CN112394117B - Method for establishing fingerprint spectrum of oyster powder and fingerprint spectrum thereof - Google Patents
Method for establishing fingerprint spectrum of oyster powder and fingerprint spectrum thereof Download PDFInfo
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Abstract
The invention discloses an oyster powder fingerprint establishing method and an oyster powder fingerprint. The establishment method of the fingerprint comprises the following steps: step 1: preparing a reference peak solution; step 2: preparing a oyster powder test solution; and 3, step 3: precisely absorbing quantitative reference peak solution and sample solution, respectively, injecting into a liquid chromatograph, and recording chromatogram; and 4, step 4: and (3) exporting the fingerprint instrument of the oyster powder obtained in the step (3), importing the fingerprint instrument into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, selecting chromatographic peaks existing in the chromatograms of the test samples of the oyster powder of different batches as common peaks, generating a contrast fingerprint of the oyster powder by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak. The method for establishing the fingerprint has the advantages of simplicity, convenience, stability, high precision, good reproducibility and the like. The fingerprint spectrum of the oyster powder provided by the invention can comprehensively and objectively characterize the quality of the oyster powder and is beneficial to comprehensively monitoring the quality of the medicine.
Description
Technical Field
The invention relates to an establishing method of oyster powder fingerprint and an oyster powder fingerprint established by the method, belonging to the technical field of traditional Chinese medicine quality identification.
Background
The traditional Chinese medicine fingerprint is an effective means of comprehensive analysis for identifying the truth of traditional Chinese medicinal materials or Chinese patent medicines by using a modern analysis technology and evaluating the consistency and stability of the quality of raw material medicines, semi-finished products and finished products. Compared with the existing identification of single effective component or index component, the fingerprint spectrum technology of traditional Chinese medicine integrates various effective chemical information, so that the result is more scientific and reasonable. At present, the traditional Chinese medicine fingerprint has become an important method for controlling the quality of traditional Chinese medicines acknowledged at home and abroad, and the research and establishment of the fingerprint have important significance for improving the quality of a traditional Chinese medicine compound and promoting the modernization of the traditional Chinese medicines.
Mu Li san was recorded in Tai Ping Hui Min He Ji Ju Fang, one of the typical prescriptions for exterior-consolidating antiperspirant in astringents of all ages. The whole prescription consists of four medicines of calcined oyster, astragalus root, ephedra root and light wheat, and the main efficacy is as follows: for deficiency, sudden deficiency of new diseases, unconsolidated body fluid, spontaneous perspiration, frequent lying on night, shortness of breath, marasmus, short breath, restlessness and fatigue. The traditional Chinese medicine composition is clinically applied more, such as addition and subtraction treatment of primary hand sweating disease, brain trauma operation sweating disease, radiotherapy middle sweating disease and the like, and has obvious curative effect, and is expected to be developed into a new antiperspirant with marine characteristics.
However, the ingredients of the oyster powder are complex, the qualitative and quantitative analysis of one or two ingredients is difficult to fully reflect the full information of the compound recipe, and no related research on the fingerprint of the oyster powder exists so far.
Disclosure of Invention
In view of the above problems, the first objective of the present invention is to provide a fingerprint spectrum establishment method of oyster powder with high precision, good repeatability and good stability, which is used for quality control of oyster powder.
The invention also aims to provide the oyster powder fingerprint spectrum established by the method.
The invention adopts the specific technical scheme that:
the method for establishing the fingerprint spectrum of the oyster powder is characterized by comprising the following steps of:
step 1: dissolving calycosin glycoside, formononetin and calycosin reference substances in methanol to obtain reference peak solution;
step 2: taking oyster powder test samples of different batches, placing the oyster powder test samples in a container, adding water for dissolution, and pretreating by using a C18 solid phase extraction column to prepare an oyster powder test sample solution;
and step 3: precisely absorbing the reference peak solution and the sample solution respectively, injecting into a liquid chromatograph, and collecting a chromatogram;
and 4, step 4: guiding the integral signal of the fingerprint of the oyster powder test solution obtained in the step 3 into software of a traditional Chinese medicine chromatography fingerprint similarity evaluation system; selecting chromatographic peaks existing in chromatograms of different batches of oyster powder as common peaks, generating a control fingerprint of the oyster powder by using an average value calculation method, and calculating relative retention time and relative peak area of each common peak;
as a preferred scheme, in the above method for establishing a fingerprint of oyster powder, the pretreatment process using a C18 solid phase extraction column in step 2 is as follows: activating with methanol, balancing water, loading, eluting with methanol aqueous solution of different concentrations, eluting with methanol formate, collecting eluate, and collecting N 2 Blow-drying, re-dissolving the residue with methanol, centrifuging, and collecting supernatant.
