CN109342579B - HPLC fingerprint detection method for traditional Chinese medicine for relaxing bowel - Google Patents
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Abstract
The invention discloses a method for detecting a fingerprint of a traditional Chinese medicine for relaxing bowel, which searches for the conditions of an instrument, a chromatographic column, a preparation method of a test solution, a mobile phase, detection wavelength and the like for detecting the fingerprint, establishes liquid-phase fingerprint detection conditions, performs methodology investigation, establishes a standard fingerprint of the traditional Chinese medicine according to a plurality of batches of large-scale production samples, can effectively guide feeding and strictly standardize production operation in the production process, really ensures the safety, effectiveness and reliability of clinical medication, and has the advantages of convenient and quick operation and the like compared with other detection methods.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine detection, and relates to an HPLC fingerprint detection method for a traditional Chinese medicine for relaxing bowel.
Background
The fingerprint is a quantifiable method for detecting the traditional Chinese medicinal materials and the traditional Chinese medicinal preparations, and is mainly used for identifying the truth and evaluating the uniformity and the stability of the quality of the raw medicinal materials, semi-finished products and the preparations. Compared with a quality analysis method for measuring the content of index components, the fingerprint can comprehensively reflect the types and the quantity of chemical components of the traditional Chinese medicine, can realize comprehensive evaluation on the internal quality of the traditional Chinese medicine and effective control on the whole substances of the traditional Chinese medicine under the condition that the effective components of a compound preparation of the traditional Chinese medicine are not completely clarified, and is the most effective means for controlling the quality of the traditional Chinese medicine and the preparation thereof at present. The detection method of traditional Chinese medicine fingerprint spectrum includes spectrometry, etc. The chromatography mainly comprises thin-layer chromatography, high performance liquid chromatography, gas chromatography, capillary electrophoresis and the like, wherein the high performance liquid chromatography has the characteristics of high efficiency, rapidness, sensitivity and good reproducibility, and is the mainstream method for the traditional Chinese medicine fingerprint spectrum research.
Zhishu Runchang granule is six kinds of Chinese medicine developed by Jianmin pharmaceutical industry group GmbH, and is prepared from four Chinese medicinal materials including immature bitter orange, white atractylodes rhizome, cassia seed and bitter apricot seed. At present, the quality control of the traditional Chinese medicine compound preparation for relaxing bowel generally adopts the qualitative or quantitative control of single components, and the product quality is difficult to be comprehensively and effectively controlled, so that the curative effect cannot be ensured.
Disclosure of Invention
The invention aims to comprehensively and effectively control the product quality and establish an HPLC fingerprint detection method for relaxing bowel.
In order to achieve the purpose, the invention adopts the following technical means:
an HPLC fingerprint detection method of a traditional Chinese medicine for relaxing bowel comprises the following components in parts by weight: 15-30 parts of immature bitter orange, 50-100 parts of bighead atractylodes rhizome, 15-30 parts of cassia seed and 10-20 parts of bitter apricot seed, and the detection method comprises the following steps:
(1) grinding the Chinese medicinal materials, extracting with organic solvent, separating the extract with chromatography column, eluting with water, collecting eluate, and making into test solution;
(2) Precisely absorbing a test solution, detecting by a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 250-300nm, and the stationary phase of the high performance liquid chromatographIs C18A chromatographic column, wherein the mobile phase is a mixed solution of acetonitrile and buffer salt, gradient elution is adopted, the flow rate is 0.5-2ml/min, the column temperature is 25-35 ℃, and the number of theoretical plates is not less than 4000;
(3) recording a chromatogram of the test solution, and performing data import, multipoint correction and data matching on the chromatogram by using a Chinese medicine chromatogram fingerprint similarity evaluation system of the State pharmacopoeia Committee to obtain a standard fingerprint and performing similarity analysis on the standard fingerprint and a fingerprint of a sample to be detected.
Preferably, the organic solvent is 40-90% by volume methanol, preferably 50% methanol.
Preferably, the extraction is ultrasonic extraction.
Preferably, the chromatography column is a polyamide column.
Preferably, the set detection wavelength of the ultraviolet detector is 280 nm.
Preferably, in the gradient elution, the volume ratio of the buffer salt phase in each time period and the mobile phase is respectively: buffering the salt phase for 0-7 min and 92%; 7-20 min, and 92-80% of buffer salt phase; 20-36 min, and 80% of buffer salt phase; 36-55 min, and 80-52% of buffer salt phase; 55-70 min, and 52-38% of buffer salt phase.
Preferably, the buffer salt is: 0.8g of monopotassium phosphate, 1.0g of sodium dodecyl sulfate and 1ml of glacial acetic acid are dissolved and diluted to 1000ml by adding water, and the pH value is 3.0.
