CN108760936B - Detection method of GC fingerprint of mass-eliminating pill preparation - Google Patents
Detection method of GC fingerprint of mass-eliminating pill preparation Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a method for detecting GC fingerprint of a mass-eliminating pill preparation, which comprises the steps of preparing a test solution; preparing a reference substance solution; measuring with gas chromatography, calculating the relative retention time of common peak with reference peak retention time of 1, and obtaining GC fingerprint or GC characteristic spectrum of the pill. The method establishes 8 characteristic peaks of volatile components in the pill, selects alpha-cyperone as reference peak, and determines relative retention time of each characteristic peak. The GC fingerprint detection method for volatile components in the mass-eliminating pill preparation provided by the invention has high sensitivity and precision, and good stability and repeatability, and compared with the existing quality standard, the method can control the quality of the volatile components in the mass-eliminating pill preparation more objectively, comprehensively and accurately, and has important significance for ensuring clinical efficacy.
Description
Technical Field
The invention relates to a method for establishing a fingerprint of a traditional Chinese medicine, in particular to a method for detecting a GC fingerprint of a mass-eliminating pill preparation.
Background
The mass eliminating pill is prepared by adding and reducing bupleurum root liver soothing powder in Ming dynasty Zhang Geng Bing from old traditional Chinese medicine Zhao Jun Xue student of medical institute in south Henan China, according to the pathogenesis of hyperplasia of mammary glands, and has definite curative effect on hyperplasia of mammary glands after being clinically applied for decades. The formula consists of eleven medicinal materials of radix bupleuri, rhizoma cyperi, rheum officinale, pericarpium citri reticulatae viride, ligusticum wallichii, angelica sinensis, radix paeoniae alba, curcuma zedoary, ground beeltle, thunberg fritillary bulb and cowherb seed, and has the effects of soothing liver, promoting qi circulation, activating blood circulation, reducing phlegm, softening hardness and dissipating stagnation. Nowadays, the 'mass-eliminating pill' is taken as a unique product circulation market of the limited company of the leiyu pharmaceutical industry group, and brings good news to the majority of patients with hyperplasia of mammary glands.
The existing quality standard of the mass-eliminating pill is the standard of the State food and drug administration, and the standard number is as follows: YBZ00932010, identification item for thin-layer identification of 6 Chinese medicinal materials including bupleuri radix, rhizoma Cyperi, radix et rhizoma Rhei, pericarpium Citri Reticulatae viride, Bulbus Fritillariae Thunbergii, and radix Paeoniae alba, and content determination item for HPLC content control of 5 emodin components including aloe-emodin, rhein, emodin, chrysophanol, and physcion. The method only measures the content of one medicinal material, and is not enough to control the quality of the compound preparation.
Comparison document 1: a fingerprint detection method (patent number: 201410126546.1) of a pill preparation for treating disease, discloses an HPLC fingerprint detection method of the pill preparation for treating disease, but the HPLC fingerprint of the pill preparation for treating disease obtained by the method is mainly a fingerprint of a non-volatile component, the characteristic peaks of the fingerprint are mainly non-volatile components of paeoniflorin, ferulic acid and emodin, and because monarch drug-rhizoma cyperi, minister drug-pericarpium citri reticulatae viride, ligusticum wallichii, adjuvant drug-angelica, rhizoma zedoariae and the like of the prescription all contain volatile pharmacological active components such as alpha-cyperone, ligustilide and the like, the existing HPLC fingerprint can not carry out comprehensive quality control on various volatile components in the pill for treating disease, and the detection method of GC fingerprint of the pill preparation for treating disease is not reported at present.
Therefore, in order to make up for the above disadvantages, more comprehensively and effectively control the quality of the mass-eliminating pill preparation, improve the quality control level, and ensure the safety and curative effect of clinical administration, it is necessary to establish a method for detecting the GC fingerprint of the volatile components of the mass-eliminating pill preparation based on the prior art.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a method for detecting a GC fingerprint of an anti-mass pill preparation, aims to overcome the defects in the prior art, and provides a fingerprint detection method which is high in precision, good in repeatability and stability, capable of objectively, comprehensively and accurately evaluating volatile components in the anti-mass pill preparation, capable of more effectively ensuring the quality of a finished product and having an important effect on controlling the quality of the anti-mass pill preparation.
