1 PCA analysis of H NMR fingerprint.
FIG. 5B shows 10 batches of red sage root injection 1 HCA analysis of H NMR fingerprint.
FIG. 6-1 is a bar graph showing valine content of 10 batches of Saviae Miltiorrhizae radix injection;
FIG. 6-2 is a bar graph of threonine content in 10 batches of red sage root injections;
FIG. 6-3 is a bar graph of alanine content in 10 batches of red sage root injection;
FIGS. 6-4 are bar graphs showing the gamma-aminobutyric acid content in 10 batches of red sage root injections;
FIGS. 6-5 are bar graphs of acetic acid content in 10 batches of red sage root injections;
FIGS. 6-6 are bar graphs of proline content in 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-7 are bar graphs of pyroglutamic acid content in 10 batches of red sage root injections;
FIGS. 6-8 are bar graphs of malonic acid content in 10 batches of red sage root injections;
FIGS. 6-9 are bar graphs of glucose content in 10 batches of red sage root injections;
FIGS. 6-10 are bar graphs of fructose content in 10 batches of red sage root injections;
FIGS. 6-11 are bar graphs of galactose content in 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-12 are bar graphs of sucrose content in 10 batches of red sage root injection;
FIGS. 6-13 are bar graphs of raffinose content in 10 batches of red sage root injection;
FIGS. 6-14 are bar graphs of uridine content in 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-15 are bar graphs of the lithospermic acid content of 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-16 are bar graphs of salvianolic acid B content in 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-17 are bar graphs of the content of tanshinol in 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-18 are bar graphs of rosmarinic acid content in 10 batches of red sage root injections;
FIGS. 6-19 are bar graphs of formic acid content in 10 batches of Saviae Miltiorrhizae radix injection;
FIGS. 6-20 are bar graphs of protocatechuic aldehyde content in 10 batches of Saviae Miltiorrhizae radix injection;
FIG. 7 is a flow chart of the quality evaluation of the salvia miltiorrhiza injection and the intermediate.
Detailed Description
Further description will be made with reference to the drawings and examples.
Example 1
Based on 1 A method for evaluating the quality of the salvia miltiorrhiza injection by H NMR technology comprises the following steps:
(1) Preparing a test sample of the salvia miltiorrhiza injection: accurately transferring 540 μl of radix Salviae Miltiorrhizae injection and 60 μl of deuterated water containing 0.05% TSP into 2ml centrifuge tube, and mixing to obtain test sample.
(2) External standard sample preparation: accurately weighing 13.12mg of calcium formate in a 10ml volumetric flask, adding deuterated water to a scale, fully dissolving and uniformly mixing, accurately transferring 1ml of deuterated water containing calcium formate in the 10ml volumetric flask, and using deionized water to a scale and uniformly mixing to obtain an external standard sample of calcium formate with the concentration of 1.01 mmol/L.
(3) 1 Acquisition of H NMR spectrum: respectively placing the sample to be tested and the external standard sample into a nuclear magnetic resonance instrument for testing, and pressing residual water peaks by using a presaturated water peak pressing pulse sequence to obtain the sample to be tested and the external standard sample 1 H NMR spectrum. 1 The H NMR acquisition instrument was a Bruker Advanced III 600 nuclear magnetic resonance spectrometer (Bruker, germany, 24-bit autosampler with 5mm BBO probe, topspin workstation). 1 The H NMR data acquisition requires tuning, probe matching and shimming, and corresponding 90 ° pulse width measurements.
1 The H NMR acquisition parameters were: the temperature of the probe is 288K; a pulse sequence NOESYGPPR1D; with solvent 90% H 2 O+10％D 2 O locking a field; a spectral width of 9.9974ppm; the center frequency was 4.696ppm; relaxation delay time is 15.0s; the acquisition time is 2.27s; the mixing time was 50ms; the collection times are 32 times; gain value is 40.3; the detected data point was 64K.
