CN107748217B - A kind of method for building up of Pisces particle GC quantitative finger print atlas and its application - Google Patents

Abstract

The invention discloses a kind of method for building up of Pisces particle GC finger-print, include: to prepare test solution: taking Pisces particle, it is finely ground, Pisces particle powder is placed in flask, using water and n-hexane as Extraction solvent, is extracted using volatile oil extractor, divide and takes n-hexane layer, filtering, takes subsequent filtrate to get test solution；At least gas chromatographic detection will be injected by 10 batches of Pisces particle test solutions respectively, chromatogram is recorded, chromatogram is analyzed using similarity software, calibrates 12 common characteristic peaks, obtains Pisces particle finger-print.The method of the present invention has good precision, linear relationship, stability, repeatability, and the rate of recovery is high, good tolerance；The content assaying method of the Pisces particle volatile ingredients fingerprint of foundation and 2 kinds of ingredients provides foundation for the quality control of Pisces particle；This method is simple and feasible, quick and precisely, can be used as the effective ways of evaluation Pisces granular mass.

Description

A kind of method for building up of Pisces particle GC quantitative finger print atlas and its application

Technical field

The invention belongs to chemical analysis fields, are related to a kind of method for building up of Pisces particle GC finger-print.

Background technique

Pisces particle (SG) prescription is made of cordate houttuynia, honeysuckle, radix paeoniae rubra, folium artemisiae argyi and 5 taste medicine of peppermint, is Jiangsu Kang Yuan medicine 6 kind new medicine of Chinese medicine compound prescription that industry limited liability company develops, has the effect of relieving the exterior syndrome with drugs pungent in flavor and cool in property, clearing heat and detoxicating, is used for affection of exogenous wind-heat Type common cold symptoms include fever and headache, Muscular stiffness, nasal obstruction, runny nose, cough, expectoration, throat itch, dry and it is thirsty, throat is red The diseases such as swollen pain, the red, rapid pulse of tongue.

Pisces particle is compound preparation, and complex chemical composition, effective component includes glycoside, monoterpenes, phenolic acid class, volatilization Oil, and such as beta-cyclodextrin containing a large amount of auxiliary materials.Currently, in Pisces granular mass standard to volatile component eucalyptol, menthol and Methylnonanone has carried out gas phase identification, and has carried out HPLC assay to chlorogenic acid and Paeoniflorin.In view of only to 2 ingredients Content control is carried out, is ground so evaluated based on the Pisces granular mass of fingerprint map analyzing and multicomponent simultaneous quantitative Study carefully, which is mainly that HPLC is used to carry out finger-print and assay research to the big middle polarity ingredient in this product.So And only identify control for the volatile component in this product, it is controlled without assay index and globality.Due to Pisces Grain detection target component is complicated, and again by auxiliary material beta-cyclodextrin inclusion compound.It is not possible to current detection method in Pisces particle Volatile effective component carry out the control of effectively quantitative and globality.

Summary of the invention

The object of the present invention is to provide a kind of method for building up of Pisces particle GC quantitative finger print atlas, mainly to volatility at Divide and carry out finger-print research, assay has been carried out to main active eucalyptol therein and menthol, to reach whole The purpose of body control.

The purpose of the present invention is what is be achieved through the following technical solutions:

A kind of method for building up of Pisces particle GC finger-print, comprising the following steps:

Step (1) prepares test solution: Pisces particle is taken, it is finely ground, and Pisces particle powder is placed in flask, with water It is Extraction solvent with n-hexane, is extracted using volatile oil extractor, self-boiling starts timing 60~120 minutes, and it is cooling, divide and takes just Hexane layer is rinsed volatile oil extractor 2~3 times with n-hexane, is merged n-hexane, is added n-hexane to be settled to 25mL, shake up, mistake Filter, takes subsequent filtrate to get test solution；Wherein, the amount ratio of the Pisces particle powder, water and n-hexane is 10g: 250mL:2~5mL；

Preparation reference substance solution: eucalyptol, menthol, methylnonanone and n-hexane are mixed to get reference substance solution；

Reference substance solution and at least 10 batches of Pisces particle test solutions are injected gas chromatographic detections respectively by step (2), Chromatogram is recorded, chromatogram is analyzed, 12 common characteristic peaks are calibrated, obtains Pisces particle finger-print；

Wherein, GC conditions are as follows: Agilent DB-1 capillary column (30m × 0.32mm, 0.25 μm)；Sample volume is 1μL；Split ratio is 5:1~50:1；Injector temperature is 230 DEG C；Nitrogen buffer gas, 0.5~0.7L/min of nitrogen flow rate； Fid detector temperature is 250 DEG C；Temperature program are as follows: column temperature: 78~82 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, It is kept for 4 minutes, then rises to 120 DEG C with 2 DEG C/min, kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

In step (1), it is preferred that the test solution the preparation method comprises the following steps: taking the Pisces under content uniformity item Grain mixes, finely ground, takes 10.0g, accurately weighed, is placed in 500mL flask, and bead number is added, and is to mention with water and n-hexane Take solvent；It is cooling after self-boiling starts timing 60~90 minutes, divide and take n-hexane layer, rinses volatile oil extractor 2 with n-hexane It is secondary, merge n-hexane in 25mL measuring bottle, add n-hexane to be settled to scale, shake up, filters, take subsequent filtrate molten to get test sample Liquid；Wherein, the amount ratio of the Pisces particle powder, water and n-hexane is 10g:250mL:3mL.

