CN109765322A - The characteristic spectrum construction method and quality determining method of schizonepeta - Google Patents

Abstract

The present invention relates to Chinese medicine detection technique fields, and in particular to the characteristic spectrum construction method and quality determining method of schizonepeta, by establishing schizonepeta involatile constituent characteristic spectrum to liquid phase chromatogram condition, flowing phase composition and the control of elution program；By selecting suitable GC conditions, schizonepeta volatile component characteristic spectrum is established；And the characteristic peak of marker characteristic map；The content of hesperidin in schizonepeta is measured by the adjustment to liquid chromatogram elution program；Simultaneously, establish the method for quality control of schizonepeta, schizonepeta is evaluated including the use of aforementioned schizonepeta characteristic spectrum, and measure the content of aurantiamarin, it further include thin-layered chromatography identification, heavy metal and harmful element inspection, persticide residue measurement, determination of extractives, quality evaluation is carried out to schizonepeta from validity and safety perspective, which has many advantages, such as that simple and quick, reliable and stable, precision is high, favorable reproducibility, is easy to grasp.

Description

The characteristic spectrum construction method and quality determining method of schizonepeta

Technical field

The present invention relates to Chinese medicine detection technique fields, and in particular to the construction method of the characteristic spectrum of schizonepeta and quality testing Method.

Background technique

Schizonepeta, slightly warm in nature, acrid flavour have effects that inducing diaphoresis relieve heat, promoting eruption and the sore that disappears, for catching a cold, having a headache, morbilli, wind Rash and sore are one of common Chinese medicinal materials.Quality monitoring in today of the quality growing interest to drug, to Chinese medicine It is extremely important.With the development of chromatographic technique, schizonepeta quality of medicinal material standard not only includes simple character, identification, routine inspection It further include the measurement of high-efficiency liquid-phase fingerprint with determination of extractives etc..Traditional Chinese medicine fingerprint is a kind of synthesis, can measure The identification means of change are the quality control models for currently meeting characteristics of Traditional Chinese Medicine, evaluation Chinese medicine authenticity, stability and consistency One of.

The effect of the finger-print mainly homogeneity and stabilization of the Chinese medicine of reflection complicated ingredient and its preparation inherent quality Property.Chromatography expert think should separating degree the higher the better, the peak separated is The more the better；Information processing expert also thinks the number provided According to The more the better.But the problem of preci-sion and accuracy of the international coordination meeting about quantitative analysis, says that " result of analysis is not More more accurate, more accurate better, but reach the requirement of analysis object ", i.e. the peak of finger-print is not The more the better, Achieve the purpose that constitute fingerprint characteristic, and as drug, on the basis of guaranteeing safety, effective component can be monitored just Have reached purpose.

It is reported that schizonepeta principle active component has volatile oil, a variety of flavone compounds, monoterpene glycosides etc., wherein volatile oil Main component is pulegone, menthones, can be used as the representative species of volatile oil；Content of hesperidin is higher in flavones ingredient, And there is certain pharmacological action.Existing China's document " content that HPLC method measures 4 kinds of ingredients in schizonepeta granule simultaneously " (Journal of Chinese Medicinal Materials, in October, 2016, the 10th phase 2285-2287 of volume 39) is open It is a kind of to use principle active component caffeic acid, aurantiamarin, Rosmarinic acid in high performance liquid chromatography detection schizonepeta granule With the content of pulegone, this method can provide a kind of schizonepeta product comprehensively and effectively quality by the measurement of active constituent content Control.But the above method uses granule, to control from source from raw material schizonepeta medicinal material or medicine materical crude slice, and There are in involatile constituent, some is present in volatile oil effective component ingredient pulegone a part, and thin Lotus ketone is also the effective component of schizonepeta, and menthones is then primarily present in volatile oil, therefore the method can not be from effective to schizonepeta Ingredient carries out comprehensive quality control.

And it is further improved the quality control of schizonepeta medicinal material, and in addition to effective component, heavy metal and harmful element, pesticide residue The safety indexes such as amount have important influence in actual application, influence drug safety, therefore, Bindery security control Traditional Chinese medicine quality control the control of effeciency of Chinese materia medica will be made to have more realistic meaning, establish schizonepeta comprehensively, quality effectively and safely Control method.

Summary of the invention

Therefore, the invention solves first technical problem be to overcome involved by schizonepeta characteristic spectrum in the prior art The not comprehensive enough defect of effective component, to provide a kind of construction method of schizonepeta characteristic spectrum, this feature map construction side Method includes the building of non-volatile effective component characteristic spectrum and volatile effective component characteristic spectrum, to Chemical Constituents From Schizonepeta The building of Characteristic chromatographic peak is more comprehensively.

The invention solves second technical problem be to overcome the prior art cannot be from two sides of validity and safety The defect that overall quality control is carried out in face of schizonepeta, establishes a kind of schizonepeta quality determining method.

The invention discloses a kind of construction method of schizonepeta characteristic spectrum, the building including involatile constituent characteristic spectrum The construction method of method and volatile component characteristic spectrum:

A, the construction method of the involatile constituent characteristic spectrum, includes the following steps:

The preparation of test solution I: schizonepeta test sample is taken to prepare test solution I；

The preparation of reference solution I: schizonepeta control medicinal material is taken to prepare control medicinal material reference solution I；Aurantiamarin is taken to compare Product prepare reference substance reference solution I；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Compose column packing；Using acetonitrile as mobile phase A, eluted using the phosphate aqueous solution that volume fraction is 0.2% as Mobile phase B；

Measuring method: it is accurate respectively to draw reference solution I and test solution I, inject liquid chromatograph, measurement to get； With

B, the construction method of the volatile component characteristic spectrum, includes the following steps:

The preparation of test solution II: taking the volatile oil prepared by schizonepeta test sample, prepares test solution II；

The preparation of reference solution II: pulegone and menthones is taken to prepare the reference solution II of reference substance respectively；

Chromatographic condition and system suitability: measuring according to gas chromatography, using N2 as carrier gas, using PEG as stationary phase, and control Injector temperature, detector temperature, column temperature and flow velocity processed；

Measuring method: accurate absorption reference solution II and test solution II respectively, injection gas chromatograph, measurement, i.e., ?.

Preferably, further include the building of compare feature map, utilize the chromatographic fingerprints of Chinese materia medica of Chinese Pharmacopoeia Commission Similarity evaluation system analyzes obtained at least 15 batches of test solution I characteristic spectrums, generates involatile constituent pair According to characteristic spectrum；And/or

At least 15 batches confessions using the similarity evaluation of Chinese Pharmacopoeia Commission, to obtaining The gas phase characteristic map of test sample solution II is analyzed, and volatile component compare feature map is generated.