Preferably, the fingerprint spectrum has 14 characteristic peaks, and the peak 1 is synephrine and the peak 9 is aurantio-obtusin after comparison with a reference substance spectrum.
The invention has the beneficial effects that:
the traditional Chinese medicine is applied under the guidance of the theory of the traditional Chinese medicine, any single active component cannot reflect the whole curative effect embodied by the traditional Chinese medicine, particularly the compound preparation exerts the curative effect through the mutual synergistic effect among multiple components and multiple target points, and one or more index components which are singly measured cannot comprehensively reflect the internal quality of the traditional Chinese medicine compound. The fingerprint can comprehensively reflect chemical information contained in the traditional Chinese medicine, in the invention, an HPLC fingerprint of the Zhishu intestine-moistening granules is established, 10 batches of the Zhishu intestine-moistening granules are simultaneously measured, 14 common peaks and a standard fingerprint are marked, and the similarity of the obtained chromatogram is more than 0.96; calculating stability, repeatability and precision by using relative retention time and relative peak area, wherein RSD values of the relative retention time are less than 2.0%, and RSD values of the relative peak area are less than 5.0%; of the 14 components, 2 components were identified, synephrine and aurantio-obtusin, respectively.
The Zhishu intestine moistening granule fingerprint standard provided by the invention can effectively guide feeding and strictly standardize production operation in the production process, really ensures the safety, effectiveness and reliability of clinical medication, and has the advantages of convenience and quickness in operation and the like compared with other detection methods.
Drawings
FIG. 1 is an HPLC chromatogram of a sample to be tested after methanol ultrasonic extraction.
FIG. 2 is an HPLC chromatogram of a sample to be tested after ethanol ultrasonic extraction.
FIG. 3 is an HPLC chromatogram after ultrasonic extraction of n-butanol of a sample to be detected.
FIG. 4 is an HPLC chromatogram after reflux extraction of a sample to be tested.
FIG. 5 HPLC chromatogram of a sample after passing through a neutral alumina column.
FIG. 6 acetonitrile-sodium dihydrogen phosphate mobile phase HPLC chromatogram.
FIG. 7 is an HPLC chromatogram obtained at a detection wavelength of 210 nm.
FIG. 8 is an HPLC chromatogram obtained at a detection wavelength of 250 nm.
FIG. 9 is an HPLC chromatogram obtained at a detection wavelength of 280 nm.
FIG. 10 is an HPLC chromatogram obtained at a detection wavelength of 300 nm.
FIG. 11 is an HPLC chromatogram obtained at a detection wavelength of 330 nm.
Fig. 12 is an HPLC standard fingerprint of Zhishu Runchang granule.
Detailed Description
The present invention will be described in detail below with reference to specific examples.
The Zhishu Runchang granule used in the following examples is provided by Jianmin pharmaceutical industry group GmbH.
Prescription: 266.67g of immature bitter orange, 1000g of bighead atractylodes rhizome, 200g of bitter apricot seed, 266.67g of cassia seed
The preparation method comprises the following steps: decocting the four medicines in water, extracting, concentrating the extracting solution until the relative density is 1.06-1.08, cooling to room temperature, adding ethanol for alcohol precipitation, taking supernatant obtained after alcohol precipitation, concentrating under reduced pressure to obtain thick paste, adding sugar powder, dextrin, fruit essence and sucralose into the thick paste, mixing uniformly, granulating, and preparing into 1000g of granules.
1. Instrument and reagent
A Waters e2695 high performance liquid chromatograph, a quaternary pump, an online vacuum degassing system, an automatic sample injector, a column incubator and an ultraviolet detector; AB204-E electronic balance (Shanghai Merle-Torledo instruments, Inc.).
10 batches of Zhishu intestine-moistening granules (180601-05, 180701-05, 10 batches in total); synephrine reference (for testing, purchased by China institute for food and drug testing, lot number 110727-201608); an orange cassia tora essence reference substance (for content detection, purchased by China food and drug testing research institute, batch number: 111900-201605); acetonitrile is chromatographic grade, and water is purified water.
2. Method step
(1) Preparation of a test solution: grinding the intestine-moistening particles of the trifoliate orange to be detected, taking 1g, precisely weighing, precisely adding 50ml of 50% methanol, weighing, carrying out ultrasonic treatment for 40 minutes (power 250W and frequency 40kHz), cooling, weighing again, complementing the lost weight with 50% methanol, shaking up, filtering, precisely weighing 20ml of subsequent filtrate, evaporating to dryness, adding 25ml of water into residues to dissolve, passing through a polyamide column (60-90 meshes, 2.5g, the inner diameter of 1.5cm, loading into the column by a dry method), eluting with 25ml of water, collecting eluent, transferring into a 25ml measuring flask, adding water to the scale, and shaking up to obtain the traditional Chinese medicine.
