CN112748211A - Detection method of gas-phase fingerprint spectrum of vital qi tablet and application thereof - Google Patents
Detection method of gas-phase fingerprint spectrum of vital qi tablet and application thereof Download PDFInfo
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- 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
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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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Abstract
The invention provides a method for detecting a gas-phase fingerprint spectrum of a qi tablet, which comprises the following steps: 1) preparing a test solution; 2) preparing a reference substance solution; 3) and (4) measuring. The invention further provides application of the gas-phase fingerprint spectrum of the qi-strengthening tablet in quality detection of components in the qi-strengthening tablet and a quality detection method thereof. The invention further provides a method for screening the gas-phase fingerprint spectrums of the multiple medicinal materials in the Zhengqi tablets. The gas-phase fingerprint spectrum of the vital qi tablet is established, the attribution of each single medicinal material chromatographic peak in the vital qi tablet is confirmed, the quality of the raw medicinal materials can be effectively monitored from the source of the raw materials, the quality of the raw medicinal materials is strictly controlled, and the relative stability of the amount of the effective components in the components of the vital qi tablet is ensured, so that the safety and the effectiveness of clinical medication are ensured.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component detection, relates to a detection method of a gas-phase fingerprint of a vital qi tablet and application thereof, and particularly relates to a detection method of a gas-phase fingerprint of a vital qi tablet and application thereof in quality detection.
Background
The disease of COVID-19 has the typical pathogenic characteristics of damp toxin, belongs to the category of epidemic diseases in traditional Chinese medicine, and is characterized by dampness encumbering the spleen and closing the lung, lung loss and dispersion, qi depression and fever, white and greasy tongue coating, loose stool, longer course of disease than common respiratory tract infection diseases, and extremely easy gas consumption and yin impairment, so the dampness resolving formula is adopted for auxiliary treatment. The seventh version of the diagnosis and treatment scheme (trial) for coronavirus pneumonia is released in 3 months and 3 days in 2020, wherein a series of agastache healthy qi preparations including an oral liquid of agastache healthy qi are used as prevention and recommendation medicines for patients with hypodynamia and gastrointestinal discomfort in a medical observation period, and the healthy qi tablet also belongs to a series of agastache healthy qi products, has the effects of dispersing wind cold, resolving dampness and regulating the middle warmer, and is used for treating common cold, headache, chest distress, vomiting, diarrhea and abdominal distension.
The formula of ZHENGQI tablet comprises ten Chinese medicinal materials, including oleum herba Pogostemonis, Perilla leaf oil, radix aucklandiae, rhizoma Atractylodis, Glycyrrhrizae radix, Poria, pericarpium Citri Tangerinae, rhizoma Pinelliae Preparada, cortex Magnolia officinalis and rhizoma Zingiberis recens. Because the prescription of the qi-strengthening tablet has more medicinal materials and complicated chemical components, the integrity and the complexity of the material basis and the chemical component group of the qi-strengthening tablet can not be comprehensively reflected only by identifying or measuring the content of a single compound, and the internal quality of the qi-strengthening tablet is difficult to be comprehensively controlled. The traditional Chinese medicine fingerprint has the characteristics of integrity, macroscopicity, fuzzy analysis and the like, and can achieve the purpose of integral quality control by describing the integral characteristics of the traditional Chinese medicine and adopting a proper fuzzy processing mode, thereby becoming an effective means for the quality control of the traditional Chinese medicine. The chromatographic fingerprint analysis can visualize the overall characteristics of various chemical components contained in the traditional Chinese medicine, thereby revealing the quality problem which is difficult to find by the conventional inspection. The fingerprint is a modern important quality control method from the perspective of 'full ingredients' according to the characteristics of the overall comprehensive action of multiple components and multiple target points of the traditional Chinese medicine, and the quality control of the non-single-component medicine is more comprehensive.
The medicine composition of the qi-tonifying tablets contains a large amount of volatile components, and the qualitative research on the system of the volatile components of the qi-tonifying tablets is lacked at present, so that the gas phase fingerprint spectrum of the qi-tonifying tablets needs to be established, the qualitative analysis of the components in the qi-tonifying tablets can be completed, and the quality of the qi-tonifying tablets can be effectively controlled.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a method for detecting a gas-phase fingerprint of a positive gas tablet and an application thereof, wherein a pretreatment of optimized conditions and a gas chromatography method are adopted to establish a gas-phase fingerprint of the positive gas tablet, and contributions of different chemical components to a fingerprint system of the positive gas tablet are highlighted from different side faces, so that detection of all chemical components of the positive gas tablet is realized, the current situation of each component in the positive gas tablet is more comprehensively reflected, and a reference basis is provided for overall control and evaluation of the quality of the positive gas tablet.
In order to achieve the above and other related objects, a first aspect of the present invention provides a method for detecting a gas phase fingerprint of a positive gas film, comprising the steps of:
1) preparation of a test solution: adding absolute ethyl alcohol into a positive gas tablet sample, performing ultrasonic extraction, centrifuging, and filtering to obtain a test solution;
2) preparation of control solutions: dissolving costunolide, dehydrocostuslactone and atractylodin as reference substances in anhydrous ethanol to obtain reference solution;
3) and (3) determination: respectively measuring the test solution and the reference solution by adopting a Gas Chromatography (GC) method with the same chromatographic conditions to obtain a fingerprint of the test solution and a fingerprint of the reference solution, comparing the fingerprint of the test solution with the fingerprint of the reference solution, and performing attribution positioning on index components in the fingerprint of the test solution, thereby obtaining the gas fingerprint of the positive gas tablet.
Preferably, in step 1), the positive gas tablet sample is a powder sample obtained by crushing a positive gas tablet.
Preferably, in step 1), the ratio of the weight (g) of the added positive gas tablet sample to the volume (mL) of the added absolute ethanol is 10: 15-25.
More preferably, the ratio of the weight (g) of the positive gas tablet sample addition to the volume (mL) of the absolute ethanol addition is 10: 20.
when the positive gas chip sample and the absolute ethyl alcohol are weighed, the positive gas chip sample and the absolute ethyl alcohol are precisely weighed.
Preferably, in the step 1), the ultrasonic extraction time is 20-40 min. More preferably, the ultrasound extraction time is 30 min.
Preferably, in step 1), the positive gas tablet sample is precisely weighed after being added with absolute ethyl alcohol.
Preferably, in step 1), the ultrasonic extraction is followed by re-weighing and the weight loss is compensated with absolute ethanol.
Preferably, in step 1), the centrifugation conditions are: centrifuging time: 15-25 min; centrifugal rotating speed: 2000-4000 r/min. More preferably, the centrifugation conditions are: centrifuging time: 20 min; centrifugal rotating speed: 3000 r/min.
Preferably, in the step 1), the filtration mode is a filter membrane filtration mode. More preferably, the pore size of the filter is 0.22 μm.
Preferably, in step 2), the CAS number of costunolide is 553-21-9, the CAS number of dehydrocostuslactone is 74299-48-2, and the CAS number of atractylodin is 55290-63-6.
Preferably, in step 2), the content ranges of the components in the control solution are as follows: costunolide is 50-200 μ g/mL, dehydrocostuslactone is 50-200 μ g/mL, and atractylodin is 50-200 μ g/mL.
More preferably, the content range of each component in the control solution is as follows: costunolide 100 μ g/mL, dehydrocostuslactone 100 μ g/mL, and atractylodin 100 μ g/mL.
Preferably, in the step 2), the reference solution is prepared by stepwise dilution.
