CN111983092A - Method for detecting fructus amomi medicinal material fingerprint - Google Patents
Method for detecting fructus amomi medicinal material fingerprint Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N30/68—Flame ionisation detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention discloses a method for detecting a gas chromatography fingerprint spectrum of a fructus amomi medicinal material, which comprises the following steps: preparing a test solution; respectively injecting the sample solution and the normal paraffin standard solution into a gas chromatograph to obtain characteristic peak information, performing qualitative determination on the characteristic peak by adopting a retention index method, and further determining the characteristic peak by adopting a qualitative standard product; introducing 10 batches of sample solution chromatograms into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and establishing a fructus amomi medicinal material comparison fingerprint; and obtaining a chromatogram of the test sample, introducing the chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and comparing the chromatogram with a reference fingerprint to calculate the similarity of the fingerprint of the sample to be detected. The system has the advantages of good applicability, strong sensitivity and specificity, good repeatability and stability and more chromatographic peak information, can be effectively applied to identification of quality and authenticity of the fructus amomi medicinal material and evaluation of stability and genuine characteristics, and has the advantages of simple and convenient operation and reliable result.
Description
Technical Field
The invention relates to the technical field of fructus amomi medicinal material detection and analysis, in particular to a method for detecting a fructus amomi medicinal material fingerprint.
Background
Fructus amomi is one of the famous four-southern medicine in China, can be eaten or used as a medicine, has the efficacy of resolving dampness and stimulating appetite, warming spleen and checking diarrhea, regulating qi and preventing miscarriage, and has wide application value in clinical traditional Chinese medicine. However, the kinds and contents of the components are easily affected by many factors such as producing area, variety, harvesting, etc. Due to the special selection of fructus amomi to the ecological environment, difficult pollination and other factors, the yield is low, the supply of commodities is not sufficient, the price is high, so that various counterfeit products appear in the market, and seeds or fruits of closely related plants such as cardamom, amomum tsao-ko and katsumadai seeds which are similar in appearance and character are often mixed with fructus amomi.
In the fructus amomi variety, the quality of the fructus amomi produced in Guangdong spring is the best at present, the fructus amomi is called by genuine medicinal materials, green shell sand is mainly produced in Vietnam and Myanmar, Hainan sand is mainly produced in Hainan, and the three fructus amomi are listed as genuine fructus amomi by pharmacopoeia. However, in the pharmacopoeia, the content measurement only measures one component of bornyl acetate to control the quality state of fructus amomi, and the overall appearance of the fructus amomi medicinal material cannot be completely reflected, so that a detection method which can provide rich compound information is needed to comprehensively characterize the quality of the fructus amomi medicinal material.
At present, the truth and quality control of the fructus amomi are mainly determined by methods such as character identification, microscopic identification, thin-layer chromatography identification, volatile oil content identification, high performance liquid chromatography fingerprint identification and the like, and the defects of inaccurate identification or complex operation and requirement of precise instruments exist when only one method is adopted. Therefore, a simpler, effective and reasonable quality evaluation method is needed to ensure the safety and effectiveness of the use of fructus amomi.
The traditional Chinese medicine fingerprint is characterized by the chemical characteristics of the traditional Chinese medicine on the whole through a characteristic chromatogram or a spectrogram by applying a certain comprehensive technology, is known as one of the most effective technical means for comprehensively evaluating the overall quality of the traditional Chinese medicine, and related patents about the fingerprint detection of the fructus amomi medicinal material are few at present.
Disclosure of Invention
The invention aims to provide a method for detecting a fingerprint of a fructus amomi medicinal material, which can simply, conveniently and quickly obtain the fingerprint of the fructus amomi medicinal material, realize quick and accurate qualification of 11 characteristic peaks including alpha-pinene, camphene, beta-myrcene, D-limonene, camphor, borneol acetate, eugenol and caryophyllene in the fructus amomi medicinal material, quickly and effectively evaluate the quality of a sample by comparing and calculating the similarity of the fingerprint of a test sample and a reference sample of the fructus amomi medicinal material, and has the characteristics of stable baseline, less noise interference, good detection repeatability, more chromatographic peak information and high sensitivity.