The technical scheme is as follows: in order to achieve the purpose, the invention provides a detection method of GC fingerprint of an anti-abdominal mass pill preparation, which comprises the following steps:
(1) preparation of a test solution: taking the content of the pill, extracting volatile oil by adding ethyl acetate from the upper end of a volatile oil extractor according to volatile oil determination method, and collecting ethyl acetate layer after extraction is completed;
(2) preparation of control solutions: respectively taking one or more of alpha-cyperone, D-limonene, zedoary turmeric ketone, ligustilide, butenyl phthalide and ethyl oleate reference substances, precisely weighing, and adding ethyl acetate to respectively prepare reference substance solutions containing 0.1-0.5 mg per 1ml to obtain the final product;
(3) precisely absorbing 1-10 mu l of each of the reference solution and the sample solution, measuring by using a gas chromatograph, collecting a chromatogram, calculating the relative retention time of each characteristic peak by taking the relative retention time of the reference peak as 1, and generating a GC fingerprint or a GC characteristic spectrum.
Preferably, the preparation scheme of the test solution in the step (1) is as follows: placing the contents of the pill in a glass mortar, adding a small amount of water, soaking overnight, grinding, transferring to a round bottom flask with water, extracting volatile oil by adding ethyl acetate from the upper end of a volatile oil extractor, collecting ethyl acetate layer, washing volatile oil analyzer with ethyl acetate, and mixing ethyl acetate solutions.
Preferably, the gas chromatography conditions are as follows: the chromatographic column is a capillary column; adopting a non-flow-splitting or flow-splitting sample introduction mode, wherein the flow rate of the column is 1.0-2.0 ml/min; the column temperature is programmed temperature rise;
the gas chromatography temperature programming mode in the step (3) is as follows: the initial temperature is 35-55 ℃, the temperature is kept for 1-10 min, the temperature is raised to 140-150 ℃ at the speed of 1-10 ℃/min, the temperature is kept for 10-20 min, the temperature is raised to 180-200 ℃ at the speed of 20-40 ℃/min, the temperature is kept for 1-25 min, the temperature is raised to 200-300 ℃ at the speed of 10-40 ℃/min, and the temperature is kept for 1-15 min;
more preferably, the gas chromatography temperature programming mode in the step (3) is as follows: the initial temperature is 45 ℃, the temperature is kept for 5min, the temperature is increased to 150 ℃ at the speed of 5 ℃/min, the temperature is kept for 16min, the temperature is increased to 190 ℃ at the speed of 30 ℃/min, the temperature is kept for 21min, the temperature is increased to 260 ℃ at the speed of 20 ℃/min, and the temperature is kept for 13 min.
More preferably, the gas chromatography temperature programming method in step (3) may further include: the initial temperature is 45 ℃, the temperature is kept for 5min, the temperature is increased to 150 ℃ at the speed of 5 ℃/min, the temperature is kept for 16min, the temperature is increased to 190 ℃ at the speed of 30 ℃/min, the temperature is kept for 5min, the temperature is increased to 260 ℃ at the speed of 30 ℃/min, and the temperature is kept for 5 min.
Preferably, the specifications of the chromatographic column are as follows: a capillary column using crosslinked 5% phenylmethylpolysiloxane as a stationary phase has a column length of 30m, an inner diameter of 0.25mm, and a membrane thickness of 0.25 μm.
Preferably, the gas chromatograph is a FID detector or a mass spectrometer, the FID detector temperature is 280 ℃, and the mass spectrometer conditions are as follows: ion source temperature 230 ℃, transmission line temperature 250 ℃, EI: 70ev, and a mass range of 30-500 amu.
Preferably, the GC characteristic map of the mass-eliminating pill preparation is that the reference peak is alpha-cyperone. The theoretical plate number is not less than 100000 calculated by alpha-cyperone.
The GC fingerprint similarity of the mass eliminating pill preparation obtained by the method is more than 0.9, and 13 common peaks are calibrated. The 13 common peaks define the medicinal flavor attribution of 8 peaks, which are No. 3 peaks from green tangerine peel, No. 4, 5, 8 and 10 peaks from nutgrass galingale rhizome, No. 6 peaks from zedoary, No. 7 peaks and No. 9 peaks from Chinese angelica, No. 9 peaks from szechuan lovage rhizome, and No. 11 peaks from Chinese insect wax (auxiliary material). The chemical structures of 11 peaks are determined in 13 common peaks, wherein the peak 3 is D-limonene, the peak 6 is zedoarone, the peak 9 is Z-ligustilide, the peak 10 is alpha-cyperone, and the peak 13 is ethyl oleate through comparison of quality spectra. The GC control fingerprint of the mass-eliminating pill preparation is shown in figure 1, and the GC fingerprint of 11 samples is shown in figure 2.
The GC characteristic spectrum of the mass-eliminating pill preparation obtained by the method shows 8 characteristic peaks in total, wherein the peak 1 is D-limonene, the peak 4 is zedoary ketone, the peak 5 is butenyl phthalide, the peak 7 is Z-ligustilide, and the peak 8 is alpha-cyperone. Wherein the peak from pericarpium Citri Reticulatae viride is No. 1, the peak from rhizoma Cyperi is No. 2, 3, 6, 8, the peak from Curcumae rhizoma is No. 4, the peak from radix Angelicae sinensis is No. 5 and No. 7, and the peak from rhizoma Ligustici Chuanxiong is No. 7. The GC profile is shown in FIG. 3.