More preferably, the stepwise dilution of the control solution comprises the steps of:
A1) adding anhydrous ethanol into costunolide, dehydrocostuslactone and atractylodin reference substances for dilution and volume fixing to prepare reference substance stock solution;
A2) and adding absolute ethyl alcohol into the reference substance storage solution for dilution and constant volume to prepare the required reference substance solution.
Further preferably, in step A1), the concentration of each component in the control stock solution is 0.5 mg/mL.
Further preferably, in step a1), the control stock solution has a storage temperature of 0-5 ℃.
Most preferably, the control stock solution has a storage temperature of 4 ℃. The control stock solution was stored in a refrigerator protected from light.
Preferably, in the step 3), the fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peak in the fingerprint of the test solution is identified through the relative retention time according to the known characteristic peak in the fingerprint of the reference solution, so as to perform attribution positioning on the index component in the fingerprint of the test solution.
Preferably, in step 3), the chromatographic column used in the gas chromatography is an Agilent HP-5 chromatographic column (30 m.times.0.32 mm, 0.25 μm).
Preferably, in step 3), the detector used in the gas chromatography is a Flame Ionization Detector (FID).
Preferably, in the step 3), the temperature rising program adopted in the gas chromatography is as follows: the initial temperature is 118-; heating to 158-162 ℃ at the speed of 1-3 ℃/min, and keeping the temperature for 1-3 min; heating to 198-202 deg.C at a rate of 1-3 deg.C/min, and maintaining for 1-3 min; heating to 218-222 ℃ at the speed of 1-3 ℃/min, and keeping for 1-3 min; raising the temperature to 238-242 ℃ at the speed of 1-3 ℃/min, and keeping the temperature for 4-6 min.
More preferably, in the gas chromatography, the temperature rising program used is: maintaining the initial temperature at 120 deg.C for 2min, heating to 130 deg.C at 2 deg.C/min, and maintaining for 2 min; heating to 160 deg.C at a rate of 2 deg.C/min, and maintaining for 2 min; heating to 200 deg.C at a rate of 2 deg.C/min, and maintaining for 2 min; heating to 220 deg.C at a rate of 2 deg.C/min, and maintaining for 2 min; the temperature was raised to 240 ℃ at a rate of 2 ℃/min and held for 5 min.
Preferably, in step 3), the gas chromatography further comprises the following detection conditions:
the temperature of a sample opening is 260-300 ℃; the sample amount is 1-5 muL; the carrier gas is high-purity helium, and the purity of the carrier gas is more than or equal to 99.999 percent; the flow rate of the carrier gas is 0.5-2.0 mL/min; the sample injection mode is split sample injection, and the split ratio is 20-40: 1.
More preferably, the gas chromatography further comprises the following detection conditions:
the sample port temperature was 280 ℃; the sample injection amount is 2 mu L; the carrier gas is high-purity helium, and the purity of the carrier gas is more than or equal to 99.999 percent; the flow rate of the carrier gas is 1.0 mL/min; the sample injection mode is split sample injection, and the split ratio is 30: 1.
The invention provides a method for detecting a gas-phase fingerprint spectrum of a qi-tonifying tablet, and application of the method in quality detection of components in the qi-tonifying tablet.
The third aspect of the invention provides a quality detection method of a qi tablet, which comprises the steps of obtaining a gas-phase fingerprint of the qi tablet by adopting the detection method of the gas-phase fingerprint of the qi tablet, and comparing the similarity of the obtained gas-phase fingerprint of the qi tablet with a standard gas-phase fingerprint of the qi tablet obtained under the same fingerprint detection condition.
Preferably, when the similarity of the measured gas-phase fingerprint of the zhengqi tablet and the standard gas-phase fingerprint of the zhengqi tablet is compared, the similarity is compared by using software 2009 (evaluation system for traditional Chinese medicine chromatography fingerprint similarity) issued by the national pharmacopoeia committee.
More preferably, the similarity between the gas phase fingerprint spectrum of the qi-tonifying tablet measured by the method and the standard gas phase fingerprint spectrum of the qi-tonifying tablet is more than or equal to 0.95.
Preferably, the standard gas phase fingerprint of the qi-tonifying tablet is obtained by the same conditions as the detection method of the gas phase fingerprint of the qi-tonifying tablet, wherein the standard gas phase fingerprint of the qi-tonifying tablet comprises 15 common fingerprint peaks, and the relative retention time of the 8 th peak is the reference peak (S peak, retention time is 1.000), and the other 14 common fingerprint peaks are the 1 st peak (0.243 ± 0.002), the 2 th peak (0.532 ± 0.003), the 3 rd peak (0.559 ± 0.003), the 4 th peak (0.790 ± 0.004), the 5 th peak (0.801 ± 0.004), the 6 th peak (0.812 ± 0.004), the 7 th peak (0.931 ± 0.005), the 9 th peak (1.115 ± 0.005), the 10 th peak (1.295 ± 0.006), the 11 th peak (1.11 ± 0.006), the 12 th peak (1.931 ± 0.448), the 13 th peak (1.391), the 14.008), the 14.010 ± 0.045 (15.010), and the 15.045).
More preferably, the standard gas phase fingerprint of the zhengqi tablet comprises 15 common fingerprint peaks, wherein the reference peak (S peak, retention time is 1.000) is the No. 8 peak, and the relative retention times of the other 14 common fingerprint peaks are the No. 1 peak (0.243 +/-0.001), the No. 2 peak (0.532 +/-0.001), the No. 3 peak (0.559 +/-0.001), the No. 4 peak (0.790 +/-0.002), the No. 5 peak (0.801 +/-0.002), the No. 6 peak (0.812 +/-0.003), the No. 7 peak (0.931 +/-0.003), the No. 9 peak (1.115 +/-0.003), the No. 10 peak (1.295 +/-0.004), the No. 11 peak (1.391 +/-0.004), the No. 12 peak (1.448 +/-0.004), the No. 13 peak (1.753 +/-0.005), the No. 14 peak (2.045 +/-0.006) and the No. 15 peak (2.006 +/-0.006).
The specific data of the standard gas-phase fingerprint spectrum of the Zhengqi tablet are shown in figure 1.
More preferably, as shown in fig. 3, the standard gas-phase fingerprint of the zhengqi tablet is compared with the gas-phase fingerprint of the control solution, and the peak No. 8 is located and determined to be the fingerprint of atractylodin, the peak No. 9 is the fingerprint of costunolide, and the peak No. 11 is the fingerprint of dehydrocostus lactone.
The fourth aspect of the invention provides a method for screening gas-phase fingerprint spectrums of a plurality of medicinal materials in a zhengqi tablet, which comprises the following steps:
A) preparing a medicinal material sample solution: preparing any one or more of 10 medicinal material samples of patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, liquorice, poria cocos, dried orange peel, pinellia ternate, mangnolia officinalis and ginger in the Zhengqi tablets according to the step 1) of the detection method of the gas-phase fingerprint spectrum of the Zhengqi tablets to respectively obtain at least one single medicinal material sample solution;
B) preparation of a mixed solution of the lacking negative samples: removing any one 1 medicinal material from 10 medicinal material samples of patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, liquorice, poria cocos, dried orange peel, pinellia ternate, mangnolia officinalis and ginger, adding auxiliary materials with corresponding mass for filling, and preparing according to the step 1) of the detection method of the gas-phase fingerprint of the zhengqi tablet to respectively obtain 10 sample-lacking negative sample mixed solutions;
C) and (3) determination: respectively measuring the single medicinal material sample solution and the 10 sample-lacking negative sample mixed solutions by adopting a Gas Chromatography (GC) with the same chromatographic conditions in the step 3) of the detection method of the gas fingerprint of the zheng qi tablet to respectively obtain the gas fingerprints of the single medicinal material sample solution and the 10 sample-lacking negative sample mixed solutions;
D) obtaining a standard gas phase fingerprint spectrum: obtaining a standard gas-phase fingerprint of the qi tablet by adopting the same steps as the detection method of the gas-phase fingerprint of the qi tablet;
E) and (3) quality detection: and respectively comparing the gas-phase fingerprint of the single-medicinal-material sample solution and the gas-phase fingerprint of the 10 sample-lacking negative-sample mixed solutions with the standard gas-phase fingerprint of the positive gas piece, and identifying the corresponding characteristic peaks of the single-medicinal-material sample solution in the standard gas-phase fingerprint of the positive gas piece through relative retention time, so as to perform attribution positioning on the characteristic peaks in the gas-phase fingerprint of the single-medicinal-material sample solution.