Preferably, the involatile constituent characteristic spectrum includes 6 characteristic peaks, characteristic peak are as follows: and No. 1 peak is aurantiamarin, 2 Number peak is Rosmarinic acid, and No. 3 peaks, No. 4 peaks, No. 5 peaks are common characteristic peaks, and No. 6 peaks are pulegone；It is reference peak with No. 1 peak, Each peak relative retention time is respectively as follows: No. 1 peak: 1.00；No. 2 peaks: 1.07；No. 3 peaks: 1.09；No. 4 peaks: 1.20；No. 5 peaks: 1.25；No. 6 peaks: 1.64；And/or

The volatile component characteristic spectrum includes 3 characteristic peaks, characteristic peak are as follows: No. 1 peak is menthones, and No. 2 peaks are recklessly Menthones, No. 3 peaks are common characteristic peaks；It is referring to peak with No. 2 peaks, each peak relative retention time is respectively as follows: No. 1 peak: 0.67； No. 2 peaks: 1.00；No. 3 peaks: 1.50.

Preferably, in the construction method of the involatile constituent characteristic spectrum, type of elution selects gradient elution side Formula, gradient elution program are as follows: 0-15min, mobile phase A: the volume ratio of Mobile phase B is 8-12%:92-88% → 12-16%: 88-84%；15-40min, mobile phase A: the volume ratio of Mobile phase B is 12-16%:88-84% → 24-28%:76-72%； 40-55min, mobile phase A: the volume ratio of Mobile phase B is 24-28%:76-72% → 58-62%:42-38%；55-60min, Mobile phase A: the volume ratio of Mobile phase B is 58-62%:42-38% → 83-87%:17-13%；60-65min, mobile phase A: stream The volume ratio of dynamic phase B is 83-87%:17-13% → 83-87%:17-13%；

Preferably, the gradient elution program are as follows: 0-15min, mobile phase A: the volume ratio of Mobile phase B is 10%:90% → 14%:86%；15-40min, mobile phase A: the volume ratio of Mobile phase B is 14%:86% → 26%:74%；40-55min, Mobile phase A: the volume ratio of Mobile phase B is 26%:74% → 60%:40%；55-60min, mobile phase A: the volume of Mobile phase B Than for 60%:40% → 85%:15%；60-65min, mobile phase A: the volume ratio of Mobile phase B is 85%:15% → 85%: 15%；

Preferably, chromatographic condition are as follows: column temperature is 25-40 DEG C；Flow velocity is 0.8-1.2ml/min；Detection wavelength is 278- 288nm；

Preferably, chromatographic condition are as follows: column temperature is 40 DEG C；Flow velocity is 1ml/min；Detection wavelength is 283nm.

Preferably, in the construction method of the volatile component characteristic spectrum, control column temperature heats up in gradient, the ladder Spend temperature program are as follows: initial temperature is 55-65 DEG C, keeps 1-3min；Temperature rises to 115-125 DEG C from 55-65 DEG C, then keeps 4-6min, heating rate are 1.5-2.5 DEG C/min；Temperature rises to 155-165 DEG C from 115-125 DEG C, heating rate 2.5-3.5 ℃/min；Temperature rises to 235-245 DEG C from 155-165 DEG C, and heating rate is 4.5-5.5 DEG C/min；

Preferably, the gradient increased temperature program are as follows: initial temperature is 60 DEG C, keeps 2min；Temperature rises to 120 from 60 DEG C DEG C, 5min is kept, heating rate is 2 DEG C/min；Temperature rises to 160 DEG C from 120 DEG C, and heating rate is 3 DEG C/min；Temperature from 160 DEG C rise to 240 DEG C, and heating rate is 5 DEG C/min；

Preferably, injector temperature is 245-255 DEG C, and detector temperature is 245-255 DEG C, total flow 3.5-3.9ml/ min；

Preferably, injector temperature is 250 DEG C, and detector temperature is 250 DEG C, total flow 3.7ml/min.

Preferably, the preparation method of the test solution I or control medicinal material reference solution I include: to take schizonepeta for examination Powder is made in sample or schizonepeta control medicinal material, and precision weighs 0.5-1g, solubilizer 15-30ml, and close plug extracts processing, puts It is cold, the weight of less loss is supplied with the solvent, is shaken up, supernatant is taken to filter, and takes subsequent filtrate to get test solution I or control Medicinal material reference solution I；

Preferably, the preparation method of the reference substance reference solution I includes: that aurantiamarin reference substance is taken to add the solvent molten The reference substance reference solution I of the g/ml aurantiamarin of μ containing 55-65 is made in solution；

Preferably, the test sample is selected from schizonepeta crude drug or schizonepeta medicine materical crude slice；

Preferably, the solvent is methanol, ethyl alcohol or water；

Preferably, the extraction process is using ultrasound 30-90min, reflux 30-120min or dipping 12-24h；

Preferably, the reference substance reference solution I is the reference substance reference solution I containing 60 μ g/ml aurantiamarins.

Preferably, the reference solution II is the reference substance containing 3-6mg/ml pulegone and 3-6mg/ml menthones Solution, solvent are ethyl acetate；

Described for II preparation method of reagent material solution includes: to take schizonepeta test sample, using " People's Republic of China's medicine Allusion quotation " the first legal systems of 2015 years four 2204 determination of volatile oil of general rule of version obtains volatile oil, and precision draws 25-50ul, adds ethyl acetate It is settled to 2ml, water removal is drying to obtain.

The present invention also provides a kind of schizonepeta quality determining methods, including use above-mentioned schizonepeta characteristic spectrum construction method pair Schizonepeta product carries out quality testing.

Preferably, schizonepeta product to be measured, obtained chaste tree are detected with method operation according to above-mentioned schizonepeta characteristic spectrum construction method 6 spies are presented in mustard involatile constituent characteristic spectrum and schizonepeta volatile component characteristic spectrum, the non-volatile characteristic spectrum Peak is levied, and should be corresponding with 6 characteristic peaks in control medicinal material object of reference characteristic spectrum, wherein peak 1, peak 6 should join with reference substance It is consistent according to object peak retention time；3 characteristic peaks are presented in the volatile component characteristic spectrum, wherein peak 1, peak 2 should with compare Product object of reference peak retention time is consistent.

Preferably, further include the steps that carrying out assay to schizonepeta, include the following steps:

Test solution III: schizonepeta test sample is taken to prepare test solution III；

Reference substance solution: taking aurantiamarin reference substance to be dissolved in solvent, prepares reference substance solution；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Compose column packing；Using acetonitrile as mobile phase A, gradient is carried out by Mobile phase B of the phosphate aqueous solution that volume fraction is 0.2% and is washed De-, carry out gradient elution: 0-28min according to following procedure, mobile phase A: the volume ratio of Mobile phase B is 17-19%:83-81% → 17-19%:83-81%；28-29min, mobile phase A: the volume ratio of Mobile phase B is 17-19%:83-81% → 84-86%: 16-14%；29-39min, mobile phase A: the volume ratio of Mobile phase B is 84-86%:16-14% → 84-86%:16-14%；Column Temperature is 25-35 DEG C；Flow velocity is 0.8-1.2ml/min；Detection wavelength is 278-288nm；Number of theoretical plate is not less than based on aurantiamarin 3000；

Measuring method: accurate absorption reference substance solution and test solution III respectively, injection liquid chromatograph, measurement, i.e., ?.