Preferably, the GC characteristic spectrum of the mass-eliminating pill preparation is that when the relative retention time of the chromatographic peak of alpha-cyperone (peak No. 8) is 1, each characteristic peak is:
peak No. 1: relative retention time of 0.30-0.36,
Peak No. 2: relative retention time of 0.55-0.68,
Peak No. 3: relative retention time of 0.61-0.75,
Peak No. 4: relative retention time of 0.71-0.86,
Peak No. 5: relative retention time of 0.78-0.96
Peak No. 6: relative retention time of 0.81-0.98
Peak No. 7: relative retention time of 0.88-0.99
Peak No. 8: relative retention time 1.000
The relative deviation of each characteristic peak to the retention time is +/-5% -10%.
More preferably, the GC characteristic spectrum of the mass-eliminating pill preparation is obtained by taking the relative retention time of the chromatographic peak (peak 8) of alpha-cyperone as 1, and the characteristic peaks are as follows:
peak No. 1: relative retention time of 0.33,
Peak No. 2: the relative retention time is 0.62,
Peak No. 3: the relative retention time is 0.68,
Peak No. 4: relative retention time of 0.79,
Peak No. 5: the relative retention time is 0.87,
Peak No. 6: the relative retention time is 0.91,
Peak No. 7: the relative retention time is 0.98,
Peak No. 8: relative retention time 1.000
The relative deviation of each characteristic peak from retention time was ± 5%.
Compared with the prior art, the detection method of the GC fingerprint spectrum of the mass-eliminating pill preparation has the following advantages:
(1) the detection method of the GC fingerprint spectrum of the mass-eliminating pill preparation can comprehensively reflect the quality level of volatile components in the mass-eliminating pill preparation and fill up the blank of the existing research.
(2) The precision test, stability test and repeatability test results of the detection method of the GC fingerprint of the mass-eliminating pill preparation show that the method is high in accuracy, stability and repeatability.
(3) The detection method of the GC fingerprint of the mass-eliminating pill preparation can be used for qualitative and quantitative determination of volatile components in the mass-eliminating pills, can be suitable for monitoring the quality of raw medicinal materials and intermediate semi-finished products of the mass-eliminating pills and the quality control of a production process, has important significance for ensuring the quality of the mass-eliminating pills, and has wide application range.
Drawings
FIG. 1 shows GC-based fingerprint of Xiaozheng pill.
FIG. 2 shows GC fingerprints of 11 batches of Xiaozheng pill samples.
FIG. 3 is a GC fingerprint of the mass eliminating pill.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
Example 1: detection method of GC fingerprint of mass-eliminating pill preparation
1. Instrument and reagent
1.1 instruments
GC-MS (Agilent 7000B gas chromatograph-mass spectrometer, MZ016-00E autosampler, triple quadrupole mass analyzer); HP-5MS capillary column (30m m X0.25 mm X0.25 μm); electronic balance of model Mettler Toledo XSE205, Switzerland (one in ten thousandth); electronic balance model BS21S, Sartorius germany (one hundred thousand); electronic balance model Mettler Toledo XPR2 switzerland (parts per million); merk Millipore ultrapure water preparation instrument; high speed universal mills (Tester instruments, Inc. of Tianjin); a temperature-regulating electric heater (manufactured by Shenzhou electric heating factory of Nantong city), a volatile oil detector and a reflux condenser tube device;
1.2 reagent
Comparison products: zedoary turmeric ketone (Shanghai Yuan leaf Biotechnology Co., Ltd., lot number: B30751, purity ≧ 98%), ligustilide (China food and drug testing institute, lot number: 111737-201406 for identification), alpha-cyperone (China food and drug testing institute, lot number: 110748-201714, purity: 99.4%). Other reference substances are prepared by laboratories (purity is ≧ 98%).
Volatile oil of reference medicinal materials: volatile oil of rhizoma Cyperi, pericarpium Citri Reticulatae viride, rhizoma Ligustici Chuanxiong, Curcumae rhizoma, and radix Angelicae sinensis as reference materials is provided by Lei's pharmaceutical Limited company.
Negative sample volatile oil: volatile oil of rhizoma Curcumae, radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, rhizoma Cyperi, pericarpium Citri Reticulatae viride and radix Angelicae sinensis and rhizoma Ligustici Chuanxiong double-yin sample is provided by Leying pharmaceutical industry group, Inc.