(2) Precisely absorbing 10 mu l of sample solution, and detecting by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 280nm, and the stationary phase of the high performance liquid chromatograph is C18The mobile phase of the chromatographic column was acetonitrile and buffer salt (potassium dihydrogen phosphate 0.8g, sodium dodecyl sulfate 1.0g,glacial acetic acid 1ml, adding water to dissolve and dilute to 1000ml, pH 3.0), and gradient eluting at flow rate of 1ml/min and column temperature of 30 deg.C with theoretical plate number not less than 4000;
(4) methodology investigation: performing precision test, stability test and repeatability test according to the requirement of the fingerprint;
(5) and (3) sample determination: taking several batches of semen hoveniae intestine moistening particles, respectively preparing 10 parts of test sample solution according to the method in the step (1), precisely absorbing 10 mu l of each test sample solution, determining by adopting the chromatographic condition in the step (3), recording a chromatogram for 0-70min, and calculating relative retention time and relative peak area by taking an orange obtusin peak (peak No. 9) as a reference peak in the chromatogram;
(6) establishing a fingerprint and analyzing peak attribution: taking batches of semen hoveniae intestine moistening particle samples respectively, preparing a test solution according to the step (1), determining according to the chromatographic conditions of the step (3), recording a chromatogram for 70min, concentrating all chromatographic peaks within 70min, comparing the chromatograms of the batches of samples, determining 14 common peaks, and comparing the chromatograms with a reference substance to confirm that No. 1 is synephrine and No. 9 is aurantio-obtusin; under the condition of the finished product chromatogram fingerprint, performing fingerprint research on each medicinal material in the prescription, and performing peak attribution analysis;
(7) Calculating the similarity of the samples: and (3) introducing the 10 batches of Zhishu intestine-moistening granule chromatograms into a 2004 edition of Chinese medicine chromatogram fingerprint similarity evaluation system issued by the State pharmacopoeia Committee, selecting a time window with the width of 0.1min, generating a chromatogram fingerprint common mode through multi-point correction and data matching, and carrying out similarity analysis on the fingerprints of 10 batches of samples.
3. Preparing a test solution: the main purpose of extracting the test sample is to reduce the influence of detected impurities (including protein, tannin and other traditional Chinese medicine impurities and auxiliary materials in the preparation) on the peak shape of the characteristic peak, so that the characteristic peak has higher separation degree and peak area. The main factors affecting the extraction effect are the extraction solvent, the extraction method and the purification method. According to the prescription and the preparation characteristics of the Zhishu intestine-moistening granules, ethanol, methanol and n-butanol are respectively considered as extraction solvents, and through analysis and comparison, a test solution obtained by methanol extraction can comprehensively reflect main components in a finished product, and has good peak shape and repeatability, so that methanol is determined as the extraction solvent (figures 1, 2 and 3); the extraction method (ultrasonic and heating reflux) is examined, and the peak area and the peak number of the sample fingerprint (figure 4) extracted by reflux are both smaller than those of ultrasonic extraction; in addition, the purification method is also examined, and the discovery shows that when the polyamide column is used for chromatographic separation and purification, the impurity peaks can be further reduced, the peak shape of the characteristic peak can be improved, meanwhile, the peak area and the number of the peak of the characteristic peak can not be influenced, and the purification effect is better than that of a neutral alumina column (figure 5).
Finally, the preparation method of the sample is determined as follows: grinding the intestine-moistening particles of the trifoliate orange to fine powder, taking about 1g of the intestine-moistening particles, precisely weighing, precisely adding 50ml of 50% methanol, weighing, ultrasonically treating for 40min (power 250W and frequency 40kHz), cooling, complementing the loss weight with 50% methanol, shaking up, filtering, precisely weighing 20ml of subsequent filtrate, evaporating to dryness, adding 25ml of water into residues to dissolve, passing through a polyamide column (60-90 meshes, 2.5g, the inner diameter of 1.5cm, and filling the column by a dry method), eluting with 25ml of water, collecting eluent, transferring to a 25ml measuring flask, adding water to the scale, and shaking up to obtain the traditional Chinese medicine.
4. Establishment of detection method
4.1 column: agilent ZORBAX SB-C184.6X 250mm, 5 μm; detection wavelength: 280 nm; the sample amount is 10 mul; column temperature: 30 ℃; flow rate: 1 ml/min.