The technical scheme of the invention is as follows:
a method for detecting a fructus amomi medicinal material fingerprint spectrum comprises the following steps:
1) freezing and preserving fructus amomi, crushing and sieving, and mixing with an extraction solvent according to the proportion of 1-5 g to 10-30 mL to form a sample solution;
2) ultrasonically extracting the sample liquid obtained in the step 1) for 10-45 min, and mixing the sample liquid with a purifying agent according to the proportion of 1ml to 4-50 mg; the purifying agent is a mixture of PSA and graphitized carbon, and the mass ratio of the PSA to the graphitized carbon is 1: 1-5;
3) filtering the supernatant obtained in the step 2) by using a microporous filter membrane to obtain a test solution;
4) carrying out gas chromatography analysis on the test solution obtained in the step 3) to obtain a chromatogram and retention time of a characteristic peak;
5) c is to be8-C20Performing gas chromatography analysis on the normal alkane standard solution under the same chromatographic conditions as the step 4) to obtain a chromatogram and C8-C20Retention time of normal alkanes;
6) the retention time of the characteristic peak in the step 4) and the retention time of the characteristic peak C in the step 5)8-C20Substituting the retention time of the normal alkane into a formula, and calculating the retention index of the characteristic peak;
7) comparing the retention index obtained in the step 6) with a theoretical retention index, and determining the characteristic peak;
8) dissolving the characteristic peak compound obtained in the step 7) with ethyl acetate to prepare a reference substance mixed solution;
9) performing gas chromatography analysis on the reference substance mixed solution obtained in the step 8) under the same chromatographic conditions as those in the step 4) to obtain a reference substance chromatogram and retention time;
10) qualitatively confirming the characteristic peak of the step 4) by using the reference chromatogram and the retention time obtained in the step 9);
11) analyzing 10 batches of the same-variety fructus amomi medicinal materials in different batches according to the steps 1) to 4), identifying characteristic peaks, introducing chromatograms into a traditional Chinese medicine fingerprint database for similarity evaluation, and generating comparison fingerprints;
12) analyzing a fructus amomi medicinal material sample to be detected according to the steps 1) to 4), identifying the characteristic peak, introducing the chromatogram into a traditional Chinese medicine fingerprint database for similarity evaluation, comparing the similarity with the comparison fingerprint obtained in the step 11), and calculating the similarity.
Preferably, the extraction solvent in the step 1) is ethyl acetate, and the fructus amomi medicinal material powder and the ethyl acetate are mixed according to the proportion of 5g to 25 mL.
Preferably, ultrasonic extraction is carried out for 30min in the step 2), and the mass ratio of the volume of the extraction solvent ethyl acetate to the purifying agent is 25mL:550 mg; the mass ratio of PSA to graphitized carbon is 1: 1.75.
Preferably, the gas chromatography analysis parameters in the step 4) comprise that the stationary phase of the chromatographic column is (5% -phenyl) -methyl polysiloxane; injection port temperature of 250 ℃, carrier gas: nitrogen with the purity of more than or equal to 99.999 percent, the split ratio of 10:1, a constant flow mode, the flow rate of 1.0mL/min and the sample volume of 1 microliter; the initial column temperature is 80 deg.C, maintained for 1min, increased to 180 deg.C at 5 deg.C/min, increased to 270 deg.C at 30 deg.C/min, and maintained for 9 min; a detector: hydrogen flame ionization detector, temperature 260 ℃.
Preferably, the fingerprint in the step 4) has 11 characteristic peaks, and the relative retention time of the characteristic peaks and the reference peak No. 8 is as follows: peak No. 1: 0.3752 + -0.0003; peak No. 2: 0.3960 + -0.0004; peak No. 3: 0.4324 + -0.0004; peak No. 4: 0.4399 + -0.0003; peak No. 5: 0.5048 + -0.0003; peak No. 6: 0.7195 + -0.0002; peak No. 7: 0.7558 plus or minus 0.0004; peak No. 8 is the reference peak (P8): 1.0000; peak No. 9: 1.1389 +/-0.0001; peak No. 10:
1.1845 + -0.0003; peak No. 11: 1.2766 + -0.0003.
Preferably, the retention index is calculated as RI ═ 100 × { n + [ t (x) -t (n))/[ t (n +1) -t (n)) ], where RI is the retention index; n is the number of carbon atoms of the normal alkane; t (n +1) and t (n) respectively represent retention time (min) of n-alkanes with carbon numbers n +1 and n; t (x) is the retention time (min) of the component to be detected, and t (n +1) > t (x) t (n). Through calculation and comparison of retention indexes, the No. 1 to No. 11 peaks in the test solution in the step 4) are respectively No. 1 peaks: alpha-pinene; peak No. 2: camphene; peak No. 3: beta-pinene; peak No. 4: beta-myrcene; peak No. 5: d-limonene, peak No. 6: camphor; peak No. 7: borneol; peak No. 8: bornyl acetate; peak No. 9: eugenol; peak No. 10: alpha-copaene; peak No. 11: caryophyllene.
Compared with the prior art, the invention has the following beneficial effects:
(1) the fructus amomi medicinal material powder is extracted by ethyl acetate, so that the extraction effect of the medicinal material components is better and the solvent toxicity is lower;
(2) the ethylenediamine-N-propyl silane and the graphitized carbon are adopted for purification, so that the pollution risk of a sample to instrument parts such as a liner tube and the like is greatly reduced on the premise of not influencing the characteristic peak response, and the running cost of the instrument is reduced;
(3) the characteristic peak is determined by adopting a normal paraffin retention index method, and the method has the advantages of large number of identification peaks, convenience, rapidness and low cost;
(4) the method has the advantages of simple and convenient operation, low detection limit and reliable result, and can quickly and accurately identify the quality, authenticity, stability and genuine characteristics of the amomum villosum medicinal material.