Xiaozhen Wan preparation: the total 11 batches are provided by lei ying pharmaceutical industry group ltd, and the national medicine standard Z20100057 is pills, and each pill weighs 0.2 g. The batch numbers are as follows:
TABLE 1 Xiaozheng pill sample information Table (n ═ 11)
| Numbering | Unique number | Batch number | Date of |
| 1 | JS2017YT0033 | OB38009 | 2015.06.04 |
| 2 | JS2017YT0034 | OB38010 | 2015.09.22 |
| 3 | JS2017YT0035 | PB38012 | 2016.12.12 |
| 4 | JS2017YT0085 | QB38001 | 2017.03.06 |
| 5 | JS2017YT0086 | QB38002 | 2017.03.20 |
| 6 | JS2017YT0087 | PB38004 | 2016.05.11 |
| 7 | JS2017YT0088 | PB38008 | 2016.06.30 |
| 8 | JS2017YT0191 | PB38001 | 2016.01.06 |
| 9 | JS2017YT0192 | PB38009 | 2016.07.19 |
| 10 | JS2017YT0193 | QB38004 | 2017.06.29 |
| 11 | JS2017YT0194 | QB38005 | 2017.08.04 |
2. Method and results
2.1 chromatographic conditions
Gas chromatography conditions: HP-5MS capillary column (30 m.times.0.25 mm.times.0.25 μm); FID detector temperature 280 ℃. The sample was injected without split flow, and the column flow rate was 1.5 ml/min. Temperature programming: the initial temperature is 45 ℃, the temperature is kept for 5min, the temperature is increased to 150 ℃ at the speed of 5 ℃/min, the temperature is kept for 16min, the temperature is increased to 190 ℃ at the speed of 30 ℃/min, the temperature is kept for 21min, the temperature is increased to 260 ℃ at the speed of 20 ℃/min, and the temperature is kept for 13 min.
Mass spectrum conditions: ion source temperature 230 ℃, transmission line temperature 250 ℃, EI: 70ev, and a mass range of 30-500 amu.
2.2 preparation of test solution: the content of the mass-eliminating pill preparation, 4.0g, is placed in a glass mortar, a small amount of water is added, the mixture is soaked overnight, and after being properly ground, the mixture is transferred into a round bottom flask by times with the total amount of water of about 400 ml. According to a volatile oil determination method A (2204 of the general rule of the four parts of the Chinese pharmacopoeia 2015 edition), 2ml of ethyl acetate is added from the upper end of an extractor for volatile oil extraction, an ethyl acetate layer is taken after complete extraction, the volatile oil determination device is washed by ethyl acetate for a proper amount of times, ethyl acetate liquid is combined, anhydrous sodium sulfate is added into a 10ml measuring flask, ethyl acetate is added to the scale, and the mixture is shaken up, so that the volatile oil is obtained.
2.3 preparation of control solutions: weighing appropriate amount of alpha-cyperone, zedoarone, ligustilide, D-limonene, butenyl phthalide, and ethyl oleate, and adding ethyl acetate to obtain solution containing 0.1mg per 1 ml.
Preparation of reference drug solution: taking 1mL of volatile oil samples of pericarpium Citri Reticulatae viride, rhizoma Cyperi, Curcumae rhizoma, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong respectively, diluting with ethyl acetate, and diluting to 100mL to obtain corresponding reference medicinal solution.
Preparation of negative sample solution: taking 1mL of each of the volatile oil samples of the zedoary, the angelica, the ligusticum wallichii, the rhizoma cyperi and the pericarpium citri reticulatae viride, and the double negative volatile oil samples of the angelica and the ligusticum wallichii, adding ethyl acetate for dilution, and fixing the volume to 100mL to prepare corresponding negative solutions.
2.4GC fingerprint establishment
Preparing 11 batches of mass-eliminating pill samples into test solution, precisely absorbing 1 mu l of each of the reference solution and 11 batches of test solution respectively, measuring by using a gas chromatograph, collecting a chromatogram, converting a data file into an AIA file, introducing the AIA file into a Chinese medicinal chromatographic fingerprint similarity evaluation system (2012.130723), matching 11 pictures according to Mark peaks to generate a reference fingerprint (S1), and cutting a solvent peak 5min before cutting, wherein the reference fingerprint is shown in figure 1. As a result: a total of 13 peaks were normalized.
2.4.1 fingerprint spectrum 13 common peaks of medicinal odor attribution
Respectively sampling the test solution of the mass eliminating pill, the negative volatile oil solution and the positive volatile oil solution of 5 medicines, and performing spectrogram comparison to determine the attribution of 8 peaks of medicines, wherein the 8 peaks are from 5 medicines of pericarpium Citri Reticulatae viride, rhizoma Cyperi, Curcumae rhizoma, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong, and the No. 11 peak is attributed to the adjuvant Chinese insect white wax, and the rest peaks are not attributed. The common peak herb flavor assignments are shown in Table 2.