Preferably, further include the steps that the step of thin-layered chromatography identification, heavy metal and harmful element measure and/or leaching The step of object measures:

C, the involatile constituent thin-layered chromatography identifies step, includes the following steps:

The coarse powder and schizonepeta control medicinal material coarse powder 0.5-1.0g for taking schizonepeta test sample respectively, add ethyl acetate 15-25ml, Ultrasonic treatment 15-25 minutes, filtration, filtrate is waved to 1ml, then each to draw as test solution IV and control medicinal material solution 1-5ul puts respectively on same lamellae, is unfolded in solvent, takes out, dries, and setting wavelength is the ultraviolet of 361nm-371nm It is inspected under light；

Preferably, the lamellae selects silica G plate or H plate；

Preferably, the solvent selects chloroform-ethyl acetate-glacial acetic acid mixed solution, and volume ratio is (9- 11):1:(0.3-0.8)；And/or

D, the volatile component thin-layered chromatography identifies step, includes the following steps:

Take schizonepeta test sample coarse powder appropriate, according to determination of volatile oil method " Pharmacopoeia of People's Republic of China 2015 version four General rule 2204 " measurement, divides and takes 0.1~0.3ml of volatile oil, add ethyl acetate to be diluted to 1.5~4.5ml, as test solution Ⅴ.(-)-menthones, pulegone's reference substance are separately taken, adds ethyl acetate that the solution that every 1ml contains 5~30mg is made, as control Product solution.It is each to draw 1-5ul, it is put respectively on same lamellae, is unfolded in solvent, taken out, dry, sprayed with 2%~8% It is clear to be heated to spot development at 105 DEG C for vanillin-sulfuric acid ethanol solution；

Preferably, the lamellae selects silica G plate or H plate；

Preferably, the solvent selects petroleum ether (60~90 DEG C)-ethyl acetate, and volume ratio is (11-13): 1； And/or；

E, the measurement of the heavy metal and harmful element includes the assay of heavy metal lead, cadmium, arsenic, mercury, copper, including such as Lower step:

The persticide residue measurement includes the assay of total six six six, total DDT and pentachloronitrobenzene；And/or

F, the step of determination of extractives includes:

The determination of extractives operating procedure are as follows: take schizonepeta for reagent material coarse powder 2-5g, it is accurately weighed, set 250~300ml Conical flask in, accurate solubilizer 100ml, close plug, cold soaking constantly shakes in first 6 hours, then stands 18 hours, is filtered with dry Device filters rapidly, and precision measures subsequent filtrate 20ml, sets and has dried into the evaporating dish of constant weight, after being evaporated in water-bath, in 105 DEG C It is 3 hours dry, 30 minutes cooling in drier, rapid accurately weighed weight is set, extract in test sample is calculated with dry product Content (%)；Wherein, solvent is ethyl alcohol, and volume ratio score is 50-90%

Technical solution of the present invention has the advantages that

1, the construction method of schizonepeta characteristic spectrum provided by the invention, including including non-volatile effective component characteristic spectrum With the building of volatile effective component characteristic spectrum, on the one hand, the non-volatile effective component characteristic spectrum include aurantiamarin, Rosmarinic acid, pulegone Characteristic chromatographic peak, the volatile effective component characteristic spectrum includes menthones and pulegone Characteristic chromatographic peak, wherein pulegone can coexist with non-volatile effective component but also coexist with volatile effective component, By liquid-phase chromatographic analysis and gas chromatographic analysis, the pulegone in detection medicinal material can be effectively evaluated, while can specific aim Ground is to effective components other in schizonepeta: Rosmarinic acid, aurantiamarin and menthones carry out complete detection, have both included non-volatile Effective component includes volatile effective component again, and chromatographic peak separation is completely, and peak shape is good and noiseless.

On the other hand, the building of characteristic spectrum is carried out to the non-volatile effective component of schizonepeta using high performance liquid chromatography, Separation efficiency is high, and sensitivity is good, and speed is fast,, can be complete with -0.2% phosphoric acid water (B) of acetonitrile (A) for mobile phase by test of many times Fully separating target substance Rosmarinic acid, aurantiamarin and pulegone；It is mended mutually using gas chromatography with high performance liquid chromatography It fills, analyzes the volatile effective component in schizonepeta, the chromatographic condition and temperature program used by test of many times cooperates, and makes It obtains menthones and pulegone is separated well, the combination of gas chromatography analysis method and liquid phase chromatography analytical method, energy It is completely separating the non-volatile effective component and volatile effective component of schizonepeta, the effective component of schizonepeta is enable to obtain comprehensively Separation and detection, effective component chromatographic peak are sufficiently separated, and peak shape is good and noiseless, and this feature map detection method has simply Quickly, reliable and stable, precision is high, favorable reproducibility, is easy to the advantages that grasping.

2, the construction method of schizonepeta characteristic spectrum provided by the invention is used as using schizonepeta crude drug and schizonepeta medicine materical crude slice for examination Product effectively can monitor quality of medicinal material from the source of medicinal material, improve the practical application value of characteristic spectrum；Liquid-phase chromatographic analysis is surveyed The pre-treatment of examination method test sample has adjusted solvent and extraction operation, and preferred embodiment uses methanol as solvent, and reflux is used as and mentions Extract operation dramatically increases the amount of dissolution, and preparation process is easy to filter and handle, and increases color so that effective component solute effect is good The sensitivity of spectrum analysis；The present invention takes the volatile component preparation method in pharmacopeia, and method is mature, volatile component obtained Purity is high carries out the dissolved dilution of ethyl acetate, obtains the suitable concentration suitable for analysis, ethyl acetate as background solvent, Interference is not generated to test result, vapor detection result is accurate, high sensitivity, and speed is fast.

3, schizonepeta characteristic spectrum construction method provided by the invention during liquid-phase chromatographic analysis, passes through test of many times, second - 0.2% phosphoric acid water (B) of nitrile (A) is mobile phase, and the Detection wavelength of use is in 278nm-288nm, in this wave-length coverage, chaste tree The non-volatile effective component separating degree of mustard is good, cooperates gradient elution program, and each effective component that can separate schizonepeta well is special Chromatographic peak is levied, and obtained characteristic spectrum baseline is steady, peak shape is good, during gas chromatographic analysis, the temperature program of use It is to be obtained by multiple test assessment, under the temperature program, gas-chromatography peak separating degree is good, simple and quick.