Drawings
FIG. 1 is a chromatogram of a test solution of example 1;
FIG. 2 shows a graph of C in example 18-C20A chromatogram of the normal alkane standard solution;
FIG. 3 is a chromatogram of the control mixed solution in example 1;
FIG. 4 is a chromatogram of the ethanol-extracted test sample solution of example 2;
FIG. 5 is a chromatogram of the test solution obtained by extraction of ethyl acetate in example 2;
FIG. 6 is a chromatogram of a test solution under the purification conditions in example 3;
FIG. 7 is a chromatogram of a test solution under unpurified conditions in example 3;
FIG. 8 is a chromatogram of an empty solvent control solution from the specificity test in example 4;
FIG. 9 is a chromatogram of 10 batches of fructus Amomi crude drugs in example 5;
FIG. 10 is a comparison fingerprint of 10 batches of the fructus Amomi crude drugs in example 5;
FIG. 11 is a graph comparing the fingerprints of the samples (S11, S12) and the control fingerprint in example 6.
Detailed Description
The present invention provides a method for detecting a fingerprint of fructus amomi medicinal material, a fingerprint and an application thereof, which are described in detail below with reference to the following embodiments, but the present invention should not be construed as being limited by the methods. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Example 1
Identification of peaks characteristic of chromatogram
Preparing a test solution: weighing 5g of fructus amomi medicinal material powder in a 50mL centrifuge tube, adding 25mL ethyl acetate, carrying out ultrasonic extraction for 30min, adding 200mg of ethylenediamine-N-propylsilane and 350mg of graphitized carbon for purification, and filtering supernate by using a 0.22 mu m microporous membrane to obtain a test solution.
C8-C20Normal paraffin standard solution: purchased from Shanghai' an spectral laboratory science and technology Co., Ltd.
Mixing the sample solution with C8-C20Normal alkane standard solution is injected into gas phase under the same chromatographic condition
Chromatograph, chromatogram of sample solution are shown in figure 1 and C8-C20The normal alkane chromatogram is shown in FIG. 2.
And (3) calculating the retention index of the characteristic peak by adopting a formula, comparing the retention index with the retention index reported in the literature, and determining the nature of the characteristic peak, wherein the result is shown in table 1.
The retention index calculation formula is RI ═ 100 × { n + [ t (x) -t (n)) ]/[ t (n +1) -t (n)) ], wherein RI is the retention index; n is the number of carbon atoms of the normal alkane; t (n +1) and t (n) respectively represent retention time (min) of n-alkanes with carbon numbers n +1 and n; t (x) is the retention time (min) of the component to be detected, and t (n +1) > t (x) t (n).
TABLE 1 characteristic peak retention index and qualitative results in Amomum villosum L.
The results show that: the difference between the index measured value and the literature reference value is kept to be less than or equal to 10, and the identification result is reliable.
Weighing compound standard substances in the qualitative manner in the table 1, and preparing a reference substance mixed solution by using ethyl acetate, wherein the concentrations of alpha-pinene, camphene, beta-myrcene, D-limonene, camphor, borneol acetate, eugenol and caryophyllene are 79.40mg/L, 6.12mg/L, 17.21mg/L, 19.02mg/L, 32.49mg/L, 15.22mg/L, 175.88mg/L, 498.95mg/L and 36.22mg/L respectively.
Injecting the reference substance mixed solution into a gas chromatograph under the same chromatographic conditions as the test solution to obtain a reference substance mixed solution chromatogram as shown in figure 3, and further performing qualitative confirmation according to the relative retention time, wherein the result is consistent with a retention index method.
Example 2
Experiment for optimizing extraction solvent in preparation of test solution
Weighing 5g of fructus amomi medicinal material powder in a 50mL centrifuge tube, respectively adding 2 parts of ethanol and ethyl acetate in 25mL, carrying out ultrasonic extraction for 30min, adding 200mg of ethylenediamine-N-propylsilane and 350mg of graphitized carbon for purification, filtering supernate with a 0.22 mu m microporous filter membrane to obtain a test solution, and carrying out gas chromatography. The chromatogram of the ethanol extraction sample is shown in figure 4, and the chromatogram of the ethyl acetate extraction sample is shown in figure 5.
According to the test results shown in fig. 4 to 5, when ethyl acetate is used as the extraction solvent, the chromatographic peak signal intensity is high, and the extraction effect is better than that of ethanol, so that ethyl acetate is preferably used as the extraction solvent.
Example 3
Optimization experiment of purification conditions in preparation of test solution
Weighing 5g of fructus amomi medicinal material powder in a 50mL centrifuge tube, adding 2 parts, adding 25mL ethyl acetate, carrying out ultrasonic treatment for 30min, adding 200mg of ethylenediamine-N-propylsilane and 350mg of graphitized carbon into one part, purifying the other part, not adding the other part, and filtering the supernatant by using a 0.22 mu m microporous filter membrane for gas chromatography analysis. The chromatogram of the sample subjected to purification treatment is shown in FIG. 6, and the chromatogram of the sample subjected to non-purification treatment is shown in FIG. 7.