Preferably, in step a), the Patchouli oil (Patchouli oil) and Perilla leaf oil (Perilla oil) should be precisely weighed. Preferably, in the step A), the Radix Aucklandiae (Aucklandiae Radix), Rhizoma Atractylodis (Atractylodes rhizome), Glycyrrhrizae Radix (Glycyrrhiza Radix et Rhizoma), Poria (Poria), Pericarpium Citri Tangerinae (Citri Reticulatum), Rhizoma Pinelliae (Pinelliae Rhizoma), Cortex Magnolia officinalis (Magnolia officinalis), and Rhizoma Zingiberis Recens (Zingiberaceae Rhizoma Recens) should be pulverized into powder and precisely weighed. The pinellia ternate is prepared pinellia ternate, and the magnolia officinalis is ginger magnolia officinalis. The rhizoma Zingiberis recens is fresh rhizoma Zingiberis recens.
The oleum herba Pogostemonis is extracted from plants of Pogostemon of Labiatae. The perilla leaf oil is extracted from annual upright herb of Labiatae family Talaroides dicotyledonae family Labiatae. The radix aucklandiae is dried root of radix aucklandiae of Compositae. Rhizoma Atractylodis is dried rhizome of Atractylodes lancea or Atractylodes chinensis of Compositae. The Glycyrrhrizae radix is dried root and rhizome of Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat or Glycyrrhiza glabra L of Leguminosae. Poria is dried sclerotium of Poria cocos (Schw.) wolf of Polyporaceae. The pericarpium Citri Tangerinae is dried mature pericarp of Rutaceae plant fructus Citri Tangerinae and its cultivar. The rhizoma Pinelliae is dried tuber of rhizoma Pinelliae of Araceae. The cortex Magnolia officinalis is dry bark, root bark and branch bark of Magnolia officinalis or Magnolia officinalis of Magnoliaceae. Ginger is the fresh rhizome of a perennial herb ginger of the family zingiberaceae. Preferably, in step a), the volume ratio of the patchouli oil to the absolute ethyl alcohol is 40: 1500-2500. More preferably, the volume ratio of the patchouli oil to the absolute ethyl alcohol is 40: 2000.
preferably, in step a), the volume ratio of the perilla leaf oil to the absolute ethyl alcohol is 20: 1500-2500. More preferably, the volume ratio of the patchouli oil to the absolute ethyl alcohol is 20: 2000.
preferably, in step a), the ratio of the weight (g) of added costus root to the volume (mL) of added absolute ethanol is 4.5: 15-25. More preferably, the ratio of the weight (g) of added aucklandia root to the volume (mL) of added absolute ethanol is 4.5: 20.
preferably, in step a), the ratio of the weight (g) of said poria cocos added to the volume (mL) of said anhydrous ethanol added is 4.5: 15-25. More preferably, the ratio of the weight (g) of said poria cocos added to the volume (mL) of said anhydrous ethanol added is 4.5: 20.
preferably, in step a), the ratio of the weight (g) of the rhizoma atractylodis added to the volume (mL) of the absolute ethyl alcohol added is 3.0: 15-25. More preferably, the ratio of the weight (g) of the rhizoma atractylodis added to the volume (mL) of the absolute ethyl alcohol added is 3.0: 20.
preferably, in step a), the ratio of the weight (g) of the added tangerine peel to the volume (mL) of the added absolute ethyl alcohol is 3.0: 15-25. More preferably, the ratio of the weight (g) of added pericarpium citri reticulatae to the volume (mL) of added absolute ethanol is 3.0: 20.
preferably, in step a), the ratio of the weight (g) of the pinellia tuber added to the volume (mL) of the absolute ethanol added is 3.0: 15-25. More preferably, the ratio of the weight (g) of the pinellia tuber addition to the volume (mL) of the absolute ethanol addition is 3.0: 20.
preferably, in the step A), the ratio of the added weight (g) of the magnolia officinalis to the added volume (mL) of the absolute ethyl alcohol is 3.0: 15-25. More preferably, the ratio of the weight (g) of magnolia bark added to the volume (mL) of absolute ethanol added is 3.0: 20.
preferably, in step a), the ratio of the weight (g) of ginger added to the volume (mL) of absolute ethanol added is 3.0: 15-25. More preferably, the ratio of the weight (g) of ginger added to the volume (mL) of absolute ethanol added is 3.0: 20.
preferably, in step a), the ratio of the weight (g) of the licorice added to the volume (mL) of the absolute ethanol added is 1.5: 15-25. More preferably, the ratio of the weight (g) of the licorice added to the volume (mL) of the absolute ethanol added is 1.5: 20.
preferably, in step B), the auxiliary material is selected from one or two of magnesium stearate or talcum powder.
Preferably, in the step E), the attribution of the characteristic peaks of the gas-phase fingerprint spectrum of the single-drug sample solution, the gas-phase fingerprint spectrum of the 10 sample-deficient negative sample mixed solutions and the standard gas-phase fingerprint spectrum of the vital-qi tablet are located, the analysis and the processing are performed by using software of 2009 edition of traditional Chinese medicine chromatography fingerprint spectrum similarity evaluation system issued by the national pharmacopoeia committee, and the attribution of the characteristic peaks of each single drug of the vital-qi tablet is confirmed by the relative retention time of each characteristic peak on the standard gas-phase fingerprint spectrum of the vital-qi tablet.
Preferably, in the step E), the fingerprint spectrum of the patchouli oil sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are peak No. 3 and peak No. 5. Specific results are shown in table 1.
Preferably, in the step E), the fingerprint spectrum of the perilla leaf oil sample solution comprises 1 common fingerprint peak, and the 1 common fingerprint peak is peak number 1. Specific results are shown in table 1.
Preferably, in step E), the fingerprint spectrum of the costustoot sample solution comprises 7 common fingerprint peaks, wherein the 7 common fingerprint peaks are peak number 1, peak number 2, peak number 5, peak number 6, peak number 9, peak number 10 and peak number 11. Specific results are shown in table 1.
Preferably, in the step E), the fingerprint spectrum of the rhizoma atractylodis sample solution includes 11 common fingerprint peaks, and the 11 common fingerprint peaks are peak No. 1, peak No. 2, peak No. 4, peak No. 5, peak No. 6, peak No. 7, peak No. 8, peak No. 12, peak No. 13, peak No. 14 and peak No. 15. Specific results are shown in table 1.
Preferably, in the step E), the fingerprint spectrum of the magnolia bark sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are a No. 2 peak, a No. 4 peak and a No. 13 peak. Specific results are shown in table 1.
Preferably, in the step E), the fingerprint of the ginger sample solution includes 1 common fingerprint peak, and the 1 common fingerprint peak is peak No. 2. Specific results are shown in table 1.