4, schizonepeta quality determining method provided by the invention, safety quality testing and the inspection of validity quality including schizonepeta It surveys, safety quality testing includes heavy metal and harmful element inspection, persticide residue measurement, is examined by the method in pharmacopeia It surveys, the detection method standard, detection sensitivity and accuracy are good；Validity quality testing non-volatile has including the use of above-mentioned The characteristic spectrum of component detection method and volatile effective component detection method test sample is imitated, and establishes characteristic spectrum conduct pair According to the characteristic spectrum has Characteristic chromatographic peak, is used as the control of schizonepeta medicinal material characteristic spectrum to be controlled, passes through similar performance Easily evaluate the effective component situation of untested medicinal material；Validity quality testing further includes the assay of aurantiamarin, with feature The detection project of map complements each other, and qualitative and quantitative quality evaluation is carried out to schizonepeta sample, further includes thin-layered chromatography mirror Not and determination of extractives can be comprehensively and targetedly to schizonepeta medicinal material progress quality by this series of detection project Control, control method is simple and easy to do, and stability is reproducible.

5, schizonepeta quality determining method provided by the invention, wherein content of hesperidin measuring method, using test solution I Preparation process prepare assay test solution III, adjust elution program, obtain the liquid chromatogram peak of aurantiamarin best Presentation so that content of hesperidin testing result is accurate, high sensitivity.

6, the thin-layered chromatography in schizonepeta quality determining method provided by the invention identifies, and optimizes existing side in pharmacopeia Method uses ethyl acetate as solvent, and chloroform-ethyl acetate-glacial acetic acid is solvent, does not use color developing agent, uses instead glimmering Light is inspected, and the fluorescence spot obtained under fluorescence is clear, and separating degree is good, and the discrimination method is reliable and stable, reproducible, and should Processing method have many advantages, such as it is easy to operate, save the time.

7, the volatile oil thin-layered chromatography in schizonepeta quality determining method provided by the invention identifies, and is with Herba Schizonepetae volatile oil Test sample, petroleum ether (60~90 DEG C)-ethyl acetate are solvent, and vanillin-sulfuric acid ethanol solution is color developing agent, and colour developing obtains Clear spot, separating degree is good, can clearly identify the effective components such as menthones, pulegone, and the discrimination method is stablized Reliably, reproducible.

8, the determination of extractives in schizonepeta quality determining method provided by the invention is made using method existing in pharmacopeia Use the ethyl alcohol of 50-90% as solvent, effective component can dissolve out well under the solvent, increase determination of extractives accuracy and Sensitivity.

Detailed description of the invention

It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.

Fig. 1 is the characteristic spectrum of control medicinal material reference solution I in embodiment 1；

Fig. 2 is the characteristic spectrum of aurantiamarin reference substance reference solution I in embodiment 1；

Fig. 3 is the characteristic spectrum of test solution I in embodiment 1；

Fig. 4 is the characteristic spectrum of test solution II in embodiment 1；

Fig. 5 is schizonepeta involatile constituent compare feature map in embodiment 4；

Fig. 6 is schizonepeta volatile component compare feature map in embodiment 4；

Fig. 7 is the liquid chromatogram of aurantiamarin reference substance solution in embodiment 5；

Fig. 8 is the liquid chromatogram of test solution III in embodiment 5；

Regression curve when Fig. 9 is content of hesperidin measurement in embodiment 5；

Figure 10 is thin-layer chromatogram described in embodiment 8；

Figure 11 is thin-layer chromatogram described in embodiment 11；

Figure 12 is the thin-layer chromatogram obtained in experimental example using official method；

Figure 13 is the low-temperature study thin-layer chromatogram of thin-layered chromatography detection method in experimental example；

Figure 14 is that 18% humidity of thin-layered chromatography detection method in experimental example investigates thin-layer chromatogram；

Figure 15 is that 72% humidity of thin-layered chromatography detection method in experimental example investigates thin-layer chromatogram；

Figure 16 is that the specificity of thin-layered chromatography detection method in experimental example investigates thin-layer chromatogram；

Figure 17 is that the durability of thin-layered chromatography detection method in experimental example investigates Qingdao Haiyang chemical industry subsidiary factory silica gel g thin-layer plate Thin-layer chromatogram；

Figure 18 is that investigate Yantai Chemical Industry Research Inst.'s silica G thin for the durability of thin-layered chromatography detection method in experimental example Laminate thin-layer chromatogram；

Figure 19 is that the reproducibility of thin-layered chromatography detection method in experimental example investigates the thin layer that an experimenter operates Chromatogram；

Figure 20 be experimental example in thin-layered chromatography detection method reproducibility investigate another experimenter operate it is thin Layer chromatography figure；

Figure 21 is the low-temperature study thin-layer chromatogram of volatile oil thin-layered chromatography detection method in experimental example；

Figure 22 is that 18% humidity of volatile oil thin-layered chromatography detection method in experimental example investigates thin-layer chromatogram；

Figure 23 is that 72% humidity of volatile oil thin-layered chromatography detection method in experimental example investigates thin-layer chromatogram；

Figure 24 is that the specificity of volatile oil thin-layered chromatography detection method in experimental example investigates thin-layer chromatogram；

Figure 25 is that the durability of volatile oil thin-layered chromatography detection method in experimental example investigates Qingdao Haiyang chemical industry subsidiary factory silica G Lamellae thin-layer chromatogram；

Figure 26 is that the durability of volatile oil thin-layered chromatography detection method in experimental example investigates Yantai Chemical Industry Research Inst. Silica gel g thin-layer plate thin-layer chromatogram；

Figure 27 is that reproducibility one experimenter of investigation of volatile oil thin-layered chromatography detection method in experimental example operates to obtain Thin-layer chromatogram；

Figure 28 investigates another experimenter for the reproducibility of volatile oil thin-layered chromatography detection method in experimental example and operates The thin-layer chromatogram arrived；

Figure 29 is the liquid chromatogram that elution process 1 obtains in the investigation of assay liquid phase chromatogram condition in experimental example；

Figure 30 is the liquid chromatogram that elution process 2 obtains in the investigation of assay liquid phase chromatogram condition in experimental example；

Figure 31 is the liquid chromatogram that elution process 3 obtains in the investigation of assay liquid phase chromatogram condition in experimental example；

Figure 32 is the liquid chromatogram that elution process 4 obtains in the investigation of assay liquid phase chromatogram condition in experimental example；

Figure 33 is schizonepeta DAD full wavelength scanner map in experimental example；

Figure 34 is the liquid phase color that elution process 1 obtains in the investigation of involatile constituent HPLC liquid phase chromatogram condition in experimental example Spectrogram；

Figure 35 is the liquid phase color that elution process 2 obtains in the investigation of involatile constituent HPLC liquid phase chromatogram condition in experimental example Spectrogram；

Figure 36 is the liquid phase color that elution process 3 obtains in the investigation of involatile constituent HPLC liquid phase chromatogram condition in experimental example Spectrogram；

Figure 37 is experimental example involatile constituent compare feature map；

Figure 38 is that volatile component gas chromatography detects the gas chromatogram that temperature program 1 obtains in test example；

Figure 39 is that volatile component gas chromatography detects the gas chromatogram that temperature program 2 obtains in test example；

Figure 40 is that volatile component gas chromatography detects the gas chromatogram that temperature program 3 obtains in test example；

Figure 41 is that volatile component gas chromatography detects the gas chromatogram that temperature program 4 obtains in test example；

Figure 42 is that volatile component gas chromatography detects the gas chromatogram that temperature program 5 obtains in test example；

Figure 43 is volatile component compare feature map in experimental example.