(4) 1 HNMR raw data preprocessing: the acquired original spectrum is processed by taking an exponential function of 0.30Hz as a window function, fourier transformation of the FID signal is carried out, and then the spectrum is imported into MestRenova software to take TSP (0.0) as chemical displacement reference calibration, so that baseline and phase correction are automatically carried out.
(5) Red sage root injection 1 H NMR spectrum assignment and fingerprint construction: red sage root injection 1 The structure of the H NMR spectrum is shown in FIG. 1, and the compounds indicated by each number in FIG. 1 are shown in Table 1.
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; delta=0.99±0.02, d, j=6.9 Hz), threonine (delta=1.33±0.02, d, j=6.6 Hz), alanine (delta=1.48±0.02, d, j=7.3 Hz), proline (delta=2.28 to 2.38, m), glutamic acid (delta=2.44 to 2.52, m), glutamine (delta=2.11 to 2.18, m), pyroglutamic acid (delta=2.39 to 2.43, m), aspartic acid (delta=2.93 to 2.99, m), asparagine (delta=4.01±0.02, dd, j=9.4, 5.5 Hz), tryptophan (delta=3.30±0.02, dd, j=15.6, 8.4 Hz), malonic acid (delta=3.17±0.02, s), gamma-aminobutyric acid (delta=1.88 to 1.96, m), lactic acid (delta=1.40±0.2, d=2.39 to 2.43, m), aspartic acid (delta=2.93 to 2.99, d), asparagine (delta=4.01±0.02, dd, j=0.5.02, j=5.5 Hz), tryptophan (delta=3.30±0.0.0.02, j=0.8 Hz); delta=3.25±0.02, dd, j=9.6, 8.0 Hz), galactose (delta=5.27±0.02, d, j=3.8 Hz), fructose (delta=4.11 to 4.16, m), sucrose (delta=5.41±0.02, d, j=3.8 Hz), raffinose (delta=5.43±0.02, d, j=3.8 Hz), salvianolic acid B (delta=6.39±0.02, d, j=8.1 Hz; delta=6.21±0.02, d, j=2.1 Hz), salvianolic acid a (delta=7.05±0.02, d, j=1.9 Hz; delta=6.73±0.02, d, j=2.0 Hz) Rosmarinic acid (δ=7.45±0.02, d, j=16.2 Hz), alkanoic acid (δ=7.14±0.02, d, j=8.6 Hz), protocatechuic acid (δ=9.62±0.02, s), protocatechuic acid (δ=6.94±0.02, d, j=8.3 Hz), danshensu (δ=6.78±0.02, d, j=2.78±0.02, d, j=14.1, 7.7 Hz), uridine (δ=7.86±0.02, d, j=8.0 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, d, j=0.02, j=3.02, j=0.02 Hz), cytidine (δ=8.0.02±3, 3 Hz).
(6) Integration of characteristic peaks and quantification of chemical components; the total of 20 chemical components in the red sage root injection is higher than the quantitative limit, and the chemical components are respectively as follows: valine, threonine, alanine, gamma-aminobutyric acid, acetic acid, proline, pyroglutamic acid, malonic acid, glucose, fructose, galactose, sucrose, raffinose, uridine, lithospermic acid, salvianolic acid B, danshensu, rosmarinic acid, formic acid, protocatechuic aldehyde. Characteristic peaks of the 20 chemical components described above were integrated by linear fitting in the MestReNova software and formate peaks in external standard samples were integrated, the integrated schematic is shown in fig. 3. Then, the concentrations of the above 20 chemical components were calculated according to formula (1).
Example 2
Based on 1 A method for evaluating quality of an intermediate in the red sage root process by H NMR technology comprises the following steps:
(1) Preparing a sample of the intermediate of the red sage root process: accurately transferring 200 μl of Saviae Miltiorrhizae radix ethanol precipitation into 2ml centrifuge tube, centrifuging, concentrating to completely volatilize solvent, adding 540 μl of deionized water and 60 μl of deuterated water containing 0.05% TSP, mixing, centrifuging at 10000r/min for 10min, collecting supernatant, and making into Saviae Miltiorrhizae radix process intermediate for sample.