TABLE 2 results of the common peak assignment of volatile components in Xiaozheng pills
| Numbering | Peak position (average RRT) | Attribution of herb and |
| 1 | 0.152 | — |
| 2 | 0.165 | — |
| 3 | 0.322 | |
| 4 | 0.599 | |
| 5 | 0.659 | |
| 6 | 0.773 | |
| 7 | 0.864 | |
| 8 | 0.902 | Rhizoma Cyperi |
| 9 | 0.973 | Chinese angelica root, Ligusticum wallichii |
| 10(S) | 1.000 | |
| 11 | 1.213 | Chinese insect white wax (auxiliary material) |
| 12 | 1.462 | — |
| 13 | 1.474 | — |
2.4.2 fingerprint identification of 13 common Peak Compounds
Injecting 1 μ l of each of the reference solution and the sample solution into a GC-MS combination instrument, and determining the structures of 11 peaks in 13 common peaks by NIST library retrieval and comparison of the reference quality spectrogram of the obtained primary mass spectrogram, which is shown in Table 3.
TABLE 3 information of compounds with common peaks in fingerprint spectrum of Xiaozheng pills
Note: "RRT" means relative retention time; "+" indicates compounds confirmed by comparison with control
2.4.3 fingerprint 13 common Peak-to-Peak Signal-to-noise Studies
The signal-to-noise ratio calculation is carried out on the 13 common peaks to examine the reproducibility of the positive detection result of the common peaks so as to determine the correctness of the selection of the common peaks. The data show that the signal-to-noise ratios of 13 common peaks in the characteristic spectrum of 11 batches of the Xiaozheng pills are all larger than 10, which indicates that the 13 peaks can ensure the reproducibility of the detection result as the common peaks of the fingerprint spectrum. See table 4.
TABLE 411 Mass eliminating pill fingerprint chromatogram common peak signal-to-noise ratio
2.4.4 fingerprint precision test
Taking the same test sample solution (batch number PB38001), precisely sucking 1 μ L according to chromatographic conditions, continuously injecting sample for 6 times, and determining. The relative retention time of 13 peaks is calculated by taking the No. 10 peak (alpha-cyperone) as a reference peak, and the RSD is less than 0.41 percent, which indicates that the precision of the instrument is good. See table 5.
Table 513 peak relative retention time precision test results (n ═ 6)
| |
1 | 2 | 3 | 4 | 5 | 6 | AVE | RSD/％ |
| 1 | 0.152 | 0.152 | 0.152 | 0.151 | 0.151 | 0.151 | 0.152 | 0.41 |
| 2 | 0.165 | 0.165 | 0.165 | 0.164 | 0.164 | 0.164 | 0.164 | 0.34 |
| 3 | 0.322 | 0.322 | 0.322 | 0.321 | 0.321 | 0.321 | 0.321 | 0.08 |
| 4 | 0.599 | 0.599 | 0.599 | 0.599 | 0.599 | 0.599 | 0.599 | 0.02 |
| 5 | 0.659 | 0.659 | 0.659 | 0.659 | 0.659 | 0.659 | 0.659 | 0.01 |
| 6 | 0.773 | 0.773 | 0.773 | 0.773 | 0.773 | 0.773 | 0.773 | 0.02 |
| 7 | 0.864 | 0.864 | 0.864 | 0.864 | 0.864 | 0.864 | 0.864 | 0.01 |
| 8 | 0.902 | 0.903 | 0.902 | 0.902 | 0.902 | 0.902 | 0.902 | 0.02 |
| 9 | 0.973 | 0.973 | 0.973 | 0.973 | 0.973 | 0.973 | 0.973 | 0.02 |
| 10(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 11 | 1.213 | 1.212 | 1.213 | 1.212 | 1.213 | 1.213 | 1.213 | 0.02 |
| 12 | 1.462 | 1.462 | 1.462 | 1.462 | 1.462 | 1.462 | 1.462 | 0.02 |
| 13 | 1.474 | 1.473 | 1.473 | 1.474 | 1.474 | 1.474 | 1.474 | 0.03 |
2.4.5 fingerprint stability test
Precisely sucking 1 μ L of the same sample solution (batch number: PB38001), and measuring according to chromatographic conditions at 0,2,4,6,10,14,18,24, and 30h after preparation. The relative retention time of 13 peaks is calculated by taking the peak No. 10 (alpha-cyperone) as a reference peak, and the RSD is less than 0.16 percent, which indicates that the sample has good stability within 30 hours. See table 6.
Table 613 peak relative retention time stability test results (n ═ 9)
2.4.6 fingerprint repeatability test:
6 portions of the same batch of samples (batch number: PB38001) were taken, and a test solution was prepared by the method, and 1. mu.L of the solution was precisely aspirated for measurement. The relative retention time of 13 peaks is calculated by taking the peak 10 (alpha-cyperone) as a reference peak, and the RSD is less than 0.16 percent, which indicates that the method has good repeatability. See table 7.