Specific embodiment

There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention Within protection scope.

Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition Conventional reagent product.

The schizonepeta characteristic spectrum detection method of the embodiment of the present invention is specifically described below.

1, laboratory apparatus and reagent used in the embodiment of the present invention are shown in Table 1.

1 laboratory apparatus of table and reagent statistical form

2, below with reference to embodiment, the present invention is further described:

Embodiment 1

Present embodiments provide a kind of construction method of schizonepeta characteristic spectrum, the structure including involatile constituent characteristic spectrum The construction method of construction method and volatile component characteristic spectrum, specific as follows:

A, the construction method of the involatile constituent characteristic spectrum, includes the following steps:

The preparation of test solution I: take originate from Anhui for examination schizonepeta medicinal powder (cross No. two sieve) 0.5g, set tool plug three In the bottle of angle, add methanol 25ml, close plug, weighed weight flows back 30 minutes, lets cool, the weight of less loss is supplied with methanol, is shaken up, is taken Supernatant filtration, take subsequent filtrate to get；

The preparation of reference solution I: schizonepeta control medicinal material powder (crossing No. two sieves) 0.5g is taken, sets in triangular flask, adds Methanol 25ml, close plug, weighed weight flow back 30 minutes, let cool, the weight of less loss is supplied with methanol, is shaken up, supernatant is taken to filter It crosses, takes subsequent filtrate to get control medicinal material reference solution I；It is appropriate to weigh aurantiamarin reference substance, methanol is added to dissolve, every 1ml is made The reference substance reference solution I of the 60 μ g containing aurantiamarin；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Column packing is composed, is selected Waters column (250mm × 4.6mm, 5 μm)；It is 0.2% with volume fraction using acetonitrile as mobile phase A Phosphate aqueous solution is Mobile phase B, carries out gradient elution: 0-15min according to the procedure below, mobile phase A: the volume ratio of Mobile phase B For 10%:90% → 14%:86%；15-40min, mobile phase A: the volume ratio of Mobile phase B is 14%:86% → 26%: 74%；40-55min, mobile phase A: the volume ratio of Mobile phase B is 26%:74% → 60%:40%；55-60min, mobile phase A: The volume ratio of Mobile phase B is 60%:40% → 85%:15%；60-65min, mobile phase A: the volume ratio of Mobile phase B is 85%: 15% → 85%:15%；Column temperature is 40 DEG C；Flow velocity is 1ml/min；Detection wavelength is 283nm, number of theoretical plate based on aurantiamarin not Lower than 3000；

Measuring method: it is accurate respectively to draw reference solution I and each 10 μ l of test solution I, liquid chromatograph is injected, is surveyed It is fixed to get；The characteristic spectrum of the control medicinal material reference solution I is as shown in Figure 1, the aurantiamarin reference substance reference solution The characteristic spectrum of I is as shown in Fig. 2, the characteristic spectrum of the test solution I is as shown in Figure 3.

B, the construction method of the volatile component characteristic spectrum, includes the following steps:

The preparation of test solution II: take originate from Anhui for try the schizonepeta medicinal material Pharmacopoeia of the People's Republic of China 2015 Year, the first legal system of four 2204 determination of volatile oil of general rule of version obtained volatile oil, and precision draws 50 μ l, ethyl acetate is added to be settled to 2ml, Add anhydrous sodium sulfate dry, shake up, take subsequent filtrate to obtain the final product；

The preparation of reference solution II: it is molten to be dissolved in 30ml ethyl acetate by accurately weighed pulegone and each 0.5g of menthones Liquid, then it is settled to 100ml, obtain the mixed reference substance solution containing pulegone 5mg/ml, menthones 5mg/ml；

Chromatographic condition and system suitability: it is measured according to gas chromatography, with N₂For carrier gas, with PEG (DB-WAX, It 30m*0.530mm) is stationary phase, injector temperature is 250 DEG C, and detector temperature is 250 DEG C, and control column temperature heats up in gradient, The gradient increased temperature program are as follows: initial temperature is 60 DEG C, keeps 2min；Temperature rises to 120 DEG C from 60 DEG C, then keeps 5min, Heating rate is 2 DEG C/min；Temperature rises to 160 DEG C from 120 DEG C, and heating rate is 3 DEG C/min；Temperature rises to 240 from 160 DEG C DEG C, heating rate is 5 DEG C/min；Total flow 3.7ml/min, number of theoretical plate are calculated as 3200 by pulegone peak；

Measuring method: it is accurate respectively to draw reference solution II and each 5 μ l of test solution II, gas chromatograph is injected, is surveyed It is fixed to get；The characteristic spectrum of the test solution II is as shown in Figure 4.

Embodiment 2

Present embodiments provide a kind of construction method of schizonepeta characteristic spectrum, the structure including involatile constituent characteristic spectrum It is the step of construction method of the step of construction method and volatile component characteristic spectrum, specific as follows:

A, the construction method of the involatile constituent characteristic spectrum, includes the following steps:

The preparation of test solution I: take originate from Anhui for examination schizonepeta medicinal powder (cross No. two sieve) 1g, set tool plug triangle In bottle, add methanol 15ml, close plug, weighed weight flows back 60 minutes, lets cool, the weight of less loss is supplied with methanol, is shaken up, is taken Clear liquid filtration, take subsequent filtrate to get；