4.2 selection of the Mobile phase
We have studied a plurality of groups of mobile phases together, wherein the first group of mobile phases is acetonitrile-buffer salt (0.8 g of potassium dihydrogen phosphate, 1.0g of sodium dodecyl sulfate and 1ml of glacial acetic acid are taken and dissolved in water and diluted to 1000ml, and the pH value is 3.0), the second group of mobile phases is acetonitrile-sodium dihydrogen phosphate solution (the pH value is adjusted to 4.4 by phosphoric acid), the same gradient elution program is adopted, and comparison and screening show that the liquid chromatogram obtained by the acetonitrile-buffer salt system has the advantages of stable baseline, symmetrical chromatographic peak shape, good separation degree and obvious superiority to the acetonitrile-sodium dihydrogen phosphate chromatogram (figure 6).
4.3 optimization of gradient elution procedure: through systematic exploration and optimization, the following linear gradient elution procedures are found to be capable of comprehensively detecting chemical components in the preparation and also capable of enabling the components to obtain ideal separation effects:
TABLE 1 gradient elution scheme
| Elution time | Buffer salt phase | Acetonitrile phase |
| 0～7min | 92％ | 8％ |
| 7～20min | 92→80％ | 8→20％ |
| 20～36min | 80％ | 20％ |
| 36～55min | 80→52％ | 20→48％ |
| 55～70min | 52→38％ | 48→62％ |
And completing 1 sample analysis program, wherein the recording time is 0-70 min.
4.4 determination of optimal measurement wavelength: through the comparative analysis of liquid chromatogram, the separation degree of fingerprint peaks in the range of 250-300nm is good, synephrine and aurantio-obtusin can be detected (figures 8, 9 and 10), especially, the fingerprint spectrum taking 280nm as the detection wavelength can best reflect the components of the preparation (figure 9), and the peak shape is the best. Whereas the peak separation and the number of chromatographic peaks, baseline and signal response outside the range of 250-300nm were weak (FIGS. 7, 11). Therefore, the detection wavelength was determined to be in the range of 250-300nm and the optimum detection wavelength was determined to be 280 nm.
5. Methodology investigation
5.1 stability test: taking the sample solution, measuring at 0, 4, 8, 12h and 24h according to the conditions of the chromatographic term, and observing the relative retention time of each common peak and the RSD of the relative peak area. The results show that the relative retention time of each spectrum peak is less than 2.0 percent, the relative peak area of each spectrum peak is less than 2.0 percent, and the fingerprint spectrum technical requirements are met. See tables 2-3.
TABLE 2 evaluation of the relative Retention time for Zhishu Runchang granule stability
TABLE 3 evaluation of relative peak area for stability evaluation of Zhishu Runchang granule
5.2 repeatability test: taking 6 parts of samples of the same batch, preparing 6 parts of test solution according to the test solution preparation method under item 1, measuring under the chromatographic conditions, and observing the relative retention time of each common peak and the RSD of the relative peak area. The results show that the relative retention time of each spectrum peak is less than 2.0 percent, the relative peak area of each spectrum peak is less than 5.0 percent, and the fingerprint spectrum technical requirements are met. See tables 4-5.
Table 4 relative retention time for repeatability test of Zhishu Runchang granule
TABLE 5 relative peak area for repeatability test of Zhishu Runchang granule
5.3 precision test: and taking a sample solution to be tested, continuously injecting samples for 6 times according to the conditions under the chromatographic terms, and inspecting the relative retention time of each common peak and the RSD of the relative peak area. The results show that the relative retention time of each spectrum peak is less than 0.1%, the relative peak area of each spectrum peak is less than 3.0%, and the fingerprint spectrum technical requirements are met. See tables 6-7.
TABLE 6 examination of the relative retention time of Zhishu Runchang granule for precision
TABLE 7 precision evaluation of Zhishu Runchang granule relative peak area
6. Establishing a fingerprint spectrum: respectively taking 10 batches of Zhishu intestine-moistening particle samples, preparing a test solution, measuring and recording an 85min chromatogram, concentrating all chromatogram peaks within 70min, comparing the chromatograms of the 10 batches of samples, and determining 14 common peaks. The confirmation shows that No. 1 is synephrine, No. 9 is aurantio-obtusin, and because the cassia seed is the main active ingredient of the compound intestine moistening, the aurantio-obtusin is selected as a reference peak;
6.1 sample similarity calculation: introducing 10 batches of Zhishu intestine-moistening granule chromatograms into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004 edition) issued by the State pharmacopoeia Committee, selecting a time window with the width of 0.1min, generating a comparison spectrum by adopting a sample 5, generating a chromatogram fingerprint common mode through multipoint correction and data matching, and obtaining 10 batches of sample fingerprint similarity calculation results shown in a table 8.