(2) External standard sample preparation: accurately weighing 13.12mg of calcium formate in a 10ml volumetric flask, adding deuterated water to a scale, fully dissolving and uniformly mixing, accurately transferring 1ml of deuterated water containing calcium formate in the 10ml volumetric flask, and using deionized water to a scale and uniformly mixing to obtain an external standard sample of calcium formate with the concentration of 1.01 mmol/L.
(3) Intermediate of red sage root 1 Acquisition of H NMR spectrum: respectively placing the sample to be tested and the external standard sample into a nuclear magnetic resonance instrument for testing, and pressing residual water peaks by using a presaturated water peak pressing pulse sequence to obtain the sample to be tested and the external standard sample 1 H NMR spectrum. 1 The H NMR acquisition instrument was a Bruker Advanced III 600 nuclear magnetic resonance spectrometer (Bruker, germany, 24-bit autosampler with 5mm BBO probe, topspin workstation). 1 The H NMR data acquisition requires tuning, probe matching and shimming, and corresponding 90 ° pulse width measurements. 1 The H NMR acquisition parameters were: the temperature of the probe is 300K; a pulse sequence NOESYGPPR1D; with solvent 90% H 2 O+10％D 2 O locking a field; a spectral width of 12.9836ppm; the center frequency was 4.696ppm; relaxation delay time is 30.0s; the acquisition time is 2.27s; the mixing time was 100ms; the collection times are 64 times; gain value is 57; the detected data point was 32K.
(4) 1 H NMR raw data pretreatment: the acquired original spectrum is processed by taking an exponential function of 0.30Hz as a window function, fourier transformation of the FID signal is carried out, and then the spectrum is imported into MestRenova software to take TSP (0.0) as chemical displacement reference calibration, so that baseline and phase correction are automatically carried out.
(5) Intermediate of red sage root 1 H NMR spectrum assignment and fingerprint construction: salvia alcohol precipitation liquid 1 The structure of the H NMR spectrum is shown in FIG. 2, and the compounds indicated by the numbers in FIG. 2 are shown in Table 1. The characteristic peaks include: leucine (δ=0.96±0.02, t), isoleucine (δ=1.01±0.02, d, j=7.0 Hz; δ0=0.94±0.02, t), valine (δ1=1.04±0.02, d, j=6.9 Hz; δ2=0.99±0.02, d, j=6.9 Hz), threonine (δ3=1.33±0.02, d, j=6.6 Hz), alanine (δ4=1.48±0.02, d, j=7.3 Hz), proline (δ5=2.28 to 2.38, m), glutamic acid (δ6=2.44 to 2.52, m), glutamine (δ7=2.11 to 2.18, m), pyroglutamic acid (δ8=2.39 to 2.43, m), aspartic acid (δ9=2.93 to 2.99, m), asparagine (δ=4.01±0.02, dd, j=9.4, 5.5 Hz), tryptophan (δ0=3.30±0.02, dd, j=15.6, 8.4 Hz), malonic acid (δ1.17±0.02, s), gamma-aminobutyric acid (δ2=1.88 to 1.96, m), lactic acid (δ8=3.11 to 2.18, m), pyroglutamic acid (δ8=2.39 to 2.43, m), aspartic acid (δ9=2.93 to 2.99, m), asparagine (δ=4.02, j=4.02, j=0.4, j=5.5 Hz), tryptophan (δ0.30±0.02, dd, j=5.4, j=3.5.3, 5 Hz), lactic acid (δ0.3=3.30±0.02, j=5.4, j=5.6, 8.4 Hz), lactic acid (δ0.3.3.3+0.3 to 2, 3, 5 Hz), gamma-aminobutyric acid (δ2.8, 5) 5-hydroxymethylfurfural (δ3=9.44±0.02, s), salvianolic acid B (δ4=6.39±0.02, d, j=8.1 Hz; δ5=6.21±0.02, d, j=2.1 Hz), salvianolic acid a (δ6=7.05±0.02, d, j=1.9 Hz; δ7=6.73±0.02, d, j=2.0 Hz), rosmarinic acid (δ=7.45±0.02, d, j=16.2 Hz), lithospermic acid (δ=7.14±0.02, d, j=8.6 Hz), protocatechuic acid (δ=9.62±0.02, s), protocatechuic acid (δ=6.94±0.02, d, j=8.3 Hz), danshensu (δ=6.78±0.02, d, j=2.1 Hz; δ=2.78±0.02, d)J=14.1, 7.7 Hz), uridine (δ=7.86±0.02, d, j=8.0 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) Integration of characteristic