As a preferred scheme, in the method for establishing the fingerprint spectrum of the oyster powder, in the step 3, the reference peak solutions are respectively and precisely absorbedLiquid and sample solution each 5μAnd L, injecting the mixture into a liquid chromatograph, and collecting a chromatogram within 95 minutes.
As a preferred scheme, in the above method for establishing the fingerprint of the oyster powder, in step 3, the liquid chromatography conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the column temperature is 30-40 ℃; a: water-B: acetonitrile is used as a mobile phase, gradient elution is carried out for 0-5 min, and B is 10%; 5-10 min, B10% -12%; 10-60 min, B12% -25%; 60-65min, 25% -27%; 65-80min, 27% -35%; 80-95min, 35-50%, ultraviolet detection wavelength: 210-.
Preferably, the oyster powder in step 2 is prepared by the following method: taking 3 parts of calcined oyster, 3 parts of astragalus, 3 parts of ephedra root and 2.5 parts of blighted wheat, soaking the above four materials in water for 1 hour, decocting twice for 2 hours each time, adding water with the weight 8 times of that of all decoction pieces twice, filtering decoction solutions, combining filtrate, concentrating the filtrate under reduced pressure, and freeze-drying to obtain the oyster powder test product.
The oyster powder fingerprint established by the method is characterized in that: the fingerprint comprises 12 common peaks, wherein the peak corresponding to calycosin glycoside is the No. 4 peak, the peak corresponding to formononetin is the No. 7 peak, and the peak corresponding to calycosin is the No. 9 peak.
Taking the corresponding chromatographic peak of the reference substance peak of the calycosin glycoside as an S peak, and calculating the relative retention time of each common peak to be in accordance with the following steps: 0.556-0.561 part of peak No. 1, 0.579-0.585 part of peak No. 2, 0.788-0.792 part of peak No. 3, 1.000 part of peak No. 4, 1.399-1.402 part of peak No. 5, 1.463-1.468 part of peak No. 6, 1.741-1.743 part of peak No. 7, 2.001-2.003 part of peak No. 8, 2.067-2.077 part of peak No. 9, 2.147-2.149 parts of peak No. 10, 3.059-3.076 parts of peak No. 11, and 3.143-3.152 part of peak No. 12.
Preferably, the fingerprint of the oyster powder is characterized in that the relative peak areas of the common peaks and the S peak are in accordance with: 0.059 to 0.712 of peak No. 1, 0.062 to 0.508 of peak No. 2, 0.093 to 0.563 of peak No. 3, 1.000 of peak No. 4, 0.062 to 0.635 of peak No. 5, 0.081 to 1.305 of peak No. 6, 0.202 to 0.530 of peak No. 7, 0.385 to 1.174 of peak No. 8, 0.102 to 1.469 of peak No. 9, 0.166 to 0.507 of peak No. 10, 0.06 to 0.823 of peak No. 11, and 0.118 to 1.151 of peak No. 12.
The invention has the beneficial effects that:
1. the fingerprint spectrum of the oyster powder provided by the invention has 12 common peaks, can effectively represent the quality of the oyster powder, can represent the information of the main medicinal materials of the oyster powder in one spectrum under the same wavelength, and can comprehensively and objectively represent the quality of a compound.
2. The invention adopts the calycosin glycoside reference substance as a reference peak, and has the advantages of large peak area, moderate retention time and good separation degree.
3. The method for establishing the fingerprint of the oyster powder has the advantages of simplicity, convenience, stability, high precision and good reproducibility.
Drawings
FIG. 1 shows HPLC finger print of 15 batches of oyster powder.
FIG. 2 is the HPLC control fingerprint of mu Li san.
FIG. 3 is the chromatogram of the common peak in the HPLC fingerprint.