It is further preferred that the test solution the preparation method comprises the following steps: take the Pisces particle under content uniformity item, mix It is even, it is finely ground, 10.0g is taken, it is accurately weighed, it is placed in 500mL flask, bead number is added, be that extraction is molten with water and n-hexane Agent；It is cooling after self-boiling starts timing 90 minutes, divide and take n-hexane layer, rinsed volatile oil extractor 2 times with n-hexane, is merged N-hexane adds n-hexane to be settled to scale, shakes up in 25mL measuring bottle, and filtering takes subsequent filtrate to get test solution；Wherein, The amount ratio of the Pisces particle powder, water and n-hexane is 10g:250mL:3mL.

Water of the present invention is deionized water.

The present invention uses 0.22 μm of filtering with microporous membrane.

In step (2), it is preferred that the GC conditions are as follows: Agilent DB-1 capillary column (30m × 0.32mm, 0.25 μm)；Sample volume is 1 μ L；Split ratio is 25:1；Injector temperature is 230 DEG C；Nitrogen buffer gas, nitrogen stream Fast 0.6L/min；Fid detector temperature is 250 DEG C；Temperature program are as follows: column temperature: 80 DEG C are kept for 2 minutes, then are risen to 8 DEG C/min It 112 DEG C, is kept for 4 minutes, then rises to 120 DEG C with 2 DEG C/min, kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

" similarity evaluation " 2012 that the present invention is promulgated using Chinese Pharmacopoeia Commission Version analyzes chromatogram, calibrates 12 common characteristic peaks.

The characteristic peak of the Pisces particle finger-print is each characteristic peak and its opposite when retaining referring to peak with eucalyptol Between it is as follows:

A method of the GC quantitative analysis of Pisces particle is carried out based on Pisces particle finger-print of the present invention, including Following steps:

Step (1) prepares test solution: Pisces particle is taken, it is finely ground, and Pisces particle powder is placed in flask, with water It is Extraction solvent with n-hexane, is extracted using volatile oil extractor, self-boiling starts timing 60~120 minutes, and it is cooling, divide and takes just Hexane layer is rinsed volatile oil extractor 2~3 times with n-hexane, is merged n-hexane, is added n-hexane to be settled to 25mL, shake up, mistake Filter, takes subsequent filtrate to get test solution；Wherein, the amount ratio of the Pisces particle powder, water and n-hexane is 10g: 250mL:2~5mL；

Preparation reference substance solution: taking eucalyptol reference substance, menthol reference substance, adds n-hexane that eucalyptol concentration is made and is 16.32~326.37 μ g/mL, the reference substance solution that menthol concentration is 16.32~326.37 μ g/mL；

Step (2), respectively by test solution, reference substance solution inject gas chromatographic detection, record chromatogram, with this hair The Pisces particle finger-print of bright acquisition is reference, calculates Pisces particle test sample similarity by similarity software；And use external standard One point method calculates separately the content of eucalyptol, menthol in Pisces particle；

Wherein, GC conditions are as follows: Agilent DB-1 capillary column (30m × 0.32mm, 0.25 μm)；Sample volume is 1μL；Split ratio is 5:1~50:1；Injector temperature is 230 DEG C；Nitrogen buffer gas, 0.5~0.7L/min of nitrogen flow rate； Fid detector temperature is 250 DEG C；Temperature program are as follows: column temperature: 78~82 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, It is kept for 4 minutes, then rises to 120 DEG C with 2 DEG C/min, kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

Of the invention is effective:

The method of the present invention has good precision, linear relationship, stability, repeatability, and the rate of recovery is high, and durability is good It is good；Separating degree, the reproducibility of Pisces particle volatility finger-print are preferable, and information is comprehensive, 12 characteristic peaks are indicated, to it In 3 ingredients carry out qualitative, each batch sample similarity is 0.90 or more.The Pisces particle volatile component that the present invention establishes refers to The content assaying method of line map and 2 kinds of ingredients provides foundation for the quality control of Pisces particle；This method is simple and feasible, quick Accurately, the effective ways of evaluation Pisces granular mass be can be used as.