peaks and quantification of chemical components. The total 22 chemical components in the red sage alcohol precipitation solution are higher than the quantitative limit, and are respectively: valine, threonine, alanine, gamma-aminobutyric acid, proline, pyroglutamic acid, succinic acid, malic acid, malonic acid, glucose, fructose, galactose, sucrose, mannose, stachyose, uridine, salvianolic acid A, salvianolic acid B, salvianic acid A, rosmarinic acid, formic acid, protocatechuic aldehyde. Characteristic peaks of the 22 chemical components described above were integrated by linear fitting in the MestReNova software, the integrated schematic being shown in fig. 3. Then, the concentrations of the 22 chemical components were calculated according to formula (1).
Example 3
Salvia injection and process intermediate of Salvia 1 H NMR quantitative methodology investigation:
(1) Linear relationship: (1) deionized water and deuterated water containing TSP were used at 9:1, preparing deuterated solvent according to the proportion. (2) Precisely weighing a certain amount of reference substances such as 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, danshensu, rosmarinic acid, formic acid and protocatechuic aldehyde, and using deuterated solvent to fix volume to obtain reference substance mother liquor. (3) The mother liquor of the reference substance is diluted by deuterated solvent by a multiple ratio to obtain the diluted solutions of the reference substances with the concentrations of 1/2, 1/4, 1/8 and 1/16 of the original mother liquor. (4) All control mother liquor and control dilutions were collected as described in step 3 of examples 1 and 2 1 H NMR spectra and integration of each control characteristic peak was performed as described in step 6 of examples 1 and 2. (5) Linear regression is carried out by taking the characteristic peak area of the reference substance as an ordinate and the mass concentration as an abscissa to obtain a coefficient r of a regression equation 2 All greater than 0.999, proving that NMR was instrumentalThe linear relationship is good.
(2) Specialization: the method comprises the steps of preparing a sample of a test solution from the red sage root injection and the red sage root alcohol precipitation according to the method in the step 1 in the examples 1 and 2, verifying the specificity of quantitative peaks by an HSQC method, and indicating that quantitative peaks have no impurity peak interference, thus indicating that the method has good specificity.
(3) Authenticity: (1) the concentration of characteristic peaks of each reference substance is calculated by the method in the step 6 in the examples 1 and 2, then the variation coefficient is calculated by the calculated value and the weighing value of the concentration of each reference substance, the calculated formula is shown as the formula (2), and the obtained NMR measurement variation coefficient of each reference substance is within 2.48%, so that the method has good authenticity.
Wherein c v Coefficient of variation C m For the quantitative measurement obtained by the method, C w Is the actual value of the weighing.
(4) Instrument precision: preparing a test solution sample from the red sage root injection according to the method of the step 1 in the example 1, and continuously collecting six times by using the method of the step 3 1 H NMR spectrum, and integrating each characteristic peak by the method in step 6, and calculating peak area ratio and RSD value of each chemical component and external standard, the result shows that RSD value of each characteristic peak is less than 2.07%, and the method has good instrument precision.