According to the test results shown in fig. 6-7, the purification and the non-purification have no obvious influence on the response of a chromatographic peak, but the purified sample has less pollution to an instrument, and the frequency of replacing parts of the instrument, the fault risk of the instrument and the detection cost are reduced, so that the purification treatment process is preferably selected to establish the fingerprint of the fructus amomi medicinal material.
Example 4
Methodology investigation
1. Reagent
Ethyl acetate, pure chromatography, available from national pharmaceutical group chemical reagents ltd; graphitized Carbon (GCB) was purchased from borna aiger technology; Ethylenediamine-N-Propylsilane (PSA) was purchased from Shanghai' an spectral laboratory science and technology, Inc.
2. Reference substance
Alpha-pinene, camphene, D-limonene, camphor, borneol acetate, eugenol reference: purchased from the Chinese food and drug testing institute; beta-myrcene and caryophyllene reference substances: purchased from Shanghai' an spectral laboratory science and technology Co., Ltd.
3. Preparation of control Mixed solution
Respectively weighing appropriate amount of reference substances such as alpha-pinene, camphene, beta-myrcene, D-limonene, camphor, borneol, bornyl acetate, eugenol and caryophyllene, and dissolving with ethyl acetate to obtain corresponding reference substance solution stock solution; and then transferring a proper amount of the stock solution of the reference substance solution, and adding ethyl acetate to dissolve the stock solution to obtain a reference substance mixed solution with the concentrations of alpha-pinene, camphene, beta-myrcene, D-limonene, camphor, borneol acetate, eugenol and caryophyllene of 79.40mg/L, 6.12mg/L, 17.21mg/L, 19.02mg/L, 32.49mg/L, 15.22mg/L, 175.88mg/L, 498.95mg/L and 36.22mg/L respectively.
4. Preparation of test solution
Crushing and sieving a refrigerated fructus amomi sample, weighing 5g of powder in a 50mL centrifuge tube, adding 25mL ethyl acetate, carrying out ultrasonic treatment for 30min, adding 200mg ethylenediamine-N-propylsilane and 350mg graphitized carbon for purification, and filtering the supernatant by using a 0.22 mu m microporous filter membrane for GC detection.
5. Chromatographic conditions
The gas chromatography parameter is that the stationary phase of the chromatographic column is (5% -phenyl) -methyl polysiloxane; injection port temperature of 250 ℃, carrier gas: nitrogen with the purity of more than or equal to 99.999 percent, the split ratio of 10:1, a constant flow mode, the flow rate of 1.0mL/min and the sample volume of 1 microliter; the initial column temperature is 80 deg.C, maintained for 1min, increased to 180 deg.C at 5 deg.C/min, increased to 270 deg.C at 30 deg.C/min, and maintained for 9 min; a detector: a hydrogen flame ionization detector with the temperature of 260 ℃; the hydrogen flow rate is 35mL/min, the air flow rate is 350mL/min, and the tail-blown nitrogen flow rate is 30 mL/min.
Under the preferred conditions, the control mix solution was injected. The result shows that the peak area of the borneol acetate chromatographic peak is large and is a typical index of the amomum villosum component, so the borneol acetate peak is selected as a reference peak.
And (3) system adaptability test: under the chromatographic conditions, the reference substance mixed solution was sampled 6 times, the Relative Retention Time (RRT) and the Relative Peak Area (RPA) were recorded, and RSD (%) was calculated, and the results are shown in tables 2 and 3.
TABLE 2 results of the System Adaptation test (relative Retention time)
| Numbering | P1 | P2 | P4 | P5 | P6 | P7 | | P9 | P11 | |
| 1 | 0.3777 | 0.3985 | 0.4425 | 0.5075 | 0.7211 | 0.7604 | 1.0000 | 1.1455 | 1.2837 | |
| 2 | 0.3773 | 0.3982 | 0.4423 | 0.5071 | 0.7209 | 0.7604 | 1.0000 | 1.1456 | 1.2840 | |