Preferably, in the step E), the fingerprint spectrums of the licorice sample solution, the poria cocos sample solution, the dried orange peel sample solution and the pinellia ternata sample solution do not include a common fingerprint peak. Specific results are shown in table 1.
TABLE 1 common fingerprint Peak attribution of each single herb in Zhengqi tablets
| Medicinal materials | Number of peaks (number) | Common fingerprint peak |
| Patchouli oil | ||
| 2 | 3、5 | |
| |
1 | 1 |
| Radix aucklandiae | 7 | 1、2、5、6、9、10、11 |
| |
11 | 1、2、4、5、6、7、8、12、13、14、15 |
| Licorice root, radix Glycyrrhizae | 0 | — |
| Poria cocos (Schw.) wolf | 0 | — |
| Dried orange peel | 0 | — |
| Pinellia ternata (Thunb.) Breit | 0 | — |
| Cortex Magnoliae officinalis | 3 | 2、4、13 |
| Ginger (fresh ginger) | 1 | 2 |
The water used in the invention is pure water.
As described above, the gas-phase fingerprint detection method of the positive gas tablets and the application thereof provided by the invention establish the gas-phase fingerprint of the positive gas tablets by adopting the pretreatment and gas chromatography method for optimizing reaction conditions, and the contributions of different chemical components to the positive gas tablet fingerprint system are reflected from different side surfaces, so that the detection of the whole formula of the positive gas tablets is realized, and the reference basis is provided for the quality standard of the positive gas tablets. The detection method of the gas-phase fingerprint of the positive gas tablet, which is established for the first time, has good precision and repeatability, determines 15 common peaks, the RSD of the relative retention time of each common peak is less than 0.2%, the similarity evaluation results of multiple batches of positive gas tablet samples are more than 0.95, and the good stability of a test solution can be maintained within 24 hours. The method confirms the attribution of chromatographic peaks of each single medicinal material in the tablets for regulating vital energy, and the gas-phase fingerprint embodies the characteristic peaks of patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, mangnolia officinalis and ginger. The method can realize effective monitoring from the source of raw materials, strictly control the quality of the raw materials, and ensure the relative stability of the effective components in the vital qi tablet, thereby ensuring the safety and effectiveness of clinical medication. The established gas-phase fingerprint spectrum method realizes the quality control of volatile components of the whole formula of the qi tablet for the first time, does not identify single compounds or medicinal materials, and has the advantages of simple operation, strong repeatability and convenient quality control. The gas-phase fingerprint detection method of the vital qi tablet, which is established by the invention, is based on the pharmacodynamic substance basis of the medicinal materials, and realizes that one spectrum covers the pharmacodynamic substances of a plurality of medicinal materials.
Drawings
Fig. 1 shows a gas phase fingerprint of the qi tablet of the present invention.
Fig. 2 shows a superimposed spectrum of the gas-phase fingerprint spectrum of 10 batches of positive gas tablet samples in the invention.
FIG. 3 shows a gas chromatogram of a control solution of the present invention, wherein 8 is atractylodin, 9 is costunolide, and 11 is dehydrocostus lactone.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and equipment used in the following examples are as follows:
1. reagent
Absolute ethanol (analytical grade, chemical reagents of national drug group, ltd); costunolide (purity > 98%), dehydrocostuslactone (purity > 98%), atractylodin (purity > 98%), all purchased from Duymant Biotech limited; ultrapure water (made by Milli-Q water purifier).
The "Zhengqi" tablets were prepared from Shanghai and Huangyao Co., Ltd, and were prepared into 10 batches, and the batch numbers are shown in Table 2 below.
TABLE 2 Positive air film batch number
| Numbering | Batch number | Numbering | Batch number |
| S1 | 170810 | S6 | 171027 |
| S2 | 170815 | S7 | 171110 |
| S3 | 170908 | S8 | 171117 |
| S4 | 170922 | S9 | 171201 |
| S5 | 171013 | S10 | 171215 |
The single medicinal material: patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, liquorice, poria cocos, dried orange peel, pinellia ternate, mangnolia officinalis and ginger are all conventional Chinese medicinal materials provided by Shanghai and Huangyao pharmaceutical Co Ltd and can be purchased from the market
2. Instrument for measuring the position of a moving object
7890A gas chromatograph (USA Agilent Co. ltd); Milli-Q water purifiers (Millipore co. ltd, usa); an electronic analytical balance of the AL204 type (Mettler Toledo co. ltd, switzerland); SB-5200-DTD ultrasonic cleaning machine (Ningbo Xinzhi Biotech Co., Ltd.).
The process of the detection method of the gas phase fingerprint spectrum of the vital qi tablet provided by the invention is as follows:
1. sample pretreatment
Preparation of a test solution: precisely weighing 10g of the normal gas tablet powder, placing the normal gas tablet powder in a 50mL EP tube, precisely adding 15-25mL of absolute ethyl alcohol, weighing the weight, sealing the sealing plug, ultrasonically extracting for 20-40min by using an ultrasonic instrument, weighing again, complementing the lost weight by using the absolute ethyl alcohol, centrifuging for 15-25min at 2000-4000r/min, taking supernatant, and filtering by using a filter membrane with the aperture of 0.22 mu m to obtain a sample solution.
Preparation of control solutions: accurately weighing costunolide, dehydrocostuslactone and atractylodin reference substances respectively, and placing in a volumetric flask for stepwise dilution. Firstly adding absolute ethyl alcohol to dissolve, and shaking up in a volumetric flask with constant volume to obtain a reference substance stock solution, wherein the concentration of each component in the reference substance stock solution is 0.5mg/mL, and the storage temperature of the reference substance stock solution is 0-5 ℃. And adding absolute ethyl alcohol into the reference substance stock solution for dilution and constant volume to prepare a required reference substance solution, wherein the concentration of each component in the reference substance solution is 50-200 mug/mL.
2. Chromatographic conditions
The chromatographic detection conditions of the gas chromatography are as follows: the chromatographic column is an Agilent HP-5 chromatographic column (30m × 0.32mm, 0.25 μm); the detector is a Flame Ionization Detector (FID); the temperature of a sample opening is 260-300 ℃; the sample amount is 1-5 muL; the carrier gas is high-purity helium, and the purity of the carrier gas is more than or equal to 99.999 percent; the flow rate of the carrier gas is 0.5-2.0 mL/min; the sample injection mode is divided-flow sample injection, and the division ratio is 20-40: 1; the adopted temperature-rising program is as follows: the initial temperature is 118-; heating to 158-162 ℃ at the speed of 1-3 ℃/min, and keeping the temperature for 1-3 min; heating to 198-202 deg.C at a rate of 1-3 deg.C/min, and maintaining for 1-3 min; heating to 218-222 ℃ at the speed of 1-3 ℃/min, and keeping for 1-3 min; raising the temperature to 238-242 ℃ at the speed of 1-3 ℃/min, and keeping the temperature for 4-6 min.
3. Measurement of
And (3) respectively measuring the test solution and the reference solution in the step (1) by adopting the gas chromatography under the chromatographic condition in the step (2) to obtain the gas fingerprint of the test solution and the gas fingerprint of the reference solution. And the gas-phase fingerprint of the test solution is compared with the gas-phase fingerprint of the reference solution, and the corresponding characteristic peak in the gas-phase fingerprint of the test solution is identified through relative retention time according to the known characteristic peak in the gas-phase fingerprint of the reference solution, so that the attribution positioning is carried out on the index components in the gas-phase fingerprint of the test solution, and the gas-phase fingerprint of the positive gas tablet is obtained.