TABLE 713 Peak relative Retention time repeatability test results (n ═ 6)
| |
1 | 2 | 3 | 4 | 5 | 6 | AVE | RSD/％ |
| 1 | 0.152 | 0.152 | 0.152 | 0.152 | 0.152 | 0.153 | 0.152 | 0.16 |
| 2 | 0.165 | 0.165 | 0.165 | 0.165 | 0.165 | 0.165 | 0.165 | 0.16 |
| 3 | 0.322 | 0.321 | 0.321 | 0.321 | 0.321 | 0.321 | 0.321 | 0.05 |
| 4 | 0.599 | 0.599 | 0.599 | 0.599 | 0.599 | 0.599 | 0.599 | 0.01 |
| 5 | 0.659 | 0.659 | 0.659 | 0.659 | 0.659 | 0.659 | 0.659 | 0.02 |
| 6 | 0.773 | 0.773 | 0.773 | 0.773 | 0.773 | 0.773 | 0.773 | 0.01 |
| 7 | 0.864 | 0.864 | 0.864 | 0.864 | 0.864 | 0.864 | 0.864 | 0.01 |
| 8 | 0.902 | 0.902 | 0.902 | 0.902 | 0.902 | 0.902 | 0.902 | 0.02 |
| 9 | 0.973 | 0.973 | 0.973 | 0.973 | 0.973 | 0.973 | 0.973 | 0.03 |
| 10(S) | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.00 |
| 11 | 1.213 | 1.213 | 1.213 | 1.213 | 1.212 | 1.213 | 1.213 | 0.02 |
| 12 | 1.462 | 1.463 | 1.462 | 1.463 | 1.462 | 1.463 | 1.462 | 0.04 |
| 13 | 1.474 | 1.475 | 1.474 | 1.475 | 1.474 | 1.474 | 1.474 | 0.04 |
2.4.7 fingerprint sample detection and similarity result
Taking 11 batches of samples, preparing a sample solution according to a method, and injecting a sample to obtain the fingerprint of each batch of samples. Through a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.130723), and with reference to the reference fingerprint, similarity evaluation is carried out on 11 batches of the mass-eliminating pill samples. The similarity of 11 batches of the mass-eliminating pills is 0.903-0.994, the average is 0.953, and the RSD is 3.3% (n is 11). The fingerprint control of the product is feasible. See table 8 and figure 2.
TABLE 811 batch Bolus sample similarity results (n ═ 11)
| Numbering | Unique number | Batch number | Degree of |
| 1 | JS2017YT0033 | OB38009 | 0.994 |
| 2 | JS2017YT0034 | OB38010 | 0.922 |
| 3 | JS2017YT0035 | PB38012 | 0.943 |
| 4 | JS2017YT0085 | QB38001 | 0.962 |
| 5 | JS2017YT0086 | QB38002 | 0.980 |
| 6 | JS2017YT0087 | PB38004 | 0.972 |
| 7 | JS2017YT0088 | PB38008 | 0.973 |
| 8 | JS2017YT0191 | PB38001 | 0.988 |
| 9 | JS2017YT0192 | PB38009 | 0.903 |
| 10 | JS2017YT0193 | QB38004 | 0.936 |
| 11 | JS2017YT0194 | QB38005 | 0.913 |
| Average | 0.953 | ||
| RSD(n＝11) | 3.3％ |
Example 2 detection method of GC characteristic spectrum of Xiaozheng pill preparation
1. Instrument and reagent
1.1 instruments
The same as in example 1.
1.2 reagent
Comparison products: zedoary turmeric ketone (Shanghai Yuan leaf Biotech Co., Ltd., batch No. B30751, purity ≧ 98%)
Xiaozhen Wan preparation: in total 11 batches, as in example 1.
2. Method and results
2.1 chromatographic conditions
Gas chromatography conditions: HP-5MS capillary column (30 m.times.0.25 mm.times.0.25 μm); FID detector temperature 280 ℃. The sample was injected without split flow, and the column flow rate was 1.5 ml/min. Temperature programming: the initial temperature is 45 ℃, the temperature is kept for 5min, the temperature is increased to 150 ℃ at the speed of 5 ℃/min, the temperature is kept for 16min, the temperature is increased to 190 ℃ at the speed of 30 ℃/min, the temperature is kept for 5min, the temperature is increased to 260 ℃ at the speed of 30 ℃/min, and the temperature is kept for 5 min.
Mass spectrum conditions: ion source temperature 230 ℃, transmission line temperature 250 ℃, EI: 70ev, and a mass range of 30-500 amu.