FIG. 4 is a qualitative diagram of the reference substance in HPLC fingerprint of oyster powder.
Wherein, in the figure: 4-calycosin glycoside, 7-formononetin and 9-calycosin.
FIG. 5 shows the stability test fingerprint of 20200815 batch oyster powder.
FIG. 6 shows the fingerprint of the mixed reference substance for precision test.
FIG. 7 is a fingerprint of a repeated experiment of 20200815 batch oyster powder.
Detailed Description
The invention is explained in further detail below by means of specific embodiments with reference to the drawings.
1. Instrument and reagent
Agilent 1290 ultra high performance liquid chromatograph (G7117B 1290 DAD, G7116B 1290 MCT, G7167B 1290 Multisampler, G7104A 1290 Flexible Pump), H1650-W desk-top high speed centrifuge (Hunan Changshan apparatus centrifuge, Inc.), ME2002E electronic balance (both Mettler-Torland, Inc., Switzerland), KQ-500V ultrasonic cleaner (Kunshan ultrasonic apparatus, Inc.), electric heating jacket (Shandong Hualu electric heating apparatus, Inc.). Acetonitrile is chromatographically pure, methanol and formic acid are analytically pure, and water is Wahaha purified water. Calycosin (18092601, purity not less than 98.0%, WUDOPIFIED Biotech Co., Ltd.), formononetin (16031111, 98.54%, Beijing Beinanna Chuangyi Biotech research institute), calycosin (16031110, purity 99.08%, Beijing Beinanna Chuangyi Biotech research institute).
Oyster powder (batch No. 20200801 and No. 20200815) was prepared by laboratory production.
2. Preparation of oyster powder
The prescription decoction pieces are purchased from Dayin slice companies all over the country for 15 batches respectively, and the production place information is shown in tables 1-4.
TABLE 1 radix astragali decoction pieces Source
TABLE 2 sources of decoction pieces of radix Ephedrae
TABLE 3 sources of blighted wheat decoction pieces
TABLE 4 sources of decoction pieces of radix Ephedrae
| Medicinal materials | Number of | Producing area | Manufacturer of the product |
| Calcined oyster | 1 | Liaoning medicine | ANHUI WANSHENG TRADITIONAL CHINESE MEDICINE DECOCTION PIECES Co.,Ltd. |
The preparation method of the oyster powder comprises the following steps: 3 parts of calcined oyster, 3 parts of astragalus, 3 parts of ephedra root and 2.5 parts of blighted wheat are taken, the four medicines are soaked in water for 1 hour, and are decocted twice, 2 hours each time, water with the weight 8 times of that of all decoction pieces is added in the two times, decoction liquids are filtered, filtrate is combined, the filtrate is decompressed and concentrated, and freeze-drying is carried out, thus obtaining the oyster powder sample.
Taking the decoction pieces according to the serial numbers of the decoction pieces listed in the table 1 to prepare the oyster powder, wherein the sampling method comprises the steps of taking the astragalus root No. 1, the ephedra root No. 1, the blighted wheat No. 1 and the calcined oyster No. 1 to prepare the oyster powder, and the batch number is 20200801; taking radix astragali No. 2, radix Ephedrae No. 2, fructus Tritici Levis No. 2, and Concha Ostreae preparata No. 1, and making into Concha Ostreae powder with lot number of 20200802, and so on.
The oyster powder prepared by the method has the batch number of 20200801 and 20200815. 20200801 lot number S1, 20200802 lot number S2, and so on, 20200815 lot number S15.
Example 1:
the method for establishing the fingerprint of the oyster powder in the embodiment 1 comprises the following steps:
step 1: taking calycosin glycoside, formononetin, and calycosin reference, adding methanol to obtain solutions with concentrations of 0.22, 0.31, and 0.28 mg/mL −1 Mixed control solution of (4).
Step 2: respectively taking 15 batches of oyster powder samples (batch No. 20200801 and No. 20200815), precisely weighing about 50mg, and dissolving in 2mL of water; the treatment of the Cleanert S C18 solid phase extraction column comprises the following steps: activating with methanol, balancing water, loading, eluting with 2 times of column volume of water solution and 2 times of column volume of 0.4% methanol formate, and collectingEluent, N 2 Blow-drying, adding 200 portions of residueμAnd (4) redissolving by using L methanol, centrifuging, and taking supernatant to prepare 15 batches of oyster powder test solution.