| 3 | 0.3778 | 0.3984 | 0.4425 | 0.5074 | 0.7212 | 0.7604 | 1.0000 | 1.1455 | 1.2838 | |
| 4 | 0.3772 | 0.3980 | 0.4420 | 0.5070 | 0.7209 | 0.7602 | 1.0000 | 1.1454 | 1.2836 | |
| 5 | 0.3773 | 0.3981 | 0.4423 | 0.5072 | 0.7210 | 0.7603 | 1.0000 | 1.1455 | 1.2838 | |
| 6 | 0.3771 | 0.3979 | 0.4421 | 0.5071 | 0.7210 | 0.7602 | 1.0000 | 1.1455 | 1.2838 | |
| Mean value | 0.3774 | 0.3982 | 0.4423 | 0.5072 | 0.7211 | 0.7603 | 1.0000 | 1.1455 | 1.2838 | |
| RSD(％) | 0.0704 | 0.0580 | 0.0436 | 0.0386 | 0.0151 | 0.0110 | 0.0000 | 0.0079 | 0.0109 |
TABLE 3 results of systematic suitability test (relative peak area)
| Numbering | P1 | P2 | P4 | P5 | P6 | P7 | | P9 | P11 | |
| 1 | 0.1637 | 0.1548 | 0.2010 | 0.1672 | 0.4897 | 0.1198 | 1.0000 | 0.5932 | 0.0959 | |
| 2 | 0.1638 | 0.1527 | 0.2008 | 0.1679 | 0.4887 | 0.1211 | 1.0000 | 0.5985 | 0.0962 | |
| 3 | 0.1625 | 0.1519 | 0.2020 | 0.1675 | 0.4888 | 0.1210 | 1.0000 | 0.5964 | 0.0963 | |
| 4 | 0.1648 | 0.1533 | 0.2024 | 0.1678 | 0.4885 | 0.1221 | 1.0000 | 0.5992 | 0.0969 | |
| 5 | 0.1619 | 0.1540 | 0.2006 | 0.1666 | 0.4880 | 0.1213 | 1.0000 | 0.5989 | 0.0959 | |
| 6 | 0.1648 | 0.1556 | 0.2039 | 0.1690 | 0.4902 | 0.1216 | 1.0000 | 0.5991 | 0.0959 | |
| Mean value | 0.1636 | 0.1537 | 0.2018 | 0.1677 | 0.4890 | 0.1212 | 1.0000 | 0.5976 | 0.0962 | |
| RSD(％) | 0.7326 | 0.8912 | 0.6204 | 0.4741 | 0.1693 | 0.6285 | 0.0000 | 0.3946 | 0.4139 |
The results show that: the repeatability RSD of the control mixed solution is less than 5%, and the system adaptability is good.
Specificity test: under the condition of the chromatogram, taking the reference substance mixed solution, the test substance solution and the blank solvent reference solution, and recording the chromatogram, wherein the chromatogram of the blank solvent reference solution is shown in figure 8.
The results show that: the blank solvent control has no interference, and the method has good specificity.
And (3) precision test: under the chromatographic conditions, the sample solution was continuously injected 6 times, and the relative retention time RSD (%) and the relative peak area RSD (%) of 11 characteristic peaks were calculated with reference to the bornyl acetate chromatographic peak (P8), and the results are shown in tables 4 and 5.
TABLE 4 precision test results (relative Retention time)
| |
1 | 2 | 3 | 4 | 5 | 6 | Mean value | RSD(％) |
| P1 | 0.3751 | 0.3753 | 0.3751 | 0.3751 | 0.3756 | 0.3753 | 0.3753 | 0.0472 |
| P2 | 0.3960 | 0.3960 | 0.3961 | 0.3960 | 0.3963 | 0.3963 | 0.3961 | 0.0403 |
| P3 | 0.4324 | 0.4324 | 0.4326 | 0.4325 | 0.4327 | 0.4328 | 0.4326 | 0.0356 |
| P4 | 0.4399 | 0.4400 | 0.4399 | 0.4399 | 0.4403 | 0.4402 | 0.4400 | 0.0426 |
| P5 | 0.5048 | 0.5048 | 0.5049 | 0.5047 | 0.5050 | 0.5050 | 0.5049 | 0.0267 |
| P6 | 0.7193 | 0.7195 | 0.7195 | 0.7195 | 0.7197 | 0.7198 | 0.7195 | 0.0230 |
| P7 | 0.7558 | 0.7559 | 0.7558 | 0.7559 | 0.7561 | 0.7562 | 0.7559 | 0.0240 |
| P8 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 0.0000 |
| P9 | 1.1392 | 1.1389 | 1.1389 | 1.1394 | 1.1387 | 1.1389 | 1.1390 | 0.0208 |
| P10 | 1.1845 | 1.1846 | 1.1845 | 1.1847 | 1.1843 | 1.1846 | 1.1845 | 0.0120 |
| P11 | 1.2767 | 1.2768 | 1.2763 | 1.2769 | 1.2765 | 1.2768 | 1.2767 | 0.0194 |
TABLE 5 precision test results (relative peak area)
The results show that: the relative retention time of 11 common characteristic peaks of the chromatogram and the RSD of the relative peak area are both less than 5 percent, which accords with the relevant requirements formulated by the fingerprint.
And (3) stability test: taking the same batch of samples, preparing a sample solution according to the method, placing for 0, 2, 4, 8, 12 and 24h at room temperature for sample injection, taking a bornyl acetate chromatographic peak (P8) as a reference, calculating the relative retention time and the relative peak area RSD (%) of each characteristic peak, and obtaining the results shown in tables 6 and 7.