The preparation of reference solution I: schizonepeta control medicinal material powder (crossing No. two sieves) 1g is taken, sets in triangular flask, adds first Alcohol 15ml, close plug, weighed weight flow back 60 minutes, let cool, the weight of less loss is supplied with methanol, is shaken up, supernatant is taken to filter, Take subsequent filtrate to get control medicinal material reference solution I；It is appropriate to weigh aurantiamarin reference substance, methanol is added to dissolve, every 1ml is made and contains The reference substance reference solution I of 55 μ g of aurantiamarin；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Column packing is composed, is selected AlchromBond-AQ column (250mm × 4.6mm, 5 μm)；Using acetonitrile as mobile phase A, with volumetric concentration Phosphate aqueous solution for 0.2% is Mobile phase B, carries out gradient elution: 0-15min according to the procedure below, mobile phase A: Mobile phase B Volume ratio be 8%:92% → 12%:88%；15-40min, mobile phase A: the volume ratio of Mobile phase B be 12%:88% → 24%:76%；40-55min, mobile phase A: the volume ratio of Mobile phase B is 24%:76% → 58%:42%；55-60min, stream Dynamic phase A: the volume ratio of Mobile phase B is 58%:42% → 83%:17%；60-65min, mobile phase A: the volume ratio of Mobile phase B For 83%:17% → 83%:17%；Column temperature is 35 DEG C；Flow velocity is 0.8ml/min；Detection wavelength is 278nm, and number of theoretical plate is pressed Aurantiamarin is calculated as 12035；

Measuring method: it is accurate respectively to draw reference solution I and each 10 μ l of test solution I, liquid chromatograph is injected, is surveyed It is fixed to get.

B, the construction method of the volatile component characteristic spectrum, includes the following steps:

The preparation of test solution II: take originate from Anhui for try the schizonepeta medicinal material Pharmacopoeia of the People's Republic of China 2015 Year, the first legal system of four 2204 determination of volatile oil of general rule of version obtained volatile oil, and precision draws 25 μ l, ethyl acetate is added to be settled to 2ml, Add anhydrous sodium sulfate dry, shake up, take subsequent filtrate to obtain the final product；

The preparation of reference solution II: it is molten to be dissolved in 30ml ethyl acetate by accurately weighed pulegone and each 0.3g of menthones Liquid, then it is settled to 100ml, obtain the mixed reference substance solution containing pulegone 3mg/ml, menthones 3mg/ml；

Chromatographic condition and system suitability: according to gas chromatography measure, using N2 as carrier gas, with PEG (DB-WAX, It 30m*0.530mm) is stationary phase, injector temperature is 245 DEG C, and detector temperature is 245 DEG C, and control column temperature heats up in gradient, The gradient increased temperature program are as follows: column temperature initial temperature is 55 DEG C, keeps 1min；Temperature rises to 115 DEG C from 55 DEG C, then keeps 4min, heating rate are 1.5 DEG C/min；Temperature rises to 155 DEG C from 115 DEG C, and heating rate is 2.5 DEG C/min；Temperature is from 155 DEG C 235 DEG C are risen to, heating rate is 4.5 DEG C/min；Total flow 3.5ml/min, number of theoretical plate are calculated as 4000 by pulegone peak；

Measuring method: it is accurate respectively to draw reference solution II and each 5 μ l of test solution II, gas chromatograph is injected, is surveyed It is fixed to get.

Embodiment 3

Present embodiments provide a kind of construction method of schizonepeta characteristic spectrum, the structure including involatile constituent characteristic spectrum The construction method step of construction method step and volatile component characteristic spectrum, specific as follows:

A, the construction method of the involatile constituent characteristic spectrum, includes the following steps:

The preparation of test solution I: take originate from Hebei for examination schizonepeta medicine materical crude slice powder (cross No. two sieve) 0.8g, set tool plug three In the bottle of angle, add ethyl alcohol 30ml, close plug, weighed weight flows back 120 minutes, lets cool, the weight of less loss is supplied with ethyl alcohol, is shaken up, is taken Supernatant filtration, take subsequent filtrate to get；

The preparation of reference solution I: schizonepeta control medicinal material powder (crossing No. two sieves) 0.8g is taken, sets in triangular flask, adds Ethyl alcohol 30ml, close plug, weighed weight flow back 120 minutes, let cool, the weight of less loss is supplied with ethyl alcohol, is shaken up, supernatant is taken to filter It crosses, takes subsequent filtrate to get control medicinal material reference solution I；It is appropriate to weigh aurantiamarin reference substance, methanol is added to dissolve, every 1ml is made The reference substance reference solution I of the 65 μ g containing aurantiamarin；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Column packing is composed, is selected Phenomex-Luna column (250mm × 4.6mm, 5 μm)；Using acetonitrile as mobile phase A, with 0.2% phosphoric acid Aqueous solution be Mobile phase B, carry out gradient elution: 0-15min according to the procedure below, mobile phase A: the volume ratio of Mobile phase B is 12%:88% → 16%:84%；15-40min, mobile phase A: the volume ratio of Mobile phase B is 16%:84% → 28%:72%； 40-55min, mobile phase A: the volume ratio of Mobile phase B is 28%:72% → 62%:38%；55-60min, mobile phase A: flowing The volume ratio of phase B is 62%:38% → 87%:13%；60-65min, mobile phase A: the volume ratio of Mobile phase B is 87%:13% → 87%:13%；Column temperature is 25 DEG C；Flow velocity is 1.2ml/min；Detection wavelength is 288nm, and number of theoretical plate is calculated as by aurantiamarin 10253；

Measuring method: it is accurate respectively to draw reference solution I and each 10 μ l of test solution I, liquid chromatograph is injected, is surveyed It is fixed to get.

B, the construction method of the volatile component characteristic spectrum, includes the following steps:

The preparation of test solution II: take originate from Anhui for try the schizonepeta medicinal material Pharmacopoeia of the People's Republic of China 2015 Year, the first legal system of four 2204 determination of volatile oil of general rule of version obtained volatile oil, and precision draws 80 μ l, ethyl acetate is added to be settled to 2ml, Add anhydrous sodium sulfate dry, shake up, take subsequent filtrate to obtain the final product；

The preparation of reference solution II: it is molten to be dissolved in 30ml ethyl acetate by accurately weighed pulegone and each 0.6g of menthones Liquid, then it is settled to 100ml, obtain the mixed reference substance solution containing pulegone 6mg/ml, menthones 6mg/ml；

Chromatographic condition and system suitability: according to gas chromatography measure, using N2 as carrier gas, with PEG (DB-WAX, It 30m*0.530mm) is stationary phase, injector temperature is 245 DEG C, and detector temperature is 245 DEG C, and control column temperature heats up in gradient, The gradient increased temperature program are as follows: column temperature initial temperature is 65 DEG C, keeps 3min；Temperature rises to 125 DEG C from 65 DEG C, then keeps 6min, heating rate are 2.5 DEG C/min；Temperature rises to 165 DEG C from 125 DEG C, and heating rate is 3.5 DEG C/min；Temperature is from 165 DEG C 245 DEG C are risen to, heating rate is 5.5 DEG C/min；Total flow 3.9ml/min, number of theoretical plate are calculated as 5000 by pulegone peak；

Measuring method: it is accurate respectively to draw reference solution II and each 5 μ l of test solution II, gas chromatograph is injected, is surveyed It is fixed to get.