The quality detection method of the vital qi tablet provided by the invention comprises the following steps:
the method comprises the steps of obtaining the gas-phase fingerprint of the qi tablet by adopting the detection method of the gas-phase fingerprint of the qi tablet, and comparing the similarity of the obtained gas-phase fingerprint of the qi tablet with the standard gas-phase fingerprint of the qi tablet obtained under the same fingerprint detection condition.
Specifically, when the similarity of the measured gas-phase fingerprint of the zhengqi tablet is compared with the standard gas-phase fingerprint of the zhengqi tablet, the similarity is compared by using software 2009 (evaluation system for traditional Chinese medicine chromatography fingerprint similarity) issued by the committee of national pharmacopoeia. The similarity between the gas phase fingerprint spectrum of the vital qi tablet measured by the method and the standard gas phase fingerprint spectrum of the vital qi tablet is more than or equal to 0.95.
The standard gas phase fingerprint of the Zhengqi tablet comprises 15 common fingerprint peaks, wherein the No. 8 peak is taken as a reference peak (S peak, retention time is 1.000), and the relative retention times of the other 14 common fingerprint peaks are sequentially No. 1 peak (0.243 +/-0.002), No. 2 peak (0.532 +/-0.003), No. 3 peak (0.559 +/-0.003), No. 4 peak (0.790 +/-0.004), No. 5 peak (0.801 +/-0.004), No. 6 peak (0.812 +/-0.004), No. 7 peak (0.931 +/-0.005), No. 9 peak (1.115 +/-0.005), No. 10 peak (1.295 +/-0.006), No. 11 peak (1.391 +/-0.006), No. 12 peak (1.448 +/-0.007), No. 13 peak (1.753 +/-0.008), No. 14 peak (2.010) and No. 15 peak (2.146 +/-0.010).
As shown in fig. 1, the standard gas-phase fingerprint of the zhengqi tablet is compared with the gas-phase fingerprint of the reference solution, and the peak No. 8 is located and determined to be the fingerprint of atractylodin, the peak No. 9 is the fingerprint of costunolide, and the peak No. 11 is the fingerprint of dehydrocostus lactone.
The process of the screening method of the fingerprint spectrums of the multiple medicinal materials in the Zhibai Dihuang pill provided by the invention is as follows:
A) preparing a medicinal material sample solution: preparing any one or more of 10 medicinal material samples of patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, liquorice, poria cocos, dried orange peel, pinellia ternate, mangnolia officinalis and ginger in the Zhengqi tablets according to the step 1) of the detection method of the gas-phase fingerprint spectrum of the Zhengqi tablets to respectively obtain at least one single medicinal material sample solution;
B) preparation of a mixed solution of the lacking negative samples: removing any one 1 medicinal material from 10 medicinal material samples of patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, liquorice, poria cocos, dried orange peel, pinellia ternate, mangnolia officinalis and ginger, adding auxiliary materials with corresponding mass for filling, and preparing according to the step 1) of the detection method of the gas-phase fingerprint of the zhengqi tablet to respectively obtain 10 sample-lacking negative sample mixed solutions;
C) and (3) determination: respectively measuring the single medicinal material sample solution and the 10 sample-lacking negative sample mixed solutions by adopting a Gas Chromatography (GC) with the same chromatographic conditions in the step 3) of the detection method of the gas fingerprint of the zheng qi tablet to respectively obtain the gas fingerprints of the single medicinal material sample solution and the 10 sample-lacking negative sample mixed solutions;
D) obtaining a standard gas phase fingerprint spectrum: obtaining a standard gas-phase fingerprint of the qi tablet by adopting the same steps as the detection method of the gas-phase fingerprint of the qi tablet;
E) and (3) quality detection: and respectively comparing the gas-phase fingerprint of the single-medicinal-material sample solution and the gas-phase fingerprint of the 10 sample-lacking negative-sample mixed solutions with the standard gas-phase fingerprint of the positive gas piece, and identifying the corresponding characteristic peaks of the single-medicinal-material sample solution in the standard gas-phase fingerprint of the positive gas piece through relative retention time, so as to perform attribution positioning on the characteristic peaks in the gas-phase fingerprint of the single-medicinal-material sample solution.
In the step A), the volume ratio of the patchouli oil to the absolute ethyl alcohol is 40: 1500-; the volume ratio of the perilla leaf oil to the absolute ethyl alcohol is 20: 1500-; the ratio of the added weight (g) of the costustoot to the added volume (mL) of the absolute ethyl alcohol is 4.5: 15-25; the ratio of the weight (g) of the added tuckahoe to the volume (mL) of the added absolute ethyl alcohol is 4.5: 15-25; the ratio of the weight (g) of the rhizoma atractylodis added to the volume (mL) of the absolute ethyl alcohol added is 3.0: 15-25; the ratio of the added weight (g) of the dried orange peel to the added volume (mL) of the absolute ethyl alcohol is 3.0: 15-25; the ratio of the weight (g) of the pinellia tuber added to the volume (mL) of the absolute ethyl alcohol added is 3.0: 15-25; the ratio of the added weight (g) of the magnolia officinalis to the added volume (mL) of the absolute ethyl alcohol is 3.0: 15-25; the ratio of the weight (g) of ginger added to the volume (mL) of absolute ethanol added was 3.0: 15-25; the ratio of the weight (g) of the added liquorice to the volume (mL) of the added absolute ethyl alcohol is 1.5: 15-25.
In the step E), as shown in Table 1, the fingerprint spectrum of the patchouli oil sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a peak 3 and a peak 5; the fingerprint spectrum of the perilla leaf oil sample solution comprises 1 common fingerprint peak, and 1 common fingerprint peak is a No. 1 peak; the fingerprint spectrum of the costustoot sample solution comprises 7 common fingerprint peaks, wherein the 7 common fingerprint peaks are a No. 1 peak, a No. 2 peak, a No. 5 peak, a No. 6 peak, a No. 9 peak, a No. 10 peak and a No. 11 peak; the fingerprint spectrum of the rhizoma atractylodis sample solution comprises 11 common fingerprint peaks, wherein the 11 common fingerprint peaks are a peak 1, a peak 2, a peak 4, a peak 5, a peak 6, a peak 7, a peak 8, a peak 12, a peak 13, a peak 14 and a peak 15; the fingerprint spectrum of the magnolia bark sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are a No. 2 peak, a No. 4 peak and a No. 13 peak; the fingerprint spectrum of the ginger sample solution comprises 1 common fingerprint peak, and 1 common fingerprint peak is a No. 2 peak; the fingerprint spectrums of the liquorice sample solution, the tuckahoe sample solution, the dried orange peel sample solution and the pinellia ternate sample solution do not comprise common fingerprint peaks.
Example 1
1. Sample pretreatment
Preparation of a test solution: precisely weighing 10g of vital qi tablet powder, placing in a 50mL EP tube, precisely adding 20mL of absolute ethyl alcohol, weighing, sealing, ultrasonically extracting for 30min with an ultrasonic instrument, weighing again, supplementing the lost weight with absolute ethyl alcohol, centrifuging for 20min at 3000r/min, collecting the supernatant, and filtering with a filter membrane with the aperture of 0.22 μm to obtain the sample solution.
Preparation of control solutions: accurately weighing costunolide, dehydrocostuslactone and atractylodin reference substances respectively, and diluting step by step. Firstly, 5mg of each reference substance is placed in a 10mL volumetric flask, absolute ethyl alcohol is added to dissolve the reference substances, the reference substances are uniformly shaken in the volumetric flask with constant volume, a reference substance stock solution is obtained, the concentration of each component in the reference substance stock solution is 0.5mg/mL, and the storage temperature of the reference substance stock solution is 4 ℃. And adding absolute ethyl alcohol into the reference substance stock solution for dilution and constant volume to prepare a required reference substance solution, wherein the concentration of each component in the reference substance solution is 100 mug/mL.