Detailed description of the invention

Fig. 1 is the gas chromatogram using temperature program I；

Fig. 2 is the gas chromatogram using temperature program II；

Fig. 3 is the gas chromatogram using temperature program III；

Fig. 4 is Pisces particle steam distillation gas chromatogram；

Fig. 5 is Pisces particle methylene chloride-water reflux extraction gas chromatogram；

Fig. 6 is Pisces particle EtOH Sonicate extraction method gas chromatogram；

Fig. 7 is the gas chromatogram of mixed reference substance solution；

Fig. 8 is the gas chromatogram of the test solution of Pisces particle；

Fig. 9 is the gas chromatogram for lacking peppermint feminine gender test sample；

Figure 10 is the gas chromatogram for lacking folium artemisiae argyi feminine gender test sample；

Figure 11 is the gas chromatogram for lacking peppermint and folium artemisiae argyi feminine gender test sample；

Figure 12 is Pisces particle reference fingerprint；

Figure 13 be Pisces particle finger-print dependency graph, be followed successively by from top to bottom radix paeoniae rubra, honeysuckle, cordate houttuynia, peppermint, Folium artemisiae argyi, mixed mark (81.59 μ g/mL of eucalyptol, 208.44 μ g/mL of menthol in reference substance solution), Pisces particulate samples.

Specific embodiment

Technical solution of the present invention is described further below by specific embodiment.

1 instrument and reagent

Agilent 7890A gas chromatograph, Agilent company, the U.S.；

MILLIPORE Milli-Q Century pure water meter, Millipore company, the U.S.；

BSA224S-CW type electronic analytical balance, German Sartorius company；

Mettler Toledo X6 type electronic analytical balance, Mettler company, Switzerland；

KQ-250DB type ultrasonic washing instrument, Kunshan Ultrasonic Instruments Co., Ltd.；

(National Institute for Food and Drugs Control, lot number: 110788-201506, content is with 98.4% for eucalyptol reference substance Meter)；

Menthol reference substance (National Institute for Food and Drugs Control, lot number: 110728-200506)；

Methylnonanone reference substance (National Institute for Food and Drugs Control, lot number: 110834-200502)；

N-hexane (Nanjing Chemistry Reagent Co., Ltd., lot number 15078352E are analyzed pure)；

Ethyl acetate (Nanjing Chemistry Reagent Co., Ltd., lot number 14092712072 are analyzed pure)；

Pisces particle (lot number: 161201,161202,161203,160701,160702,160801,160802, 160901,160902,161001, Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov's production).

2 chromatography conditions

2.1 reference substance solutions are prepared and are selected referring to peak

Take eucalyptol, menthol, methylnonanone reference substance appropriate respectively, it is accurately weighed, add n-hexane to be made every 1mL points Not Han 80 μ g eucalyptols, 200 μ g menthols, 50 μ g methylnonanones mixed reference substance solution.The eucalyptus oil for selecting separating degree good Essence is as referring to peak (S).

The preparation of 2.2 test solutions

Take under content uniformity item Pisces particle (lot number: 161201), mixing, take it is appropriate, it is finely ground, take about 10.0g, it is accurate It is weighed, it sets in 500mL flask, bead number is added, water 250mL is added from volatile oil extractor upper end, adds n-hexane 3mL.Self-boiling starts timing and places cooling in 30 minutes after sixty minutes, divides and takes n-hexane layer, volatile oil extractor is rinsed with n-hexane It washes 2 times, merges n-hexane in 25mL measuring bottle, adding n-hexane dilution to be settled to scale, shake up, cross 0.22 μm of miillpore filter, take Subsequent filtrate, as test solution.

2.3 temperature programs are investigated

Capillary column: Agilent DB-1 (30m × 0.32mm, 0.25 μm)；1 μ l of sample volume；Split ratio 50:1；Injection port 230 DEG C of temperature；Fid detector temperature is 250 DEG C；Nitrogen buffer gas, nitrogen flow rate 0.6L/min.

Temperature program I: column temperature: 50 DEG C are kept for 0 minute, then rise to 75 DEG C with 4 DEG C/min, are kept for 10 minutes, then with 1 DEG C/ Min rises to 100 DEG C, then rises to 150 DEG C with 8 DEG C/min, is kept for 2 minutes, then rise to 165 DEG C with 3 DEG C/min, is kept for 5 minutes, then 250 DEG C are risen to 8 DEG C/min, is kept for 3 minutes.

Temperature program II: column temperature: 50 DEG C are kept for 2 minutes, then rise to 100 DEG C with 8 DEG C/min, are kept for 10 minutes, then with 8 DEG C/min rises to 250 DEG C, it is kept for 5 minutes.