TABLE 8 Ten sample similarities
| Batch number | Degree of similarity |
| 180601 | 0.990 |
| 180602 | 0.981 |
| 180603 | 0.966 |
| 180604 | 0.988 |
| 180605 | 0.987 |
| 180701 | 0.972 |
| 180702 | 0.990 |
| 180703 | 0.991 |
| 180704 | 0.994 |
| 180705 | 0.989 |
| Comparison fingerprint | 1 |
6.2 preparation of Standard fingerprint of Zhishu Runcang granule
Taking a plurality of batches of Zhishu intestine-moistening particles, respectively preparing a test solution according to the preparation method of the test solution, establishing a high performance liquid chromatogram of the Zhishu intestine-moistening particles according to a plurality of batches of detection data obtained by detection, determining the peak retention time and the peak area value of the high performance liquid chromatogram, then collecting all detection data, calculating the average value of the relative retention time of each corresponding peak, the average value of the relative peak area, the standard deviation and the like, respectively dividing the average retention time and the average peak area of a reference peak, using the average retention time and the relative peak area value of a standard fingerprint, deriving a calculation result, making standard fingerprint data, drawing a standard fingerprint, and obtaining a standard fingerprint in a figure 12 and a result in a table 9.
TABLE 9 Standard fingerprint data
6.3 correlation between the fingerprints of the herbs and the finished products
Preparing a medicinal material test solution: extracting the raw materials in the prescription according to a granular preparation process to obtain an extract, and performing chromatographic peak assignment according to standard fingerprint spectrum conditions. The results are shown in Table 10.
TABLE 10 correlation spectra of Zhishu Runchang granule and herbs
| Peak number | Immature bitter orange | White atractylodes rhizome | Bitter apricot kernel | Cassia seed |
| 1 | + | |||
| 2 | + | |||
| 3 | + | + | + | |
| 4 | + | |||
| 5 | + | |||
| 6 | + | |||
| 7 | + | |||
| 8 | + | + | ||
| 9(s) | + | |||
| 10 | + | |||
| 11 | + | |||
| 12 | + | + | ||
| 13 | + | |||
| 14 | + |
Claims (2)
1. An HPLC fingerprint detection method of a traditional Chinese medicine for relaxing bowel is characterized in that the traditional Chinese medicine for relaxing bowel comprises the following components in parts by weight: 15-30 parts of immature bitter orange, 50-100 parts of bighead atractylodes rhizome, 15-30 parts of cassia seed and 10-20 parts of bitter apricot seed, and the detection method comprises the following steps:
(1) grinding the Chinese medicinal materials, ultrasonic extracting with 50% methanol, separating with polyamide chromatographic column, eluting with water, collecting eluate, and making into test solution;
(2) precisely absorbing a sample solution, and detecting by using a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the set detection wavelength of the ultraviolet detector is 280nm, and the stationary phase of the high performance liquid chromatograph is C18A chromatographic column, wherein the mobile phase is a mixed solution of acetonitrile and buffer salt, gradient elution is adopted, the flow rate is 0.5-2ml/min, the column temperature is 25-35 ℃, and the number of theoretical plates is not less than 4000;
(3) Recording chromatogram of the test solution, performing data import, multipoint correction and data matching on the chromatogram by using a similarity evaluation system of the chromatogram and the fingerprint of the Chinese medicinal committee of the national pharmacopoeia to obtain a standard fingerprint, performing similarity analysis on the standard fingerprint and the fingerprint of the sample to be tested,
in the gradient elution, the volume ratio of each time period and the buffer salt phase in the mobile phase is respectively as follows: buffering the salt phase for 0-7 min and 92%; 7-20 min, buffer salt phase 92 → 80%; 20-36 min, and 80% of buffer salt phase; 36-55 min, 80 → 52% of buffer salt phase; 55-70 min, buffer salt phase 52 → 38%,
the buffer salt is: 0.8g of monopotassium phosphate, 1.0g of sodium dodecyl sulfate and 1ml of glacial acetic acid, and adding water to dissolve and dilute the mixture to 1000ml, wherein the pH value is 3.0.
2. The HPLC fingerprint detection method of the traditional Chinese medicine for relaxing bowel according to claim 1, wherein the HPLC fingerprint detection method comprises the following steps: the fingerprint spectrum has 14 characteristic peaks, and the 14 characteristic peaks are compared with a reference substance spectrum, wherein the peak 1 is synephrine, and the peak 9 is aurantio-obtusin.
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