2. Chromatographic conditions
The chromatographic detection conditions of the gas chromatography are as follows: the chromatographic column is an Agilent HP-5 chromatographic column (30m × 0.32mm, 0.25 μm); the detector is a Flame Ionization Detector (FID); the sample port temperature was 280 ℃; the sample injection amount is 2 mu L; the carrier gas is high-purity helium, and the purity of the carrier gas is more than or equal to 99.999 percent; the flow rate of the carrier gas is 1.0 mL/min; the sample injection mode is divided sample injection, and the dividing ratio is 30: 1; the adopted temperature-rising program is as follows: maintaining the initial temperature at 120 deg.C for 2min, heating to 130 deg.C at 2 deg.C/min, and maintaining for 2 min; heating to 160 deg.C at a rate of 2 deg.C/min, and maintaining for 2 min; heating to 200 deg.C at a rate of 2 deg.C/min, and maintaining for 2 min; heating to 220 deg.C at a rate of 2 deg.C/min, and maintaining for 2 min; the temperature was raised to 240 ℃ at a rate of 2 ℃/min and held for 5 min.
3. Measurement of
And (3) respectively measuring the test solution and the reference solution in the step (1) by adopting the gas chromatography under the chromatographic condition in the step (2) to obtain the gas fingerprint of the test solution and the gas fingerprint of the reference solution. And the gas-phase fingerprint of the test solution is compared with the gas-phase fingerprint of the reference solution, and the corresponding characteristic peak in the gas-phase fingerprint of the test solution is identified through relative retention time according to the known characteristic peak in the gas-phase fingerprint of the reference solution, so that the attribution positioning is carried out on the index components in the gas-phase fingerprint of the test solution, and the gas-phase fingerprint of the positive gas tablet is obtained.
Example 2
The gas-phase fingerprint spectrum of the normal gas tablets of 10 batches is detected by adopting the detection method of the gas-phase fingerprint spectrum of the normal gas tablets established in the embodiment 1, so that the gas-phase fingerprint spectrum of the test solution and the gas-phase fingerprint spectrum of the reference solution are obtained, and the batch numbers of the normal gas tablets of 10 batches are shown in a table 2. The obtained gas-phase fingerprint data of the test sample is introduced into software 2009, which is published by the State pharmacopoeia Committee and is compared with the standard gas-phase fingerprint of the Zhengqi tablet under the same fingerprint detection condition, the similarity of the gas-phase fingerprint of the Zhengqi tablet and the standard gas-phase fingerprint of the Zhengqi tablet is more than or equal to 0.95 as the standard, the No. 8 peak is used as a reference peak (S peak, retention time is 1.000), the standard fingerprint of the Zhengqi tablet is determined to comprise 15 common fingerprint peaks, and the relative retention time of the other 14 common fingerprint peaks is sequentially No. 1 peak (0.243 +/-0.001), No. 2 peak (0.532 +/-0.001), No. 3 peak (0.559 +/-0.001), No. 4 peak (0.790 +/-0.002), No. 5 peak (0.801 +/-0.002), No. 6 peak (0.812 +/-0.003), No. 7 peak (0.003), No. 9 +/-0.115), No. 9 peak (1.295), No. 1.004 +/-0.004), and No. 1.004 (10.95), Peak 11 (1.391. + -. 0.004), peak 12 (1.448. + -. 0.004), peak 13 (1.753. + -. 0.005), peak 14 (2.045. + -. 0.006) and peak 15 (2.146. + -. 0.006).
Comparing the standard gas-phase fingerprint of the qi tablet with the gas-phase fingerprint of the reference solution, identifying the corresponding characteristic peak in the standard gas-phase fingerprint of the qi tablet according to the known characteristic peak in the gas-phase fingerprint of the reference solution in fig. 1 by relative retention time, and positioning and determining that the No. 8 peak is the fingerprint peak of atractylodin, the No. 9 peak is the fingerprint peak of costunolide, and the No. 11 peak is the fingerprint peak of dehydrocostuslactone.
Example 3
The gas-phase fingerprint spectrum of the normal gas tablets of 10 batches is detected by adopting the detection method of the gas-phase fingerprint spectrum of the normal gas tablets established in the embodiment 1, so that the gas-phase fingerprint spectrum of the test solution and the gas-phase fingerprint spectrum of the reference solution are obtained, and the batch numbers of the normal gas tablets of 10 batches are shown in a table 2. The obtained fingerprint data of the test sample is imported into software 2009 issued by the national pharmacopoeia committee, namely traditional Chinese medicine chromatography fingerprint similarity evaluation system, similarity comparison is carried out on the fingerprint data and a standard gas-phase fingerprint of a vital qi tablet obtained under the same fingerprint detection condition, the similarity of the gas-phase fingerprint of the vital qi tablet and the standard gas-phase fingerprint of the vital qi tablet is not less than 0.95 as a standard, an S1 sample is used as a reference spectrum, a reference spectrum is generated by an average method and is shown in figure 1, a superposed spectrum is shown in figure 2, and 15 common fingerprint peaks in the fingerprint are determined. The peak-off time of No. 8 peak is stable and moderate, so No. 8 is selected as a positioning peak (S peak).
The ratio of the retention time and the retention peak area of each spectrum peak to the retention time and the retention peak area of the S peak in the same spectrum is calculated through the gas-phase fingerprint obtained from 10 batches of normal gas tablets, and the obtained relative retention time and relative peak area are shown in Table 3. As can be seen from Table 3, the RSD of each common peak relative to the retention time is less than 0.2%, and the RSD of the relative peak area is between 2 and 42%, which indicates that the components of 10 batches of positive gas tablet samples are relatively stable.
TABLE 3 relative retention time and relative peak area of common peaks
The specific standard gas-phase fingerprint of the qi-tonifying tablet is shown in figure 1, the specific standard gas-phase fingerprint of the qi-tonifying tablet is compared with the gas-phase fingerprint of a reference substance solution, corresponding characteristic peaks in the standard gas-phase fingerprint of the qi-tonifying tablet are identified through relative retention time according to known characteristic peaks in the gas-phase fingerprint of the reference substance solution in figure 1, the No. 8 peak is positioned and determined to be the fingerprint peak of atractylodin, the No. 9 peak is the fingerprint peak of costunolide, and the No. 11 peak is the fingerprint peak of dehydrocostuslactone.
Example 4
The detection method of the gas-phase fingerprint spectrum of the qi tablet is verified by methodology, and the performance index result is as follows.
1. Precision degree
Taking 1 part of 170810 batches of qi tablet samples, detecting according to the detection method of the gas-phase fingerprint of the qi tablet in the example 1, continuously sampling for 6 times, taking the No. 8 peak (atractylodin) as a reference peak, and as a result, the relative retention time RSD of 15 common peaks is less than 0.2%, and the RSD of the relative peak area is less than 2%, which indicates that the precision of the instrument is good.
2. Repeatability of
Taking 6 parts of 170810 batches of qi-tonifying tablet samples, detecting according to the method for detecting the gas-phase fingerprint of the qi-tonifying tablet in the example 1, taking the No. 8 peak (atractylodin) as a reference peak, and as a result, the relative retention time RSD of 15 common peaks is less than 0.2%, and the RSD of the relative peak area is less than 3%, which indicates that the experiment repeatability is good and the accuracy is high.