And step 3: precisely absorbing each test sample solution and each reference peak solution respectively, injecting the test sample solution and the reference peak solution into a liquid chromatograph, and recording a chromatogram map of 95 minutes; the specific chromatographic conditions are as follows: agilent ZORBAX SB C18 (4.6 × 250mm, 5 μm), the column temperature is 30-40 ℃; a: water-B: acetonitrile, gradient elution for 0-5 min, B10%; 5-10 min, B10% -12%; 10-60 min, B12% -25%; 60-65min, 25% -27%; 65-80min, 27% -35%; 80-95min, 35% -50%, ultraviolet detection wavelength: 210-360nm, the sample injection amount is 5μL。
And 4, step 4: and (3) exporting the 15 batches of oyster vein dispersion instruments obtained in the step (3) (see figure 1), and importing the oyster vein dispersion instruments into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system A. Chromatographic peaks existing in 15 batches of anti-oyster powder fingerprints are selected as common peaks. The mean calculation was used to generate a control fingerprint, see figure 2. The relative retention time, relative peak area limit range and similarity of each common peak were calculated and the results are shown in tables 5-6.
Selection of reference peaks: the calycosin glycoside is one of the indexes of content determination of the pharmacopoeia of astragalus root decoction pieces, so that the invention selects the calycosin glycoside with higher content, moderate retention time and better separation degree as a reference substance.
TABLE 5.15 batches of oyster powder sharing peak relative retention time and relative peak area limit ranges
| Number of peak | Relative retention time | Relative peak area |
| 1 | 0.556-0.561 | 0.059-0.712 |
| 2 | 0.579-0.585 | 0.062-0.508 |
| 3 | 0.788-0.792 | 0.093-0.563 |
| S | 1.000 | 1.000 |
| 5 | 1.399-1.402 | 0.062-0.635 |
| 6 | 1.463-1.468 | 0.081-1.305 |
| 7 | 1.741-1.743 | 0.202-0.530 |
| 8 | 2.001-2.003 | 0.385-1.174 |
| 9 | 2.067-2.077 | 0.102-1.469 |
| 10 | 2.147-2.149 | 0.166-0.507 |
| 11 | 3.059-3.076 | 0.06-0.823 |
| 12 | 3.143-3.152 | 0.118-1.151 |
TABLE 6.15 fingerprint similarity results for mu san batches
| Numbering | Degree of similarity | Numbering | Degree of similarity |
| S1 | 0.951 | S9 | 0.929 |
| S2 | 0.963 | S10 | 0.924 |
| S3 | 0.966 | S11 | 0.962 |
| S4 | 0.900 | S12 | 0.965 |
| S5 | 0.948 | S13 | 0.882 |
| S6 | 0.924 | S14 | 0.882 |
| S7 | 0.919 | S15 | 0.885 |
| S8 | 0.934 |
Determining technical parameters of the fingerprint spectrum: the above experimental results show that the similarity between the fingerprints of 15 batches of oyster powder and the comparison fingerprints is calculated, and the results are all greater than 0.85, so the fingerprint standard of the tentative oyster powder is as follows: the sample fingerprint is consistent with the comparison fingerprint, the similarity of the sample fingerprint and the comparison fingerprint calculated by similarity calculation software according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system is not less than 0.85, and the relative retention time and the relative peak area of each common peak in 15 batches of fingerprints are in accordance with the limit range of the table 6.
3. Oyster powder fingerprint spectrum component source analysis
Preparing the 4 medicinal materials of the prescription into single medicinal test solution according to the preparation method of the oyster powder test solution, injecting sample, analyzing by retention time, and calibrating 12 common peaks in fingerprint, wherein the peaks 4, 7, 8, 9, 10, 11, and 12 are from radix astragali, and the peaks 1, 2, 3, 5, and 6 are from radix Ephedrae, as shown in figure 3.
4. Identification of chromatographic Peak
Comparison of the control substances for the experiment confirms that the No. 4 peak in the common peaks is calycosin glycoside, the No. 7 peak is formononetin glycoside, and the No. 9 peak is calycosin. The results of the comparison of the control are shown in FIG. 4.