2.2 preparation of test solution: the same as in example 1.
2.3 preparation of control solutions: collecting alpha-cyperone, adding ethyl acetate to obtain a solution containing about 0.1mg per 1 ml.
2.4 establishment of GC feature map:
2.4.1 characteristic peaks of characteristic spectra of compound information and medicinal odor attribution
Precisely sucking 1 μ l of each of the sample solution and the reference solution, and injecting into a gas chromatograph. And calculating the relative retention time of each characteristic peak by taking the relative retention time of the reference peak alpha-cyperone as 1, and establishing a characteristic map. As shown in fig. 3. And (3) screening characteristic peaks on the basis of the control fingerprint obtained in the step 2.2.3: as the peaks 1, 2, 12 and 13 in the table 1 are not assigned with the medicinal flavor, the peaks 1 and 2 in the table 2 are not identified by the compound, and the peak 11 is assigned with the auxiliary material insect white wax, the identification significance of the medicinal flavor of the prescription is not provided. Therefore, the peaks 1, 2, 11, 12 and 13 in the fingerprint are not taken as the characteristic peaks in the characteristic map, finally 8 peaks in total of No. 3 to No. 10 peaks are determined as the characteristic peaks in the volatile component characteristic map of the mass-eliminating pill, the peak numbers of the 8 characteristic peaks in the map are numbered again, and the corresponding relation between the peak numbers and the common peak-peak number of the fingerprint is shown in Table 9. The herb attribution and compound identification of the characteristic peaks in the characteristic spectrum are the same as the fingerprint spectrum, and the information is shown in Table 9.
TABLE 9 characteristic peaks of characteristic spectra of compound information and medicinal odor attribution
2.4.2 validation and establishment of characteristic Peak relative Retention time
Taking the 11 batches of sample solutions, respectively carrying out sample injection on a SHIMADZU GC-2010 gas chromatograph and an Agilent 7890A/7000B triple quadrupole gas chromatograph-mass spectrometer, and calculating the Relative Retention Time (RRT) of 8 characteristic peaks. According to the results of the durability tests of 11 samples and different brands of instruments, the average relative retention time of each characteristic peak was finally 0.329 (peak 1), 0.616 (peak 2), 0.678 (peak 3), 0.786 (peak 4), 0.872 (peak 5), 0.907 (peak 6), 0.975 (peak 7), 1.000 (peak 8, S), and the relative deviation of each characteristic peak on the two instruments was found to be 5% at maximum, so that the drift window was set at ± 5%. See tables 10-13.
TABLE 1011 RRT of each characteristic peak in batches (n ═ 11, Shimadzu/HP-5 MS column)
TABLE 1111 relative deviation/% (n 11, Shimadzu/HP-5 MS column) of the individual characteristic peaks RRT of the samples
TABLE 12 RRT and relative deviation of characteristic peaks on different brands of instruments
TABLE 13 determination of the respective characteristic peaks RRT
2.4.3 determination of the number of theoretical plates of the reference Peak
The theoretical plate number of alpha-cyperone in each batch of samples was calculated using alpha-cyperone as a reference peak, see table 14. As a result: the lowest theoretical plate number in the 11 samples was 661323, and the theoretical plate number in the standard was set as: the number of theoretical plates should not be less than 100000 calculated as alpha-cyperone peak.
TABLE 14 theoretical plate number of reference peak alpha-cyperone in batches
| Unique number | Shimadzu GC-2010PLUS instrument/HP-5 MS column |
| JS2017YT0033 | 710479 |
| JS2017YT0034 | 760327 |
| JS2017YT0035 | 681331 |
| JS2017YT0085 | 679279 |
| JS2017YT0086 | 754646 |
| JS2017YT0087 | 739798 |
| JS2017YT0088 | 748611 |
| JS2017YT0191 | 661323 |
| JS2017YT0192 | 744766 |
| JS2017YT0193 | 691824 |
| JS2017YT0194 | 767060 |
2.4.4 sample testing of feature profiles
See fingerprint spectrum, and 8 characteristic peaks are detected in 11 batches of samples.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. The detection method of the GC fingerprint spectrum or GC characteristic spectrum of the mass-eliminating pill preparation is characterized by comprising the following steps:
(1) preparation of a test solution: taking the content of the pill, extracting volatile oil by adding ethyl acetate from the upper end of a volatile oil extractor according to volatile oil determination method, and collecting ethyl acetate layer after extraction is completed;
(2) preparation of control solutions: precisely weighing one or more of alpha-cyperone, D-limonene, zedoary turmeric ketone, ligustilide, butenyl phthalide and ethyl oleate reference substances, and adding ethyl acetate to prepare reference substance solutions containing 0.1-0.5 mg per 1ml respectively;