TABLE 6 stability test results (relative Retention time)
| |
0 | 2 | 4 | 8 | 12 | 24 | Mean value | RSD(％) |
| P1 | 0.3750 | 0.3751 | 0.3751 | 0.3756 | 0.3758 | 0.3754 | 0.3753 | 0.0847 |
| P2 | 0.3957 | 0.3959 | 0.3961 | 0.3965 | 0.3965 | 0.3962 | 0.3962 | 0.0746 |
| P3 | 0.4321 | 0.4322 | 0.4324 | 0.4329 | 0.4329 | 0.4326 | 0.4325 | 0.0806 |
| P4 | 0.4397 | 0.4398 | 0.4398 | 0.4404 | 0.4404 | 0.4402 | 0.4401 | 0.0687 |
| P5 | 0.5045 | 0.5046 | 0.5047 | 0.5052 | 0.5051 | 0.5050 | 0.5049 | 0.0599 |
| P6 | 0.7192 | 0.7193 | 0.7193 | 0.7199 | 0.7196 | 0.7197 | 0.7195 | 0.0385 |
| P7 | 0.7556 | 0.7556 | 0.7557 | 0.7562 | 0.7561 | 0.7560 | 0.7559 | 0.0345 |
| P8 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 0.0000 |
| P9 | 1.1389 | 1.1388 | 1.1387 | 1.1389 | 1.1389 | 1.1386 | 1.1388 | 0.0112 |
| P10 | 1.1842 | 1.1847 | 1.1842 | 1.1844 | 1.1843 | 1.1841 | 1.1843 | 0.0186 |
| P11 | 1.2765 | 1.2768 | 1.2763 | 1.2764 | 1.2765 | 1.2760 | 1.2764 | 0.0210 |
TABLE 7 stability test results (relative peak area)
The test result shows that: the relative retention time and the relative peak area RSD of each common characteristic peak of the chromatogram are less than 5 percent, and the relative retention time and the relative peak area RSD accord with the related requirements formulated by the fingerprint.
And (3) repeatability test: taking the same batch of samples, preparing 6 test sample solutions according to the method, sequentially injecting samples, and calculating the relative retention time and the relative peak area RSD (%) of each common characteristic peak by taking a bornyl acetate chromatographic peak (P8) as a reference, wherein the results are shown in tables 8 and 9.
TABLE 8 repeatability test results (relative retention time)
| |
1 | 2 | 3 | 4 | 5 | 6 | Mean value | RSD(％) |
| P1 | 0.3749 | 0.3749 | 0.3752 | 0.3752 | 0.3755 | 0.3754 | 0.3752 | 0.0672 |
| P2 | 0.3956 | 0.3958 | 0.3961 | 0.3961 | 0.3965 | 0.3963 | 0.3960 | 0.0795 |
| P3 | 0.4321 | 0.4321 | 0.4325 | 0.4325 | 0.4328 | 0.4327 | 0.4324 | 0.0678 |
| P4 | 0.4397 | 0.4397 | 0.4400 | 0.4400 | 0.4402 | 0.4402 | 0.4399 | 0.0513 |
| P5 | 0.5045 | 0.5045 | 0.5047 | 0.5048 | 0.5050 | 0.5050 | 0.5048 | 0.0465 |
| P6 | 0.7194 | 0.7192 | 0.7195 | 0.7196 | 0.7195 | 0.7197 | 0.7195 | 0.0260 |
| P7 | 0.7554 | 0.7556 | 0.7558 | 0.7558 | 0.7561 | 0.7560 | 0.7558 | 0.0309 |
| P8 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 0.0000 |
| P9 | 1.1390 | 1.1388 | 1.1389 | 1.1387 | 1.1390 | 1.1387 | 1.1389 | 0.0112 |
| P10 | 1.1846 | 1.1847 | 1.1846 | 1.1842 | 1.1846 | 1.1841 | 1.1845 | 0.0208 |
| P11 | 1.2769 | 1.2770 | 1.2765 | 1.2765 | 1.2765 | 1.2765 | 1.2766 | 0.0181 |
TABLE 9 repeatability test results (relative peak area)
The test result shows that: the relative retention time and the relative peak area RSD of each common characteristic peak of the chromatogram are less than 5 percent, and the relative retention time and the relative peak area RSD accord with the related requirements formulated by the fingerprint.
Example 5
Establishment of fructus amomi medicinal material contrast fingerprint
Preparing 10 batches of representative fructus amomi medicinal materials (numbered from S1 to S10) into a test solution according to the method of the embodiment 4, injecting the test solution into a gas chromatography for analysis to obtain 10 batches of sample chromatograms, as shown in figure 9, and introducing the sample chromatograms into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system for data comparison treatment to generate a comparison fingerprint, as shown in figure 10.