Embodiment 4

Present embodiments provide a kind of construction method of schizonepeta characteristic spectrum, including schizonepeta involatile constituent compare feature The building of map and the building of volatile component compare feature map:

A, the building of schizonepeta involatile constituent compare feature map includes:

Totally 20, sample of different batches are randomly selected, according to the spy of the non-volatile effective ingredient of schizonepeta described in embodiment 1 Sign map construction method measures the feature chromatography of the non-volatile effective component of 20 schizonepeta samples, utilizes Chinese Pharmacopoeia Commission Similarity evaluation, the compare feature map of schizonepeta involatile constituent is obtained, such as Fig. 5 institute Show；

By in Fig. 6 it is found that the involatile constituent compare feature map include 6 characteristic peaks, common characteristic peaks are as follows: 1 Number peak is aurantiamarin, and No. 2 peaks are Rosmarinic acid, and No. 3 peaks, No. 4 peaks, No. 5 peaks are common characteristic peaks, and No. 6 peaks are pulegone；With No. 1 peak is referring to peak, and each peak relative retention time is respectively as follows: No. 1 peak: 1.00；No. 2 peaks: 1.07；No. 3 peaks: 1.09；No. 4 Peak: 1.20；No. 5 peaks: 1.25；No. 6 peaks: 1.64；The feature chromatogram of above-mentioned 6 characteristic peaks and control medicinal material reference solution I In 6 characteristic peaks it is corresponding, wherein when opposite in No. 1 peak and No. 6 peaks and the chromatogram of reference substance reference solution I retains Between it is consistent；

B, the building of schizonepeta volatile component characteristic spectrum includes:

Totally 20, schizonepeta sample of different batches are randomly selected, according to schizonepeta volatility effective ingredient described in embodiment 1 Characteristic spectrum detection method measures the feature chromatography of schizonepeta volatile effective component, utilizes the Chinese medicine chromatography of Chinese Pharmacopoeia Commission Fingerprint similarity evaluation system obtains schizonepeta volatile component compare feature map, as shown in Figure 6；

By in Fig. 6 it is found that the schizonepeta volatile component characteristic spectrum include 3 characteristic peaks, common characteristic peaks are as follows: No. 1 Peak is menthones, and No. 2 peaks are pulegone, and No. 3 peaks are common characteristic peaks；It is referring to peak, when each peak number retains relatively with No. 2 peaks Between be respectively as follows: No. 1 peak: 0.67；No. 2 peaks: 1.00；No. 3 peaks: 1.50；Wherein No. 1 peak and No. 2 peaks and object of reference in embodiment 1 are molten The relative retention time at object of reference peak is consistent in the characteristic spectrum of liquid II.

Embodiment 5

The quality determining method for present embodiments providing a kind of schizonepeta includes the steps that carrying out schizonepeta assay, packet Include following steps:

Test solution III: it takes originate from Anhui powder (cross No. two sieve) 0.5g is made for examination schizonepeta medicine materical crude slice, sets tool plug three In the bottle of angle, add methanol 25ml, close plug, weighed weight flows back 30 minutes, lets cool, the weight of less loss is supplied with methanol, is shaken up, is taken Supernatant filtration, take subsequent filtrate to get；

Reference substance solution: aurantiamarin (C is weighed₂₈H₃₄O₁₅) appropriate reference substance, add methanol to dissolve, every 1ml is made containing aurantiamarin 1. the reference substance solution of 68.231 μ g, then carries out serial dilution, be made concentration be respectively 34.116 μ g/ml, 17.058 μ g/ml, 8.529 μ g/ml, 4.264 μ g/ml, 2.132 μ g/ml aurantiamarin reference substance solution 2. -6.；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Column packing is composed, is selected Waters column (250mm × 4.6mm, 5 μm)；Using acetonitrile as mobile phase A, to contain volumetric concentration for 0.2% Phosphate aqueous solution be Mobile phase B carry out gradient elution, according to following procedure carry out gradient elution: 0-28min, mobile phase A: stream The volume ratio of dynamic phase B is 0-28min, and mobile phase A: the volume ratio of Mobile phase B is 18%:82% → 18%:82%；28- 29min, mobile phase A: the volume ratio of Mobile phase B is 18%:82% → 85%:15%；29-39min, mobile phase A: Mobile phase B Volume ratio be 85%:15% → 85%:15%；Column temperature is 30 DEG C；Flow velocity is 1ml/min；Detection wavelength is 283nm；It is theoretical Plate number is calculated as 9000 by aurantiamarin；

Measuring method: accurate absorption reference substance solution and test solution III10ul respectively, injection liquid chromatograph, measurement, To obtain the final product；The chromatogram of the reference substance solution 1. is as shown in fig. 7, the chromatogram of the test solution III is as shown in Figure 8；

Calculate: by reference substance solution 1. -6. detect respectively twice, peak area and average peak area are calculated separately, such as 2 institute of table Show, obtain the linear relationship of aurantiamarin concentration and average peak area, and be fitted the regression equation of test sample, aurantiamarin is in 2.132 μ Linear relationship is good within the scope of the μ of g/ml~68.231 g/ml, the regression equation be Y=21954X, r=0.9997, described time Return the curve graph of equation as shown in Figure 7；The content of aurantiamarin is calculated with one point external standard method, the content of hesperidin being calculated is seen below Table 3, this test sample are calculated by dry product, (the C containing aurantiamarin₂₈H₃₄O₁₅) 0.06 must not be less than.

2 schizonepeta aurantiamarin linear relationship of table is investigated

Concentration (μ g/ml)	Peak area 1	Peak area 2	Average peak area
				2.132	45689	47099	46394
4.264	94946	94734	94840
				8.529	190110	187972	189041
17.058	375568	347321	361444.5
				34.116	696680	755340	726010
68.231	1541295	1483502	1512399

The content of aurantiamarin in 3 sample of table

Sample name	Aurantiamarin (%)
		Sample 1	0.309

Embodiment 6

The quality determining method for present embodiments providing a kind of schizonepeta includes the steps that carrying out schizonepeta assay, packet Include following steps:

Test solution III: take originate from Anhui for examination schizonepeta medicinal powder (cross No. two sieve) 1g, set triangular flask It is interior, add methanol 15ml, close plug, weighed weight flows back 60 minutes, lets cool, the weight of less loss is supplied with methanol, is shaken up, supernatant is taken Liquid filtration, take subsequent filtrate to get；

Reference substance solution: it is prepared according to aurantiamarin reference substance solution preparation method described in embodiment 5；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Column packing is composed, is selected AlchromBond-AQ column (250mm × 4.6mm, 5 μm)；Using acetonitrile as mobile phase A, with volume fraction Phosphate aqueous solution for 0.2% is that Mobile phase B carries out gradient elution, carries out gradient elution: 0-28min, stream according to following procedure Dynamic phase A: the volume ratio of Mobile phase B is 17%:83% → 17%:83%；28-29min, mobile phase A: the volume ratio of Mobile phase B For 17%:83% → 84%:16%；29-39min, mobile phase A: the volume ratio of Mobile phase B is 84%:16% → 84%: 16%；Column temperature is 35 DEG C；Flow velocity is 0.8ml/min；Detection wavelength is 278nm；Number of theoretical plate is calculated as 11235 by aurantiamarin；

Measurement and calculating: schizonepeta described in the present embodiment is obtained for reagent material according to measurement and calculation method described in embodiment 5 Content of hesperidin, as shown in table 4.