3. Stability of
Taking 1 part of 170810 batches of qi-tonifying tablet samples, according to the detection method of the gas-phase fingerprint chromatogram of the qi-tonifying tablet in the embodiment 1, preparing a sample solution, and then respectively placing the sample solution for 0h, 4h, 8h, 12h, 16h and 24h for detection, taking the No. 8 peak (atractylodin) as a reference peak, and as a result, the relative retention time RSD of 15 common peaks is less than 0.2%, and the RSD of the relative peak area is less than 2%, which indicates that the stability of the sample in 24h is good.
Example 5
By adopting the method for detecting the gas-phase fingerprint of the qi-tonifying tablets in the embodiment 1, 10 batches of qi-tonifying tablets are subjected to gas-phase fingerprint analysis, the batch numbers of the 10 batches of qi-tonifying tablets are shown in a table 2, and the detection result is shown in a table 1. Meanwhile, the overall similarity evaluation of 10 batches of qi tablet samples is carried out by adopting software 2009 (traditional Chinese medicine chromatography fingerprint similarity evaluation system) issued by the State pharmacopoeia Committee, and the overall similarity can reflect the similarity of various components among different batches of samples in terms of types and relative contents thereof, and the result is shown in Table 4. The results in Table 4 show that the similarity of 10 batches of positive gas tablets is more than or equal to 0.95, which indicates that the similarity of 10 batches of positive gas tablets is good, the difference between batches is small, and the gas-phase fingerprint is suitable for quality control of the positive gas tablets.
TABLE 410 evaluation results of similarity of the air film batches
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | R | |
| S1 | 1.000 | ||||||||||
| S2 | 1.000 | 1.000 | |||||||||
| S3 | 1.000 | 1.000 | 1.000 | ||||||||
| S4 | 1.000 | 1.000 | 1.000 | 1.000 | |||||||
| S5 | 1.000 | 1.000 | 0.999 | 1.000 | 1.000 | ||||||
| S6 | 0.999 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | |||||
| S7 | 0.997 | 0.997 | 0.997 | 0.997 | 0.997 | 0.997 | 1.000 | ||||
| S8 | 0.997 | 0.997 | 0.997 | 0.997 | 0.997 | 0.997 | 1.000 | 1.000 | |||
| S9 | 0.991 | 0.991 | 0.990 | 0.991 | 0.992 | 0.992 | 0.981 | 0.981 | 1.000 | ||
| S10 | 0.991 | 0.991 | 0.990 | 0.991 | 0.992 | 0.992 | 0.981 | 0.981 | 1.000 | 1.000 | |
| R | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.997 | 0.997 | 0.993 | 0.993 | 1.000 |
Example 6
The sample solution was prepared by the sample pretreatment step described in example 1 above.
Respectively taking 40 mu L of patchouli oil, 20 mu L of perilla leaf oil, 4.5g of costustoot, 4.5g of poria cocos, 3g of rhizoma atractylodis, 3g of dried orange peel, 3g of pinellia ternate, 3g of mangnolia officinalis, 3g of fresh ginger and 1.5g of liquorice, and respectively preparing 10 single medicinal material sample solutions by adopting the sample pretreatment steps in the embodiment 1.
Preparing deficient sample negative samples according to the prescription and the process of the vital qi tablet, wherein the deficient sample negative samples are obtained by removing 1 medicinal material from 10 medicinal material samples, the deficient medicinal materials in each deficient sample negative sample are filled by a corresponding amount of auxiliary materials such as magnesium stearate or talcum powder, and the final weight meets the weight required by the vital qi tablet, so that 10 deficient sample negative samples are prepared. 10 kinds of missing negative sample mixed solutions were prepared by using 10.0g of each of the 10 kinds of missing negative sample powders and the sample pretreatment step in example 1.
By adopting the steps 2 and 3 of the method for detecting the gas-phase fingerprint of the vital qi tablet in the embodiment 1, the gas-phase fingerprint of the test solution, the 10 single-herb sample solutions and the 10 sample-lacking negative sample mixed solutions are respectively obtained by measuring the test solution, the 10 single-herb sample solutions and the 10 sample-lacking negative sample mixed solutions. The gas-phase fingerprints of the obtained test solution, 10 single-drug sample solutions and 10 sample-lacking negative sample mixed solutions are introduced into software of 2009, a "traditional Chinese medicine chromatography fingerprint similarity evaluation system" issued by the national pharmacopoeia committee for analysis and processing, and meanwhile, the standard gas-phase fingerprint of the zhengqi tablets is obtained by adopting the detection method of the gas-phase fingerprint of the zhengqi tablets in example 1. Comparing the gas-phase fingerprints of the test solution, the 10 single-drug sample solutions and the 10 sample-lacking negative sample mixed solutions with the standard gas-phase fingerprints of the positive gas tablets respectively, and identifying the corresponding characteristic peaks of the 10 single-drug sample solutions in the standard gas-phase fingerprints of the positive gas tablets through relative retention time, so as to perform attribution positioning on the characteristic peaks in the gas-phase fingerprints of the 10 single-drug sample solutions, wherein specific results are shown in fig. 3 and table 1. The gas-phase fingerprint spectrums of the 10 sample-lacking negative sample mixed solutions can be used for comparison and confirming the source of the characteristic peak.
As can be seen from fig. 3 and table 1, peak 3 is derived from patchouli oil only; 5 common peaks such as peak 7, peak 8, peak 12, peak 14, peak 15 and the like are only derived from rhizoma atractylodis; 3 common peaks such as peak 9, peak 10, and peak 11 are derived from radix aucklandiae; peak 1 is derived from perilla leaf oil, radix aucklandiae and rhizoma Atractylodis; peak 2 is derived from radix aucklandiae, rhizoma Atractylodis, cortex Magnolia officinalis and rhizoma Zingiberis recens; peak 4 and Peak 13 are derived from Atractylodes lancea and Magnolia officinalis; peak 5 is derived from patchouli oil, costus root and atractylodes; peak 6 is derived from Aucklandia lappa and Atractylodes lancea. The 10 medicinal materials in the Zhengqi tablet, the liquorice, the tuckahoe, the dried orange peel and the pinellia tuber do not comprise common peaks, and the chemical characteristic peaks of other 6 medicinal materials are better reflected in the fingerprint spectrum and are confirmed to belong to the same category. Meanwhile, after comparison with a reference chromatogram, peak 8 is determined to be atractylodin, peak 9 is costunolide, and peak 11 is determined to be dehydrocostuslactone.
In conclusion, the detection method and the application of the gas-phase fingerprint spectrum of the vital qi tablet provided by the invention establish the gas-phase fingerprint spectrum of the vital qi tablet, confirm the attribution of each single medicinal material chromatographic peak in the vital qi tablet, effectively monitor the source of raw materials, strictly control the quality of the raw medicinal materials, and ensure that the amount of effective components in the components of the vital qi tablet is relatively stable, thereby ensuring the safety and effectiveness of clinical medication. Therefore, the present invention overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A method for detecting a gas phase fingerprint spectrum of a vital qi tablet comprises the following steps:
1) preparation of a test solution: adding absolute ethyl alcohol into a positive gas tablet sample, performing ultrasonic extraction, centrifuging, and filtering to obtain a test solution;
2) preparation of control solutions: dissolving costunolide, dehydrocostuslactone and atractylodin as reference substances in anhydrous ethanol to obtain reference solution;
3) and (3) determination: respectively measuring the sample solution and the reference solution by adopting a gas chromatography method under the same chromatographic conditions to obtain a fingerprint of the sample solution and a fingerprint of the reference solution, comparing the fingerprint of the sample solution with the fingerprint of the reference solution, and performing attribution positioning on index components in the fingerprint of the sample solution, thereby obtaining the gas fingerprint of the positive gas tablet.