Temperature program III: column temperature: 80 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, are kept for 4 minutes, then with 2 DEG C/ Min rises to 120 DEG C, is kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

1 three kinds of temperature program separation effect comparison results of table

Shown by table 1 and Fig. 1-Fig. 3 equal using III eucalyptol of temperature program II and temperature program, the separating degree of menthol Greater than 1.5, appearance time is comprehensively considered, it is preferred to use the whole separation effect of temperature program III, each chromatographic peak is preferable.

Conclusion: GC conditions are as follows: 1 μ l of sample volume；230 DEG C of injector temperature；Fid detector temperature is 250 DEG C；It rises Warm program are as follows: column temperature: 80 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, are kept for 4 minutes, then rise to 120 with 2 DEG C/min DEG C, it is kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

3 system suitabilities

3.1 reference substance solutions are prepared

Take eucalyptol, menthol reference substance appropriate respectively, it is accurately weighed, add n-hexane that every 1mL is made and contains 830.50 μ respectively G eucalyptol, 2099.52 μ g menthols mixing reference substance mother liquor 1. contain 815.93 μ g eucalyptols, 2084.40 μ respectively with every 1mL 2. the mixing reference substance mother liquor of g menthol, takes mixing reference substance mother liquor 1. in 1mL to 10mL measuring bottle, n-hexane to be added to dilute constant volume To scale, shake up up to mixed reference substance solution 1..

The preparation of 3.2 test solutions

Take under content uniformity item Pisces particle (lot number: 161201), mixing, take it is appropriate, it is finely ground, take about 10.0g, it is accurate It is weighed, it sets in 500mL flask, bead number is added, water 250mL is added from volatile oil extractor upper end, adds n-hexane 3mL.Self-boiling starts timing and places after sixty minutes 30 minutes, divides and takes n-hexane layer, volatile oil extractor rinses 2 with n-hexane It is secondary, merge n-hexane in 25mL measuring bottle, adding n-hexane dilution to be settled to scale, shakes up, cross 0.22 μm of miillpore filter, take continuous filter Liquid, as test solution.

3.3 GC conditions

Capillary column: Agilent DB-1 (30m × 0.32mm, 0.25 μm).

1 μ l of sample volume；Split ratio 25:1；230 DEG C of injector temperature；Fid detector temperature is 250 DEG C；Nitrogen flow rate 0.6L/min；Temperature program are as follows: column temperature: 80 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, are kept for 4 minutes, then with 2 DEG C/min rises to 120 DEG C, it is kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

Under the above conditions, the 1. continuous sample introduction 5 times measurements of 3.1 lower mixed reference substance solutions are taken, are recorded to be measured at swarming Area, takes 3.2 lower test solution continuous sample introductions 5 times, records Theory of Components plate number to be measured, separating degree, symmetrical factor.Knot Fruit: eucalyptol, menthol separating degree are all larger than 1.5, have reached baseline separation.To ensure the accurate, it is specified that theoretical of quantitative analysis Plate number must not calculate by menthol peak lower than 200000.

4 durabilities are investigated

According to " 3 system suitability " item, the durability for taking test solution and reference substance solution to carry out chromatographic condition is investigated. When fixation other conditions are constant, each condition below single variation: nitrogen flow rate, split ratio, initial temperature investigate different nitrogen Influence of the variation of flow velocity, different split ratios, different initial temperatures to eucalyptol and Determination of menthol measurement result.It the results are shown in Table 2, conditions above minor change does not make significant difference to assay result.

2 durability of table investigates result

Remarks: nitrogen flow rate 0.6L/min sample, split ratio used are 25:1, and starting column temperature is 80 DEG C.

The determination of 4.1 GC conditions

Result is investigated according to durability and determines eucalyptol, Determination of menthol measurement GC conditions are as follows: Agilent DB- 1 capillary column (30m × 0.32mm, 0.25 μm), using temperature program are as follows: column temperature: 80 DEG C are kept for 2 minutes, then with 8 DEG C/min liter It to 112 DEG C, is kept for 4 minutes, then rises to 120 DEG C with 2 DEG C/min, kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min；Sample volume 1μl；Split ratio 25:1；230 DEG C of injector temperature；Fid detector temperature is 250 DEG C；Nitrogen flow rate 0.6L/min.

The preparation method of 5 test solutions is investigated

In order to extract eucalyptol, menthol in preparation completely, extracting mode, extraction time are examined respectively It examines.GC conditions are the same as " 4.1 " item.

5.1 extracting methods are investigated

Extracting method I (steam distillation): the Pisces particle under content uniformity item is taken (lot number: 161201), to mix, take In right amount, finely ground, about 10.0g is weighed, it is accurately weighed, it sets in 500mL flask, bead number is added, from volatile oil extractor Water 250mL is added in end, adds n-hexane 3mL.Self-boiling starts timing and places after sixty minutes 30 minutes, divides and takes n-hexane layer, Volatile oil extractor is rinsed 2 times with n-hexane, is merged n-hexane in 25mL measuring bottle, adding n-hexane dilution to be settled to scale, is shaken It is even, 0.22 μm of miillpore filter is crossed, subsequent filtrate is taken, as test solution I.