(5) Sample preparation repeatability: taking Saviae Miltiorrhizae radix injection and Saviae Miltiorrhizae radix ethanol precipitation, preparing 6 samples in parallel according to the method of step 1 in examples 1 and 2, and collecting six parallel samples respectively according to the method of step 3 in examples 1 and 2 1 H NMR spectra, and integrating each characteristic peak by the method described in step 6 of examples 1 and 2, and calculating the peak area ratio and RSD value of each chemical component and external standard, the result shows that the RSD value of each characteristic peak is less than 2.84%, which proves that the sample preparation method has good repeatability.
(6) Stability for 24 h: taking Saviae Miltiorrhizae radix injection and Saviae Miltiorrhizae radix ethanol precipitation, respectively preparing sample of test solution according to the method described in step 1 in examples 1 and 2The samples were collected at the 0h,2h,4h,6h,9h,12h,24h using the method described in step 3 of examples 1 and 2 1 H NMR spectra, and integrating each characteristic peak and calculating the peak area ratio and RSD value of each chemical component and external standard by the method described in step 6 in examples 1 and 2, and the results show that no new characteristic peak appears in 24 hours, and the RSD value of each characteristic peak is less than 2.47%, which proves that the stability of the sample of the test solution is good in 24 hours.
(7) Sample addition and recovery experiment: taking red sage root injection with known content, adding quantitative reference substances into three concentration groups of high, medium and low concentration groups, preparing a sample of the test solution by using the red sage root injection added with the reference substances according to the method of step 1 in example 1, measuring the content by using the method of steps 3-6 in 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 shows that the method has good quantitative accuracy on the salvia miltiorrhiza injection.
Taking the red sage alcohol precipitation solution with known content, adding quantitative reference substances into the red sage alcohol precipitation solution with three concentration groups of high, medium and low concentration groups, preparing a sample of the test solution by using the red sage alcohol precipitation solution added with the reference substances according to the method of step 1 in example 2, measuring the content by using the method of step 3-6 in example 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 shows that the method has good quantitative accuracy on the intermediate of the red sage root process.
Example 4
Based on 1 The quality one-measurement-multiple-evaluation application of the salvia miltiorrhiza injection of the H NMR technology comprises the following steps:
1. taking 10 batches of radix Salviae Miltiorrhizae injection, and obtaining radix Salviae Miltiorrhizae injection according to the method described in step 1-6 of example 1 1 H NMR fingerprint and quantifying 20 chemical components therein.
2. Fingerprint analysis of the salvia miltiorrhiza injection: the collected fingerprint of 10 batches of red sage root injection is shown in fig. 4, and the Pearson correlation coefficient method in the similarity evaluation method and the PCA and HCA method in the multivariate analysis are selected to analyze the fingerprint of the 10 batches of red sage root injection, but the analysis and evaluation method of the fingerprint is not limited to the method, and the specific steps are as follows:
(1) The fingerprint of 10 batches of red sage root injection is led out in boxes with the width of 0.02ppm by the method of 'sectional integration', and the water peak inhibition area and the two-end no-signal area are deleted to obtain a matrix X (10X 415) containing 10 observed values and 415 variables.
(2) Taking the average spectrum of 10 batches of red sage root injection fingerprints as a reference, selecting the data points of characteristic peaks of the fingerprints, and calculating Pearson correlation coefficients of 10 batches of samples as similarity evaluation indexes. The batch numbers and the similarity of the red sage root injections in each batch are shown in table 2, and the similarity evaluation results show that the similarity of the rest batches except the batch S3 is greater than 0.99, 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 the samples have higher similarity, which indicates that the consistency of 10 batches of red sage root injections is good. In addition, the six batches S5-S10 all have a similarity greater than 0.995, indicating that the six batches have a higher similarity.