TABLE 10 fructus Amomi medicinal materials similarity calculation results
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Control |
| S1 | 1.000 | 1.000 | 0.994 | 0.992 | 0.994 | 0.998 | 0.998 | 0.995 | 0.997 | 0.997 | 0.998 |
| S2 | 1.000 | 1.000 | 0.994 | 0.992 | 0.994 | 0.998 | 0.998 | 0.995 | 0.997 | 0.997 | 0.998 |
| S3 | 0.994 | 0.994 | 1.000 | 0.999 | 0.999 | 0.997 | 0.997 | 0.998 | 0.995 | 0.995 | 0.998 |
| S4 | 0.992 | 0.992 | 0.999 | 1.000 | 1.000 | 0.996 | 0.996 | 0.998 | 0.994 | 0.994 | 0.998 |
| S5 | 0.994 | 0.994 | 0.999 | 1.000 | 1.000 | 0.998 | 0.997 | 0.999 | 0.995 | 0.995 | 0.999 |
| S6 | 0.998 | 0.998 | 0.997 | 0.996 | 0.998 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S7 | 0.998 | 0.998 | 0.997 | 0.996 | 0.997 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S8 | 0.995 | 0.995 | 0.998 | 0.998 | 0.999 | 0.999 | 0.999 | 1.000 | 0.999 | 0.999 | 0.999 |
| S9 | 0.997 | 0.997 | 0.995 | 0.994 | 0.995 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 |
| S10 | 0.997 | 0.997 | 0.995 | 0.994 | 0.995 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 |
| Control | 0.998 | 0.998 | 0.998 | 0.998 | 0.999 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
11 common peaks are selected as characteristic peaks of the fingerprint, the borneol acetate chromatographic peak (P8) is taken as a reference, the similarity is calculated, and the result is shown in the table 10. The result shows that the similarity among 10 representative fructus amomi samples is greater than 0.99, which indicates that the 10 representative fructus amomi samples have consistent quality and meet the requirement of making a fingerprint spectrum for comparison.
Example 6
Application of fingerprint spectrum in quality control of fructus amomi medicinal material
Preparing a sample solution from 2 batches of fructus amomi medicinal material samples (S11 and S12) according to the method of example 4, injecting the sample solution into a gas chromatography for analysis to obtain 2 batches of sample chromatograms, introducing the sample chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (issued by the national pharmacopoeia committee), simultaneously introducing the reference fingerprint of example 5, selecting 11 common peaks as characteristic peaks of the fingerprints, taking a borneol acetate chromatographic peak (P8) as reference (S), and calculating the similarity between S11 and S12 and the reference fingerprint, wherein the similarity result is shown in Table 11.
TABLE 112 calculation of fructus Amomi crude drug similarity
| Numbering | S11 | S12 | Control |
| S11 | 1.000 | 1.000 | 0.998 |
| S12 | 1.000 | 1.000 | 0.998 |
| Control | 0.998 | 0.998 | 1.000 |
The result shows that the similarity of the medicinal materials S11 and S12 is 0.998, which indicates that the two batches of fructus amomi medicinal materials are qualified and stable in quality.
Claims (6)
1. A method for detecting a fructus amomi medicinal material fingerprint spectrum is characterized by comprising the following steps:
1) freezing and preserving fructus amomi, crushing and sieving, and mixing with an extraction solvent according to the proportion of 1-5 g to 10-30 mL to form a sample solution;
2) ultrasonically extracting the sample liquid obtained in the step 1) for 10-45 min, and mixing the sample liquid with a purifying agent according to the proportion of 1ml to 4-50 mg; the purifying agent is a mixture of PSA and graphitized carbon, and the mass ratio of the PSA to the graphitized carbon is 1: 1-5.
3) Filtering the supernatant obtained in the step 2) by using a microporous filter membrane to obtain a test solution;
4) and (3) carrying out gas chromatography analysis on the test solution obtained in the step 3) to obtain a chromatogram and retention time of a characteristic peak.
5) C is to be8-C20Performing gas chromatography analysis on the normal alkane standard solution under the same chromatographic conditions as the step 4) to obtain a chromatogram and C8-C20Retention time of normal alkanes;
6) the retention time of the characteristic peak in the step 4) and the retention time of the characteristic peak C in the step 5)8-C20Substituting the retention time of the normal alkane into a formula, and calculating the retention index of the characteristic peak;
7) comparing the retention index obtained in the step 6) with a theoretical retention index, and determining the characteristic peak;
8) dissolving the characteristic peak compound obtained in the step 7) with ethyl acetate to prepare a reference substance mixed solution;
9) performing gas chromatography analysis on the reference substance mixed solution obtained in the step 8) under the same chromatographic conditions as those in the step 4) to obtain a reference substance chromatogram and retention time;
10) qualitatively confirming the characteristic peak of the step 4) by using the reference chromatogram and the retention time obtained in the step 9);
11) analyzing 10 batches of the same-variety fructus amomi medicinal materials in different batches according to the steps 1) to 4), identifying characteristic peaks, introducing chromatograms into a traditional Chinese medicine fingerprint database for similarity evaluation, and generating comparison fingerprints;
12) analyzing a fructus amomi medicinal material sample to be detected according to the steps 1) to 4), identifying the characteristic peak, introducing the chromatogram into a traditional Chinese medicine fingerprint database for similarity evaluation, comparing the similarity with the comparison fingerprint obtained in the step 11), and calculating the similarity.
2. The detection method according to claim 1, wherein the extraction solvent in step 1) is ethyl acetate, and the fructus amomi medicinal material powder and the ethyl acetate are mixed according to a ratio of 5g to 25 mL.