Extracting method II (methylene chloride-water reflux extraction): take under content uniformity item Pisces particle (lot number: 161201) it, mixes, takes in right amount, it is finely ground, about 10.0g is weighed, accurately weighed in 150ml flask, deionized water is added in precision 30mL dissolves sample, adds methylene chloride 15mL, and water-bath 30 minutes (99.9 DEG C) of reflux divide after cooling and take methylene chloride, Water layer is extracted with dichloromethane three times, each 3mL methylene chloride, merges methylene chloride in 25mL measuring bottle, adds methylene chloride dilute It releases and is settled to scale, shake up, cross 0.22 μm of miillpore filter, subsequent filtrate is taken, as test solution II.

Extracting method III (EtOH Sonicate extraction method): the Pisces particle under content uniformity item is taken (lot number: 161201), to mix It is even, it takes in right amount, it is finely ground, about 10.0g is weighed, accurately weighed in 150mL conical flask, ethyl alcohol 25mL, 30 points of ultrasound is added in precision Clock (250W, 40KHZ), after letting cool, takes supernatant to be centrifuged, and crosses 0.22 μm of miillpore filter, subsequent filtrate is taken, as test solution Ⅲ。

3 extracting method of table is investigated

It the results are shown in Table 3 and Fig. 4-Fig. 6, it is aobvious with eucalyptol in test solution made from extracting method I and Determination of menthol It writes and is higher than other two kinds of extracting methods, and test solution is colourless, it is not easy to pollute capillary column.

5.2 extraction times were investigated

Take under content uniformity item Pisces particle (lot number: 161201), mixing, take it is appropriate, it is finely ground, weigh about 10.0g, put down 4 parts of row are accurately weighed, set in 500mL flask, and bead number is added, from volatile oil extractor upper end addition water 250mL, then plus Enter n-hexane 3mL.Self-boiling starts to place 30 minutes after timing 30,60,90,120 minutes respectively, divides and takes n-hexane layer, volatilizees Oil extractor is rinsed 2 times with n-hexane, is merged n-hexane, is placed in 25mL measuring bottle, is added n-hexane dilution to be settled to scale, is shaken It is even, 0.22 μm of miillpore filter is crossed, subsequent filtrate is taken, obtains test solution.Assay is carried out, measurement result is shown in Table 4, and display mentions It takes 60,90,120min eucalyptol and Determination of menthol variation not significant, contains in view of eucalyptol obtained by extraction 90min and menthol Amount is higher by slightly, therefore the selective extraction time is 90 minutes.

The selection of 4 extraction time of table

5.3 determine the preparation method of test solution

Determine in summary test solution the preparation method comprises the following steps: take this product under content uniformity item, mix, take it is appropriate, It is finely ground, about 10.0g is weighed, it is accurately weighed, it sets in 500mL flask, bead number is added, be added from volatile oil extractor upper end Water 250mL adds n-hexane 3mL.Self-boiling is placed 30 minutes after starting timing 90 minutes, is divided and is taken n-hexane layer, volatile oil Extractor is rinsed 2 times with n-hexane, is merged n-hexane, is placed in 25mL measuring bottle, is added n-hexane dilution to be settled to scale, is shaken up, 0.22 μm of miillpore filter is crossed, subsequent filtrate is taken, as test solution.

6 methodological studies

6.1 specificities are investigated

6.1.1 the preparation of reference substance solution

It takes and mixes reference substance mother liquor 2. under 3.1.

6.1.2 the preparation of test solution

Test solution is made in preparation method with 5.3 lower test solutions.

6.1.3 the preparation of negative test solution

Original side removes folium artemisiae argyi, peppermint medicinal material respectively and goes folium artemisiae argyi and peppermint simultaneously, and scarce folium artemisiae argyi is made respectively by preparation process, lacks Peppermint, the negative preparation for lacking folium artemisiae argyi and peppermint take above-mentioned three kinds negative preparation about 10.0g, by the preparation of test solution respectively Method, the negative preparation test solution that scarce folium artemisiae argyi is made with method, lacks peppermint and scarce folium artemisiae argyi and peppermint.