TABLE 2 batch number and similarity of 10 batches of Danshen injection
(2) The matrix X was imported into SIMCA software for PCA analysis, and the PCA score plot is shown in fig. 5 (a), from which it can be seen that lot S4 differs significantly from the other lots.
(3) Based on the analysis of HCA by SIMCA software, the HCA tree is shown in FIG. 5 (B), the fingerprint of the injection of the batch S5-S10 is high, the batch S1-S3 times is high, the relative distance between the batch S4 and other batches is the largest, the chemical composition of the injection is the largest 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 red sage root injection: application of 1 H NMR was performed on valine, threonine, alanine, gamma-aminobutyric acid, acetic acid, proline, pyroglutamic acid, malonic acid, glucose, fructose, galactose, sucrose, raffinose,the measurement results of the content of uridine, lithospermic acid, salvianolic acid B, tanshinol, rosmarinic acid, formic acid and protocatechuic aldehyde are shown in Table 3. The visualized histogram is shown in fig. 6-1 to 6-20.
Table 3 is based on 1 Quantitative results of 10 batches of red sage root injection by H NMR (unit: mg/ml)
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4. Measuring total solid content of 10 batches of Saviae Miltiorrhizae radix injection by dry constant weight method, measuring cation content by atomic absorption spectrometry, measuring anion content by anion chromatography, and calculating 1 H NMR quantifiable ratio of all chemical component content to total solids content (1% by weight) and 1 h NMR can quantify the proportion of all chemical components to the other solids content than inorganic ions (ratio 2), ratio 2 can also be regarded as 1 The H NMR can quantitatively determine the proportion of all chemical components in the total organic components in the red sage root injection. The measurement results of the total solid content and the inorganic ion content and the calculation results of the ratio 1 and the ratio 2 are shown in table 4, and the results show that the total content of the chemical components quantified by NMR 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.
TABLE 4 determination of total solids, inorganic ion content and NMR quantitative ingredient content ratio of 10 batches of Danshen injection
The quality evaluation flow chart of the salvia miltiorrhiza injection and the intermediate is shown in figure 7.
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (19)
1. Based on 1 An H NMR-based method for testing and evaluating a multi-grade salvia miltiorrhiza injection or a process intermediate thereof, comprising the steps of:
(1) Preparation of external standard samples and test samples: weighing calcium formate, and dissolving the calcium formate with 10% deuterated water to prepare an external standard sample solution of the calcium formate; taking a red sage root sample to be detected, and preparing a sample solution by using deuterated water containing TSP and deionized water;
（2） 1 h NMR spectrum determination: the pre-saturated water peak pressing pulse sequence is used for pressing water peaks, and the test sample solution and the external standard sample solution are determined 1 H NMR spectrum;
（3） 1 h NMR fingerprint construction and analysis: will be original 1 Preprocessing the H NMR spectrum and attaching signals, constructing a fingerprint spectrum by using attached characteristic peaks of chemical components, and analyzing the fingerprint spectrum; wherein the sample solution 1 The pretreatment of the H NMR spectrum is followed by attributing 40 chemical components, including 12 amino acids, 7 small molecule organic acids, 8 saccharides and degradation products thereof, 7 salvianolic acid compounds and 6 nucleoside compounds;
(4) Content measurement and overall quality evaluation: and (3) carrying out integral on characteristic peaks corresponding to chemical components exceeding a quantitative limit, absolute quantification by an external standard method, and comprehensively evaluating the quality of a sample by combining with a fingerprint.
2. The method according to claim 1, wherein the red sage root sample to be tested in step (1) is a red sage root injection or its process intermediate.
3. The method according to claim 1, wherein when the sample in step (1) is a red sage root injection, the volume ratio of the sample to the deuterated water is 9:1, mixing the materials in proportion to obtain the product; when the sample is the intermediate of the red sage root process, the intermediate of the red sage root process is centrifugally concentrated, the solvent is volatilized, and the volume ratio of the added solvent is 9:1, re-dissolving deionized water and deuterated water containing TSP, centrifuging, and taking supernatant to obtain; the total volume of the mixed liquid is 600-1000 mu l.