(3) precisely absorbing 1-10 mu l of each of the reference solution and the sample solution, measuring by using a gas chromatograph, collecting a chromatogram, calculating the relative retention time of each characteristic peak by taking the relative retention time of the reference peak as 1, and generating a GC fingerprint or a GC characteristic spectrum;
the gas chromatography conditions were: the specification of the chromatographic column is as follows: a capillary column taking crosslinked 5% phenyl methyl polysiloxane as a stationary phase, wherein the length of the column is 30m, the inner diameter of the column is 0.25mm, and the thickness of the membrane is 0.25 mu m; adopting a non-flow-splitting or flow-splitting sample introduction mode, wherein the flow rate of the column is 1.0-2.0 ml/min;
the gas chromatograph in the step (3) is characterized in that a detector is an FID detector or a mass spectrometer, the temperature of the FID detector is 280 ℃, and the conditions of the mass spectrometer are as follows: ion source temperature 230 ℃, transmission line temperature 250 ℃, EI: 70ev, mass range 30-500 amu;
the GC fingerprint gas chromatography temperature programming mode in the step (3) is as follows: the initial temperature is 45 ℃, the temperature is kept for 5min, the temperature is increased to 150 ℃ at the speed of 5 ℃/min, the temperature is kept for 16min, the temperature is increased to 190 ℃ at the speed of 30 ℃/min, the temperature is kept for 21min, the temperature is increased to 260 ℃ at the speed of 20 ℃/min, and the temperature is kept for 13 min;
the chromatogram temperature programming mode of the GC characteristic spectrum is as follows: the initial temperature is 45 ℃, the temperature is kept for 5min, the temperature is increased to 150 ℃ at the speed of 5 ℃/min, the temperature is kept for 16min, the temperature is increased to 190 ℃ at the speed of 30 ℃/min, the temperature is kept for 5min, the temperature is increased to 260 ℃ at the speed of 30 ℃/min, and the temperature is kept for 5 min.
2. The method for detecting the GC fingerprint or GC characteristic of an Xiaozheng pill preparation according to claim 1, wherein the reference peak in the step (3) is α -cyperone, and the theoretical plate number is not less than 100000 calculated by α -cyperone.
3. The method for detecting the GC fingerprint or the GC characteristic spectrum of the mass-eliminating pill according to claim 1, wherein the GC fingerprint in the step (3) is evaluated by a traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012.130723, the similarity is greater than 0.9, and 13 common peaks are calibrated.
4. The method for detecting the GC fingerprint or GC characteristic spectrum of the mass-eliminating pill preparation according to claim 1, wherein the GC characteristic spectrum in the step (3) shows 8 characteristic peaks in total, wherein the peak 1 is D-limonene, the peak 4 is zedoary turmeric ketone, the peak 5 is butenyl phthalide, the peak 7 is Z-ligustilide, and the peak 8 is alpha-cyperone; wherein the peak from pericarpium Citri Reticulatae viride is No. 1, the peak from rhizoma Cyperi is No. 2, 3, 6, 8, the peak from Curcumae rhizoma is No. 4, the peak from radix Angelicae sinensis is No. 5 and No. 7, and the peak from rhizoma Ligustici Chuanxiong is No. 7.
5. The method for detecting the GC fingerprint or GC characteristic spectrum of the mass-eliminating pill preparation according to claim 4, wherein the GC characteristic spectrum in the step (3) is characterized in that the relative retention time of the chromatographic peak of No. 8 peak alpha-cyperolone is 1, and each characteristic peak is as follows:
peak No. 1: relative retention time of 0.30-0.36,
Peak No. 2: relative retention time of 0.55-0.68,
Peak No. 3: relative retention time of 0.61-0.75,
Peak No. 4: relative retention time of 0.71-0.86,
Peak No. 5: relative retention time of 0.78-0.96%,
Peak No. 6: relative retention time of 0.81-0.98,
Peak No. 7: relative retention time of 0.88 to 0.99,
Peak No. 8: relative retention time 1.000.
6. The method for detecting the GC fingerprint or GC characteristic spectrum of the mass-eliminating pill preparation according to claim 5, wherein the GC characteristic spectrum is characterized in that the relative retention time of the chromatographic peak of No. 8 peak alpha-cyperolone is 1, and each characteristic peak is as follows:
peak No. 1: relative retention time of 0.33,
Peak No. 2: the relative retention time is 0.62,
Peak No. 3: the relative retention time is 0.68,
Peak No. 4: relative retention time of 0.79,
Peak No. 5: the relative retention time is 0.87,
Peak No. 6: the relative retention time is 0.91,
Peak No. 7: the relative retention time is 0.98,
Peak No. 8: relative retention time of 1.000,
The relative deviation of each characteristic peak from retention time was ± 5%.
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