Respectively it is accurate draw above-mentioned mixing reference substance mother liquor 2., test solution, lack folium artemisiae argyi, lack peppermint and scarce folium artemisiae argyi and thin Each 1 μ L of negative preparation test solution of lotus injects gas chromatograph, is measured according to 4.1 lower GC conditions, as a result As shown in Fig. 7-Figure 11, show that scarce peppermint feminine gender test sample is noiseless to the measurement of menthol, lacks folium artemisiae argyi and the negative of peppermint supplies Test product is noiseless to the measurement of eucalyptol.Illustrate that the method for the present invention has good specificity to menthol.It is in view of eucalyptol The shared ingredient of folium artemisiae argyi and peppermint, and interfered without other flavour of a drug, therefore eucalyptol is also included in assay research.

6.1 linear relationships are investigated

It takes and mixes reference substance mother liquor 2. under 3.1, precision measures 0.1mL, 0.2mL, 0.5mL, 1.0mL, 1.5mL, 2.0mL It is respectively placed in 5mL measuring bottle, n-hexane dilution is added and is settled to scale, shakes up up to respectively containing 16.32 μ g/mL of eucalyptol, thin 41.69 μ g/mL of lotus brain, contain 32.64 μ g/mL of eucalyptol, 83.38 μ g/mL of menthol, the respectively 81.59 μ g/ containing eucalyptol respectively ML, 208.44 μ g/mL of menthol, contain 163.19 μ g/mL of eucalyptol respectively, 416.88 μ g/mL of menthol, contain eucalyptol respectively 244.78 μ g/mL, 625.32 μ g/mL of menthol, the respectively system containing 326.37 μ g/mL of eucalyptol, 833.76 μ g/mL of menthol Column mixed reference substance solution.Above-mentioned 1 μ L of each mixed reference substance solution is drawn respectively, injects gas chromatograph, according to 4.1 lower gas phases Chromatographic condition is measured, and the results are shown in Table 5,6, and using peak area average value as ordinate (Y), reference substance concentration (μ g/mL) is cross Coordinate (X) draws standard curve and obtains regression equation:

Eucalyptol: Y=0.7185X+1.0899；R=0.9999；

Menthol: Y=0.7234X+2.6501；R=0.9999.

5 linear relationship investigation of table-eucalyptol

6 linear relationship investigation of table-menthol

The result shows that eucalyptol is in good linear relationship, menthol between 16.32~326.37 μ g/mL in concentration It is in good linear relationship between 41.69~833.76 μ g/mL in concentration.

6.4 precision test

Take mixed reference substance solution (the low mark: respectively containing 16.32 μ g/mL of eucalyptol, thin of basic, normal, high three various concentrations Acceptance of the bid: the mixed reference substance solution of 41.69 μ g/mL of lotus brain contains 81.59 μ g/mL of eucalyptol, 208.44 μ g/mL of menthol respectively Mixed reference substance solution, high standard: the mixing reference substance respectively containing 326.37 μ g/mL of eucalyptol, 833.76 μ g/mL of menthol is molten Liquid), it continuous sample introduction 6 times, 1 μ L of sample volume respectively, is measured according to 4.1 lower GC conditions, the results are shown in Table the face 7, Qi Feng Product RSD (%) is below 2%, the results showed that, instrument precision is good.It takes test solution continuous sample introduction 6 times, before shearing is removed Solvent peak after 6 minutes and 24 minutes, with finger-print obtained by the 1st sample introduction as control, fingerprint obtained by 5 sample introductions after calculating The similarity of map, as a result similarity is all larger than 0.99.The result shows that this method precision is good.

7 precision result (peak area) of table

6.5 repetitive test

Pisces particle is taken (lot number: 161201), 6 parts of test solutions to be made by sample solution preparation method, according to 4.1 Lower GC conditions are measured, and the results are shown in Table 8, eucalyptol content 0.219mg/g, Determination of menthol 0.609mg/g.Shearing Solvent peak after removing first 6 minutes and 24 minutes calculates another 5 parts of samples institute using finger-print obtained by the 1st part of sample as control The similarity of finger-print is obtained, as a result similarity is all larger than 0.99.Show that the method for the present invention repeatability is good.

The repeated result of table 8

6.6 stability test

Same test solution under 6.5 is taken to the results are shown in Table 9, test solution respectively at 0,2,4,8,12 hour sample introduction Middle eucalyptol, menthol, content RSD be respectively 1.62%, 1.48%, solvent after first 6 minutes and 24 minutes is removed in shearing Peak is that control calculates similarity with finger-print obtained by 0 hour sample introduction, and similarity result is all larger than 0.99, shows that test sample is molten Liquid is stablized in 12 hours.