5. Optimization of chromatographic conditions
5.1 selection of wavelength
The invention adopts the ultraviolet detector, compares the wavelength information of 210 plus 360nm, finds that 210nm has more chromatographic peaks, larger representable information amount and better chromatographic peak separation degree and peak type, and selects 210nm as the optimal detection wavelength.
5.2 selection of the Mobile phase
The invention inspects the conditions of mobile phases such as methanol-water, acetonitrile-0.1% formic acid and the like. The results show that the acetonitrile-water system is optimal, the separation degree of each peak is good, the peak shape is sharp, and the retention time is moderate, so that the acetonitrile-water system is selected as a chromatographic condition.
5.3 preparation of test solutions
The method comprises the following steps: grinding the product into fine powder, weighing 50mg, adding 2mL methanol, ultrasonic treating for 0.5 hr, cooling, shaking, centrifuging, collecting supernatant, and adding N 2 Blow-drying, adding 200μAnd (4) re-dissolving with L methanol, centrifuging, and taking supernatant to obtain the product.
The second method comprises the following steps: grinding the product, precisely weighing about 50mg, and dissolving in 2mL of 5% methanol; the method comprises the following steps of pretreating a Cleanert S C18 solid-phase extraction column, wherein the pretreatment process comprises the following steps: activating with methanol, balancing water, loading, eluting with 2 times of column volume of water and 2 times of column volume of 0.4% formic acid methanol, collecting eluate, and collecting N 2 Blow-drying, adding 200 portions of residueμAnd (4) re-dissolving with L methanol, centrifuging, and taking supernatant to obtain the product.
According to the investigation, the interference of the polysaccharide and other high-polarity components in the test solution prepared by the first method is serious, the interference of the polysaccharide and other high-polarity components in the test solution prepared by the second method can be effectively removed, the loss of medium and small-polarity components is not caused, and the baseline is stable, so the test solution prepared by the second method is adopted.
6. Methodology validation
6.1 stability test
A sample solution (lot number 20200815) was prepared and tested as in example 1, and analyzed at 0, 2, 4, 6, 8, 10, 12, 18, and 24h, respectively, to examine the relative retention time of the main chromatographic peaks and the consistency of the relative peak areas, and the results are shown in Table 7 and FIG. 5.
TABLE 7 stability similarity
| Time/h | Degree of |
| 0 | 0.943 |
| 2 | 0.999 |
| 4 | 0.997 |
| 6 | 0.999 |
| 8 | 0.998 |
| 10 | 0.999 |
| 12 | 0.998 |
| 18 | 0.999 |
| 24 | 0.997 |
The sample solution is placed at room temperature for 24 hours, and the similarity of the fingerprint is more than 0.94, which shows that the sample solution has good stability within 24 hours.
6.2 precision test
Taking the mixed reference substance solution, preparing the test substance solution according to the method in the example 1, detecting, continuously injecting samples for 6 times, and inspecting the relative retention time of chromatographic peaks and the consistency of relative peak areas, wherein the results are shown in the table 8 and the figure 6.
TABLE 8 precision similarity
| Numbering | Degree of similarity |
| 1 | 1.000 |
| 2 | 1.000 |
| 3 | 1.000 |
| 4 | 1.000 |
| 5 | 1.000 |
| 6 | 1.000 |
The reference substance solution is continuously injected for 6 times, the similarity of the fingerprint is 1.000, and the method has good precision.
6.3 repeatability test
6 portions of the test solution (lot 20200815) were collected, and the test solution was prepared and examined as in example 1 to examine the relative retention time of the main chromatographic peak and the consistency of the relative peak area, and the results are shown in Table 9 and FIG. 7.
TABLE 9 repeatability similarity
| Numbering | Degree of similarity |
| 1 | 0.995 |
| 2 | 0.998 |
| 3 | 0.998 |
| 4 | 0.998 |
| 5 | 0.995 |
| 6 | 0.998 |
The similarity of the fingerprint spectrums of 6 test solution is more than 0.99, which shows that the method has good repeatability.
The results show that the fingerprint spectrum method of the oyster powder established by the invention has good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the oyster powder, and has important significance for ensuring the clinical curative effect.