2. The method for detecting the gas phase fingerprint spectrum of the positive gas film according to claim 1, wherein the step 1) comprises any one or more of the following conditions:
A1) the ratio of the weight of the added positive gas tablet sample to the volume of the added absolute ethyl alcohol is 10: 15-25 g/mL;
A2) the ultrasonic extraction time is 20-40 min;
A3) the centrifugation conditions were: centrifuging time: 15-25 min; centrifugal rotating speed: 2000-4000 r/min.
3. The method for detecting the gas-phase fingerprint spectrum of the vital qi tablet according to claim 1, wherein in the step 2), the content ranges of the components in the reference solution are as follows: costunolide is 50-200 μ g/mL, dehydrocostuslactone is 50-200 μ g/mL, and atractylodin is 50-200 μ g/mL.
4. The method for detecting the gas phase fingerprint spectrum of the vital qi tablet as claimed in claim 1, wherein in the step 3), the detection conditions of the gas chromatography are as follows: the chromatographic column is an Agilent HP-5 chromatographic column, and is 30m multiplied by 0.32mm and 0.25 mu m; the detector is a flame ionization detector; the temperature of a sample opening is 260-300 ℃; the sample amount is 1-5 muL; the carrier gas is high-purity helium, and the purity of the carrier gas is more than or equal to 99.999 percent; the flow rate of the carrier gas is 0.5-2.0 mL/min; the sample injection mode is split sample injection, and the split ratio is 20-40: 1.
5. The method for detecting the gas phase fingerprint spectrum of the vital qi tablet according to the claim 1, wherein in the step 3), the temperature rising program adopted in the gas chromatography is as follows: the initial temperature is 118-; heating to 158-162 ℃ at the speed of 1-3 ℃/min, and keeping the temperature for 1-3 min; heating to 198-202 deg.C at a rate of 1-3 deg.C/min, and maintaining for 1-3 min; heating to 218-222 ℃ at the speed of 1-3 ℃/min, and keeping for 1-3 min; raising the temperature to 238-242 ℃ at the speed of 1-3 ℃/min, and keeping the temperature for 4-6 min.
6. Use of the method for detecting the gas phase fingerprint of the qi-tonifying tablet according to any one of claims 1 to 5 in quality detection of components in the qi-tonifying tablet.
7. A quality detection method of Zhengqi tablet comprises obtaining gas phase fingerprint of Zhengqi tablet by the detection method of gas phase fingerprint of Zhengqi tablet as claimed in any one of claims 1-5, and comparing the obtained gas phase fingerprint with standard gas phase fingerprint of Zhengqi tablet obtained under the same fingerprint detection condition.
8. The method for detecting the quality of an essential gas tablet according to claim 7, wherein the standard gas phase fingerprint of the essential gas tablet is obtained under the same conditions as the method for detecting the gas phase fingerprint of the essential gas tablet according to any one of claims 1 to 5, and comprises 15 common fingerprint peaks, wherein the peak 8 is taken as the reference peak S peak, the retention time is 1.000, and the relative retention times of the other 14 common fingerprint peaks are, in order, 0.241-0.245 of the peak 1, 0.529-0.535 of the peak 2, 0.556-0.562 of the peak 3, 0.786-0.794 of the peak 4, 0.797-0.805 of the peak 5, 0.808-0.816 of the peak 6, 0.926-0.936 of the peak 7, 1.110-1.120 of the peak 9, 1.035-1.301 of the peak 10, 1.385-1.289 of the peak 11, 1.441, 1.3971-1.853, 1.761 of the peak 1.3513-1.761 of the peak 1.761, 2-1.761 of the peak 3-1.761, and 2 of the peak 3, Peak 15 2.136-2.156.
9. A method for screening gas-phase fingerprint spectrums of a plurality of medicinal materials in vital qi tablets comprises the following steps:
A) preparing a medicinal material sample solution: preparing any one or more of 10 medicinal material samples of patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, liquorice, poria cocos, dried orange peel, pinellia ternate, mangnolia officinalis and ginger in the zhengqi tablet according to the step 1) of the detection method of the gas-phase fingerprint spectrum of the zhengqi tablet in any one of claims 1 to 5 to respectively obtain at least one single medicinal material sample solution;
B) preparation of a mixed solution of the lacking negative samples: removing any one 1 medicinal material from 10 medicinal material samples of patchouli oil, perilla leaf oil, costustoot, rhizoma atractylodis, liquorice, poria cocos, dried orange peel, pinellia ternate, mangnolia officinalis and ginger, adding auxiliary materials with corresponding mass for filling, and preparing according to the step 1) of the detection method of the gas-phase fingerprint of the zheng qi tablet in any one of claims 1 to 5 to respectively obtain 10 sample-lacking negative mixed solutions;
C) and (3) determination: respectively measuring the single medicinal material sample solution and the 10 sample-lacking negative sample mixed solutions by using the gas chromatography method with the same chromatographic conditions in the step 3) of the method for detecting the gas fingerprint of the vital qi tablet according to any one of claims 1 to 5 to respectively obtain the gas fingerprints of the single medicinal material sample solution and the 10 sample-lacking negative sample mixed solutions;
D) obtaining a standard gas phase fingerprint spectrum: obtaining a standard gas-phase fingerprint of the qi-tonifying tablet by adopting the same steps as the detection method of the gas-phase fingerprint of the qi-tonifying tablet of any one of claims 1 to 5;
E) and (3) quality detection: and respectively comparing the gas-phase fingerprint of the single-medicinal-material sample solution and the gas-phase fingerprint of the 10 sample-lacking negative-sample mixed solutions with the standard gas-phase fingerprint of the positive gas piece, and identifying the corresponding characteristic peaks of the single-medicinal-material sample solution in the standard gas-phase fingerprint of the positive gas piece through relative retention time, so as to perform attribution positioning on the characteristic peaks in the gas-phase fingerprint of the single-medicinal-material sample solution.
10. The method for screening the gas-phase fingerprint spectrums of the multiple medicinal materials in the zhengqi tablet as claimed in claim 9, wherein the step E) comprises any one or more of the following conditions:
B1) the fingerprint spectrum of the patchouli oil sample solution comprises 2 common fingerprint peaks, wherein the 2 common fingerprint peaks are a No. 3 peak and a No. 5 peak;
B2) the fingerprint spectrum of the perilla leaf oil sample solution comprises 1 common fingerprint peak, wherein the 1 common fingerprint peak is a No. 1 peak;
B3) the fingerprint spectrum of the costustoot sample solution comprises 7 common fingerprint peaks, wherein the 7 common fingerprint peaks are a No. 1 peak, a No. 2 peak, a No. 5 peak, a No. 6 peak, a No. 9 peak, a No. 10 peak and a No. 11 peak;
B4) the fingerprint spectrum of the rhizoma atractylodis sample solution comprises 11 common fingerprint peaks, wherein the 11 common fingerprint peaks are a peak 1, a peak 2, a peak 4, a peak 5, a peak 6, a peak 7, a peak 8, a peak 12, a peak 13, a peak 14 and a peak 15;
B5) the fingerprint spectrum of the magnolia bark sample solution comprises 3 common fingerprint peaks, wherein the 3 common fingerprint peaks are a No. 2 peak, a No. 4 peak and a No. 13 peak;
B6) the fingerprint spectrum of the ginger sample solution comprises 1 common fingerprint peak, wherein the 1 common fingerprint peak is a No. 2 peak;
B7) the fingerprint spectrums of the liquorice sample solution, the tuckahoe sample solution, the dried orange peel sample solution and the pinellia ternate sample solution do not comprise common fingerprint peaks.
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