9 stability result of table

6.7 recovery test

Take the Pisces particle (lot number: 161201, eucalyptol content 0.219mg/g, Determination of menthol under content uniformity item 0.609mg/g), mix, take it is appropriate, it is finely ground, weigh 6 parts, every part of about 5g respectively, it is accurately weighed, set in 500mL flask, respectively Precision is added mixing reference substance mother liquor 2. 1.5mL and bead number is added after reference substance mother liquor to be mixed and test sample are sufficiently mixed , be added water 250mL from volatile oil extractor upper end, then plus n-hexane 3mL, after the good condenser pipe of airtight connection, self-boiling starts It timing 90 minutes, places 30 minutes points and takes n-hexane layer, volatile oil extractor is rinsed 2 times with n-hexane, is merged n-hexane, is placed in In 25mL measuring bottle, adds n-hexane dilution to be settled to scale, shake up, cross 0.22 μm of miillpore filter, take subsequent filtrate, it is molten as test sample Liquid.Sample introduction carries out assay, the results are shown in Table 10,2 components recoveries between 95.62~107.84%, RSD% is respectively less than 2.3%, show that this method rate of recovery is good.

10 sample recovery rate result of table

The acquisition of 6.8 multiple batches of sample detections and reference fingerprint

Ten batches of Pisces particulate samples are collected, prepare test solution, sample introduction by the preparation method of 5.3 lower test solutions 1 μ L is measured, measures eucalyptol, Determination of menthol, the while " Chinese medicine promulgated using National Pharmacopeia according to 4.1 lower GC conditions Chromatographic fingerprinting similarity evaluation system " version was analyzed 2012 years, solvent after shearing is removed first 6 minutes and 24 minutes Peak is generated reference fingerprint with average method reference fingerprint method of formation (see Figure 12, Figure 13).With reference fingerprint For reference, each batch sample similarity is calculated by similarity software, the results are shown in Table 12.Each feature is calculated by object of reference of eucalyptol The relative retention time at peak, and provide the range of relative retention time.It the results are shown in Table 13.

12 Pisces particle content measuring of table and fingerprint similarity result

13 Pisces particle Qualitative fingerprint result of table and characteristic peak relative retention time

Peak number	Substance title	Relative retention time
			1	It is unknown	0.893 ± 20%
2	Eucalyptol	1
			3	It is unknown	1.321 ± 20%
4	It is unknown	1.361 ± 20%
			5	It is unknown	1.424 ± 20%
6	Menthol	1.451 ± 20%
			7	It is unknown	1.473 ± 20%
8	It is unknown	1.512 ± 20%
			9	It is unknown	1.767 ± 20%
10	Methylnonanone	2.037 ± 20%
			11	It is unknown	2.375 ± 20%
12	It is unknown	2.535 ± 20%

6.9 correlations are investigated

Extracting honeysuckle medicinal material, radix paeoniae rubra medicinal material, Herba Houttuyniae, peppermint medicinal material, folium artemisiae argyi medicinal material each about 3g, it is accurately weighed in circle In the flask of bottom, by 5.3 lower preparation method of test article, test solution is prepared, sample introduction is analyzed by 4.1 lower chromatographic conditions, sees Figure 13,1,10,11, No. 12 peak mostlys come from cordate houttuynia in 12 characteristic peaks, and No. 2 peaks mostly come from folium artemisiae argyi and peppermint, 3, 5, No. 7 peaks mostly come from folium artemisiae argyi, and 4,6, No. 8 mostly come from peppermint.

Claims (7)

1. a kind of method for building up of Pisces particle GC finger-print, it is characterised in that the following steps are included:

Step (1) prepares test solution: Pisces particle is taken, it is finely ground, and Pisces particle powder is placed in flask, Yi Shui and just Hexane is Extraction solvent, is extracted using volatile oil extractor, and self-boiling starts timing 60~120 minutes, cooling, divides and takes n-hexane Layer, is rinsed volatile oil extractor 2~3 times with n-hexane, is merged n-hexane, is added n-hexane to be settled to 25mL, shake up, and is filtered, is taken Subsequent filtrate is to get test solution；Wherein, the amount ratio of the Pisces particle powder, water and n-hexane is 10g:250mL:2 ~5mL；

Step (2) at least will inject gas chromatographic detection by 10 batches of Pisces particle test solutions respectively, record chromatogram, use Similarity software analyzes chromatogram, calibrates 12 common characteristic peaks, obtains Pisces particle finger-print；

Wherein, GC conditions are as follows: Agilent DB-1 capillary column, 30m × 0.32mm, 0.25 μm；Sample volume is 1 μ L； Split ratio is 5:1~50:1；Injector temperature is 230 DEG C；Nitrogen buffer gas, 0.5~0.7L/min of nitrogen flow rate；FID inspection Surveying device temperature is 250 DEG C；Temperature program are as follows: column temperature: 78~82 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, keep 4 Minute, then 120 DEG C are risen to 2 DEG C/min, it is kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

2. the method for building up of Pisces particle GC finger-print according to claim 1, it is characterised in that the test sample Solution the preparation method comprises the following steps: take the Pisces particle under content uniformity item, mix, it is finely ground, take 10.0g, it is accurately weighed, be placed in In 500mL flask, bead number is added, using water and n-hexane as Extraction solvent；After self-boiling starts timing 60~90 minutes, It is cooling, divide and take n-hexane layer, rinsed volatile oil extractor 2 times with n-hexane, merges n-hexane in 25mL measuring bottle, add n-hexane It is settled to scale, is shaken up, filters, takes subsequent filtrate to get test solution；Wherein, the Pisces particle powder, water and just oneself The amount ratio of alkane is 10g:250mL:3mL.