4. The method according to claim 1, wherein the concentration of the calcium formate external standard sample prepared in the step (1) is 0.8-1.2mmol/L.
5. The method according to claim 1, wherein the conditions of the measurement in the step (2) are: the temperature of the probe is 288-300K; a pulse sequence NOESYGPPR1D; the spectrum width is 9.9974-12.9836ppm; the center frequency was 4.696ppm; relaxation delay time is 14.0-30.0s; the acquisition time was 2.27s; the mixing time is 50-100ms; the collection times are 32 or 64 times; gain value is 32-57; the detected data points are 32K or 64K.
6. The method according to claim 1, wherein the conditions of the measurement in the step (2) are: the temperature of the probe is 290-295 and K; a pulse sequence NOESYGPPR1D; with solvent 90% H 2 O+10% D 2 O locking a field; the spectrum width is 11.4912-12.4967ppm; the center frequency was 4.696ppm; relaxation delay time is 14.5-15.5s; the acquisition time was 2.27s; the mixing time is 50-100ms; the collection times are 32 times; gain value is 40.3-45.2; the detected data point was 32K.
7. The method of claim 1, wherein in step (2) 1 The H NMR data acquisition requires tuning, probe matching and shimming, and corresponding 90 ° pulse width measurements.
8. The method of claim 1, wherein the profile pre-processing in step (3) comprises fourier transform, baseline correction, phase correction, and profile alignment of the raw FID signal.
9. The method of claim 8, wherein the pattern is processed as a window function with an exponential function of 0.30Hz prior to fourier transformation in step (3).
10. The method of claim 1, wherein the 40 chemical components assigned in step (3) are: 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, mannose, stachyose, 5-hydroxymethylfurfural, salvianolic acid B, salvianolic acid A, rosmarinic acid, lithospermic acid, protocatechuic acid, salvianic acid, uridine, 2' -deoxyadenosine, adenine, adenosine, guanosine and cytidine.
11. The method according to claim 1, wherein 1-2 peaks of each chemical component are selected as characteristic peaks in step (3) for constructing a fingerprint, and the characteristic peaks are required to be independent signal peaks or independent signal peaks separated by simple deconvolution.
12. The method of claim 1, wherein the fingerprint analysis in step (3) requires "binning" of the fingerprint.
13. The method according to claim 12, wherein the "binning" method in step (3) comprises "summing", "average", "piecewise integration", "median"; the analysis method comprises a similarity evaluation method and a multivariate statistical method; the similarity evaluation method comprises a Pelson 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 squares discriminant analysis and orthogonal partial least squares analysis.
14. The method according to claim 1, wherein the content measurement method in the step (4) is a pulon method using calcium formate as an external standard.
15. The method according to claim 1, wherein the chemical components of the red sage root injection in step (4) exceeding the quantitative limit are 20, which are 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, respectively.
16. The method according to claim 1, wherein the step (4) comprises 22 chemical components exceeding the quantitative limit of the intermediate sample of the red sage root process, which are respectively: valine, threonine, alanine, gamma-aminobutyric acid, proline, pyroglutamic acid, succinic acid, malic acid, malonic acid, glucose, fructose, galactose, sucrose, mannose, stachyose, uridine, salvianolic acid A, salvianolic acid B, salvianic acid A, rosmarinic acid, formic acid and protocatechuic aldehyde.
17. The method according to claim 15, wherein the total content of the 20 chemical components in the red sage root injection is 75% -80% of the total solid content, and the total content of the organic matters is 90% -95%.
18. Use of the method for multiple evaluation of a red sage root injection or its process intermediate according to any one of claims 1-17 for analytical evaluation of quality of a product containing red sage root.
19. The use according to claim 18, wherein the product is a red sage root injection or a red sage root process intermediate.
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