3. The detection method according to claim 1, wherein in the step 2), ultrasonic extraction is carried out for 30min, and the mass ratio of the volume of the extraction solvent ethyl acetate to the purifying agent is 25mL:550 mg; the mass ratio of PSA to graphitized carbon is 1: 1.75.
4. The detection method according to claim 1, wherein the gas chromatography analysis parameters in the step 4) comprise that the chromatographic column stationary phase is (5% -phenyl) -methyl polysiloxane; injection port temperature of 250 ℃, carrier gas: nitrogen with the purity of more than or equal to 99.999 percent, the split ratio of 10:1, a constant flow mode, the flow rate of 1.0mL/min and the sample volume of 1 microliter; the initial column temperature is 80 deg.C, maintained for 1min, increased to 180 deg.C at 5 deg.C/min, increased to 270 deg.C at 30 deg.C/min, and maintained for 9 min; a detector: hydrogen flame ionization detector, temperature 260 ℃.
5. The detection method according to claim 1, wherein the fingerprint in the step 4) has 11 characteristic peaks, and the relative retention time of the characteristic peaks and the reference peak No. 8 is as follows: peak No. 1: 0.3752 + -0.0003; peak No. 2: 0.3960 + -0.0004; peak No. 3: 0.4324 + -0.0004; peak No. 4: 0.4399 + -0.0003; peak No. 5: 0.5048 + -0.0003; peak No. 6: 0.7195 + -0.0002; peak No. 7: 0.7558 plus or minus 0.0004; peak No. 8 is the reference peak (P8): 1.0000; peak No. 9: 1.1389 +/-0.0001; peak No. 10: 1.1845 + -0.0003; peak No. 11: 1.2766 + -0.0003.
6. The detection method according to claim 1, wherein the retention index is calculated as RI ═ 100 x { n + [ t (x) -t (n))/[ t (n +1) -t (n)) ], where RI is the retention index; n is the number of carbon atoms of the normal alkane; t (n +1) and t (n) respectively represent retention time (min) of n-alkanes with carbon numbers n +1 and n; t (x) is the retention time (min) of the component to be detected, and t (n +1) > t (x) t (n); through calculation and comparison of retention indexes, the No. 1 to No. 11 peaks in the test solution in the step 4) are respectively No. 1 peaks: alpha-pinene; peak No. 2: camphene; peak No. 3: beta-pinene; peak No. 4: beta-myrcene; peak No. 5: d-limonene, peak No. 6: camphor; peak No. 7: borneol; peak No. 8: bornyl acetate; peak No. 9: eugenol; peak No. 10: alpha-copaene; peak No. 11: caryophyllene.
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| CN112748211A (en) * | 2020-12-01 | 2021-05-04 | 上海和黄药业有限公司 | Detection method of gas-phase fingerprint spectrum of vital qi tablet and application thereof |
| CN113462811A (en) * | 2021-08-19 | 2021-10-01 | 中国热带农业科学院南亚热带作物研究所 | Fructus amomi SSR molecular marker primer group and application thereof |
| CN114372063A (en) * | 2022-01-21 | 2022-04-19 | 华谱科仪(北京)科技有限公司 | Fault detection method based on chromatograph and electronic equipment |
| CN114755315A (en) * | 2021-07-23 | 2022-07-15 | 山东省中医药研究院 | Method for identifying amomum villosum or green-shell sand by using GC characteristic fingerprint |
| CN114858931A (en) * | 2022-02-18 | 2022-08-05 | 杭州市食品药品检验研究院(杭州市食品药品审核查验服务中心、杭州市药品与医疗器械不良反应监测中心) | Fatty acid component fingerprint spectrum-based antelope horn authenticity identification method |
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| CN112748211A (en) * | 2020-12-01 | 2021-05-04 | 上海和黄药业有限公司 | Detection method of gas-phase fingerprint spectrum of vital qi tablet and application thereof |
| CN114755315A (en) * | 2021-07-23 | 2022-07-15 | 山东省中医药研究院 | Method for identifying amomum villosum or green-shell sand by using GC characteristic fingerprint |
| CN113462811A (en) * | 2021-08-19 | 2021-10-01 | 中国热带农业科学院南亚热带作物研究所 | Fructus amomi SSR molecular marker primer group and application thereof |
| CN113462811B (en) * | 2021-08-19 | 2024-02-13 | 中国热带农业科学院湛江实验站 | Fructus amomi SSR molecular marker primer group and application thereof |
| CN114372063A (en) * | 2022-01-21 | 2022-04-19 | 华谱科仪(北京)科技有限公司 | Fault detection method based on chromatograph and electronic equipment |
| CN114372063B (en) * | 2022-01-21 | 2022-08-12 | 华谱科仪(北京)科技有限公司 | Fault detection method based on chromatograph and electronic equipment |
| CN114858931A (en) * | 2022-02-18 | 2022-08-05 | 杭州市食品药品检验研究院(杭州市食品药品审核查验服务中心、杭州市药品与医疗器械不良反应监测中心) | Fatty acid component fingerprint spectrum-based antelope horn authenticity identification method |
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