3. the method for building up of Pisces particle GC finger-print according to claim 2, it is characterised in that the test sample Solution the preparation method comprises the following steps: take the Pisces particle under content uniformity item, mix, it is finely ground, take 10.0g, it is accurately weighed, be placed in In 500mL flask, bead number is added, using water and n-hexane as Extraction solvent；It is cold after self-boiling starts timing 90 minutes But, divide and take n-hexane layer, rinsed volatile oil extractor 2 times with n-hexane, merge n-hexane in 25mL measuring bottle, adding n-hexane fixed Hold to scale, shake up, filters, take subsequent filtrate to get test solution；Wherein, Pisces particle powder, water and the n-hexane Amount ratio be 10g:250mL:3mL.

4. the method for building up of Pisces particle GC finger-print according to claim 1,2 or 3, it is characterised in that use 0.22 μm filtering with microporous membrane.

5. the method for building up of Pisces particle GC finger-print according to claim 1, it is characterised in that the gas phase color Spectral condition are as follows: Agilent DB-1 capillary column, 30m × 0.32mm, 0.25 μm；Sample volume is 1 μ L；Split ratio is 25:1；Into Sample mouth temperature is 230 DEG C；Nitrogen buffer gas, nitrogen flow rate 0.6L/min；Fid detector temperature is 250 DEG C；Temperature program Are as follows: column temperature: 80 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, are kept for 4 minutes, then rise to 120 DEG C with 2 DEG C/min, are protected It holds 6 minutes；250 DEG C are risen to again with 20 DEG C/min.

6. the method for building up of Pisces particle GC finger-print according to claim 1, it is characterised in that the Pisces The characteristic peak of grain finger-print is referring to peak with eucalyptol, and each characteristic peak and its relative retention time are as follows:

Peak number Substance title Relative retention time 1 It is unknown 0.893 ± 20% 2 Eucalyptol 1 3 It is unknown 1.321 ± 20% 4 It is unknown 1.361 ± 20% 5 It is unknown 1.424 ± 20% 6 Menthol 1.451 ± 20% 7 It is unknown 1.473 ± 20% 8 It is unknown 1.512 ± 20% 9 It is unknown 1.767 ± 20% 10 Methylnonanone 2.037 ± 20% 11 It is unknown 2.375 ± 20% 12 It is unknown 2.535 ± 20%

。

7. a kind of method for carrying out the GC quantitative analysis of Pisces particle based on Pisces particle finger-print described in claim 1, packet Include following steps:

Step (1) prepares test solution: Pisces particle is taken, it is finely ground, and Pisces particle powder is placed in flask, Yi Shui and just Hexane is Extraction solvent, is extracted using volatile oil extractor, and self-boiling starts timing 60~120 minutes, cooling, divides and takes n-hexane Layer, is rinsed volatile oil extractor 2~3 times with n-hexane, is merged n-hexane, is added n-hexane to be settled to 25mL, shake up, and is filtered, is taken Subsequent filtrate is to get test solution；Wherein, the amount ratio of the Pisces particle powder, water and n-hexane is 10g:250mL:2 ~5mL；

Preparation reference substance solution: taking eucalyptol reference substance, menthol reference substance, and adding n-hexane that eucalyptol concentration is made is 16.32 ~326.37 μ g/mL, the reference substance solution that menthol concentration is 16.32~326.37 μ g/mL；

Step (2), respectively by test solution, reference substance solution inject gas chromatographic detection, record chromatogram, with claim The 1 Pisces particle finger-print obtained is reference, calculates Pisces particle test sample similarity by similarity software；And with external standard one Point method calculates separately the content of eucalyptol, menthol in Pisces particle；

Wherein, GC conditions are as follows: Agilent DB-1 capillary column, 30m × 0.32mm, 0.25 μm；Sample volume is 1 μ L； Split ratio is 5:1~50:1；Injector temperature is 230 DEG C；Nitrogen buffer gas, 0.5~0.7L/min of nitrogen flow rate；FID inspection Surveying device temperature is 250 DEG C；Temperature program are as follows: column temperature: 78~82 DEG C are kept for 2 minutes, then rise to 112 DEG C with 8 DEG C/min, keep 4 Minute, then 120 DEG C are risen to 2 DEG C/min, it is kept for 6 minutes；250 DEG C are risen to again with 20 DEG C/min.