Claims (5)
1. The method for establishing the fingerprint spectrum of the oyster powder is characterized by comprising the following steps of:
step 1: dissolving calycosin glycoside, formononetin and calycosin reference substances in methanol to obtain reference peak solution;
step 2: taking oyster powder test samples of different batches, placing the oyster powder test samples in a container, adding water for dissolution, and pretreating by using a C18 solid phase extraction column to prepare an oyster powder test sample solution;
the pretreatment process comprises the following steps: activating with methanol, balancing water, loading, eluting with 2 times of column volume of water solution and 2 times of column volume of 0.4% methanol formate, collecting eluate, and collecting N 2 Blow-drying, re-dissolving the residue with methanol, centrifuging, and collecting supernatant;
the oyster powder is prepared by the following method: taking 3 parts of calcined oyster, 3 parts of astragalus, 3 parts of ephedra root and 2.5 parts of blighted wheat, soaking the above four materials in water for 1 hour, decocting twice for 2 hours each time, adding water with the weight 8 times of that of all decoction pieces twice, filtering decoction solutions, combining filtrate, concentrating the filtrate under reduced pressure, and freeze-drying to obtain the oyster powder test sample;
and step 3: precisely absorbing the reference peak solution and the sample solution respectively, injecting into a liquid chromatograph, and collecting a chromatogram;
and 4, step 4: guiding the integral signal of the fingerprint of the oyster powder test solution obtained in the step 3 into software of a traditional Chinese medicine chromatography fingerprint similarity evaluation system; selecting chromatographic peaks existing in the chromatogram maps of the oyster powder of different batches as common peaks, generating a comparison fingerprint of the oyster powder by using an average value calculation method, and calculating the relative retention time and the relative peak area of each common peak;
in the step 3, the liquid chromatography conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and the specification of a chromatographic column is 4.6 multiplied by 250mm and 5 mu m; the column temperature is 30-40 ℃; a: water-B: taking acetonitrile as a mobile phase, performing gradient elution for 0-5 min, and performing B10%; 5-10 min, B10% -12%; 10-60 min, B12% -25%; 60-65min, B25% -27%; 65-80min, B27% -35%; 80-95min, B35% -50%, ultraviolet detection wavelength: 210 nm.
2. The method for establishing the fingerprint of oyster powder according to claim 1, wherein in step 3, 5 μ L of each of the reference peak solution and the sample solution is precisely extracted, injected into a liquid chromatograph, and a liquid chromatogram is acquired for 95 minutes.
3. The method for establishing the fingerprint of the oyster powder according to claim 1, wherein the method comprises the following steps: the fingerprint comprises 12 common peaks, wherein the peak corresponding to calycosin glycoside is the No. 4 peak, the peak corresponding to formononetin is the No. 7 peak, and the peak corresponding to calycosin is the No. 9 peak.
4. The method for establishing the fingerprint of the oyster powder according to claim 3, wherein the method comprises the following steps: taking the corresponding chromatographic peak of the reference substance peak of the calycosin glycoside as an S peak, and calculating the relative retention time of each common peak to be in accordance with the following steps: 0.556-0.561 part of peak No. 1, 0.579-0.585 part of peak No. 2, 0.788-0.792 part of peak No. 3, 1.000 part of peak No. 4, 1.399-1.402 part of peak No. 5, 1.463-1.468 part of peak No. 6, 1.741-1.743 part of peak No. 7, 2.001-2.003 part of peak No. 8, 2.067-2.077 part of peak No. 9, 2.147-2.149 parts of peak No. 10, 3.059-3.076 parts of peak No. 11, and 3.143-3.152 part of peak No. 12.
5. The method for establishing the fingerprint of the oyster powder according to claim 4, wherein the method comprises the following steps: the relative peak area of each common peak and the S peak is in accordance with: 0.059 to 0.712 of peak No. 1, 0.062 to 0.508 of peak No. 2, 0.093 to 0.563 of peak No. 3, 1.000 of peak No. 4, 0.062 to 0.635 of peak No. 5, 0.081 to 1.305 of peak No. 6, 0.202 to 0.530 of peak No. 7, 0.385 to 1.174 of peak No. 8, 0.102 to 1.469 of peak No. 9, 0.166 to 0.507 of peak No. 10, 0.06 to 0.823 of peak No. 11, and 0.118 to 1.151 of peak No. 12.
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