The content of aurantiamarin in 4 sample of table

Sample name	Aurantiamarin (%)
		Sample	0.187

Embodiment 7

The quality determining method for present embodiments providing a kind of schizonepeta includes the steps that carrying out schizonepeta assay, packet Include following steps:

Test solution III: take originate from Hebei for examination schizonepeta medicinal powder (cross No. two sieve) 0.8g, set triangular flask It is interior, add ethyl alcohol 30ml, close plug, weighed weight flows back 120 minutes, lets cool, the weight of less loss is supplied with ethyl alcohol, is shaken up, supernatant is taken Liquid filtration, take subsequent filtrate to get；

Reference substance solution: it is prepared according to aurantiamarin reference substance solution preparation method described in embodiment 5；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as color Column packing is composed, is selected Phenomex-Luna column (250mm × 4.6mm, 5 μm)；Using acetonitrile as mobile phase A, with 0.2% phosphoric acid Aqueous solution be Mobile phase B carry out gradient elution, according to following procedure carry out gradient elution: 0-28min, mobile phase A: mobile phase The volume ratio of B is 19%:81% → 19%:81%；28-29min, mobile phase A: the volume ratio of Mobile phase B be 19%:81% → 86%:14%；29-39min, mobile phase A: the volume ratio of Mobile phase B is 86%:14% → 86%:14%；Column temperature is 33 DEG C； Flow velocity is 1.2ml/min；Detection wavelength is 288nm；Number of theoretical plate is not less than 3000 based on aurantiamarin；

Measurement and calculating: schizonepeta described in the present embodiment is obtained for reagent material according to measurement and calculation method described in embodiment 5 Content of hesperidin, as shown in table 5.

The content of aurantiamarin in 5 sample of table

Sample name	Aurantiamarin (%)
		Sample 3	0.174

Embodiment 8

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out involatile constituent thin layer to schizonepeta It is the step of chromatographic identification, specific as follows:

Take originate from Anhui for examination schizonepeta crude drug, the schizonepeta medicine materical crude slice and each 0.8g of schizonepeta control medicinal material that originate from Hebei, point It not plus ethyl acetate 20ml, is 250W with power, frequency is ultrasonic treatment 20 minutes of 40KHZ, filtration, and filtrate is waved to 1ml, obtained To 3 parts of filtrates, each accurate absorption 2ul is put respectively in same silica G plate, plate sets presaturation 20 minutes in expansion cylinder, in volume ratio To be unfolded in chloroform-ethyl acetate-glacial acetic acid Mixed Expansion agent of 10:1:0.5, takes out, dry, setting wavelength is 366nm It is inspected under ultraviolet light, the results are shown in Figure 10, and wherein serial number 1 is schizonepeta control medicinal material, and serial number 2 is schizonepeta crude drug, and serial number 3 is Schizonepeta medicine materical crude slice.

Embodiment 9

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out thin layer to schizonepeta involatile constituent It is the step of chromatographic identification, specific as follows:

Take originate from Anhui for examination schizonepeta crude drug, the schizonepeta medicine materical crude slice and each 0.5g of schizonepeta control medicinal material that originate from Hebei, point It not plus ethyl acetate 15ml, is 250W with power, frequency is ultrasonic treatment 15 minutes of 40KHZ, filtration, and filtrate is waved to 1ml, respectively Precision draws 1ul, is put respectively in same silica gel H plate, mixed in chloroform-ethyl acetate-glacial acetic acid that volume ratio is 9:1:0.3 It closes and is unfolded in solvent, take out, dry, setting wavelength is to inspect under 361nm ultraviolet light.

Embodiment 10

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out involatile constituent thin layer to schizonepeta It is the step of chromatographic identification, specific as follows:

Take originate from Anhui for examination schizonepeta crude drug, the schizonepeta medicine materical crude slice and each 1g of schizonepeta control medicinal material that originate from Hebei, respectively Add ethyl acetate 25ml, be ultrasonically treated 25 minutes, filtration, filtrate is waved to 1ml, and each accurate absorption 5ul is put respectively in same silica gel H plate is unfolded in chloroform-ethyl acetate-glacial acetic acid Mixed Expansion agent that volume ratio is 11:1:0.8, takes out, dries, set Wavelength is to inspect under 371nm ultraviolet light.

Embodiment 11

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out thin layer color to schizonepeta volatile component The step of spectrum identifies, specific as follows:

Take originate from Anhui for try schizonepeta crude drug, schizonepeta medicine materical crude slice, according to determination of volatile oil method (Pharmacopoeia of People's Republic of China Four general rules 2204 of version in 2015) measurement, divides and takes volatile oil 0.2ml, add ethyl acetate to be diluted to 3ml, it is molten to obtain 2 parts of test samples Liquid.(-)-menthones, pulegone's reference substance are separately taken, adds ethyl acetate that solution of every 1ml containing 20mg is made, obtains 2 parts of controls Product solution.Above-mentioned solution 1ul is respectively drawn, is put respectively on same silica G plate, in the petroleum ether (60~90 that volume ratio is 12:1 DEG C) be unfolded in-ethyl acetate Mixed Expansion agent, it takes out, dries, spray is heated with 5% vanillin-sulfuric acid ethanol solution at 105 DEG C Clear to spot development, as a result as shown in figure 11, wherein serial number 1 is (-)-menthones, and serial number 2 is pulegone, and serial number 3 is chaste tree Mustard medicinal material, serial number 4 are schizonepeta medicine materical crude slice.

Embodiment 12

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out thin-layer chromatography mirror to Herba Schizonepetae volatile oil Other step, specific as follows:

Take originate from Anhui for try schizonepeta crude drug, schizonepeta medicine materical crude slice, according to determination of volatile oil method (Pharmacopoeia of People's Republic of China Four general rules 2204 of version in 2015) measurement, divides and takes volatile oil 0.1ml, add ethyl acetate to be diluted to 1.5ml, obtain 2 parts of test samples Solution.Separately take (-)-menthones, pulegone's reference substance, add ethyl acetate that solution of every 1ml containing 30mg is made, obtain 2 parts it is right According to product solution.Respectively draw above-mentioned solution 2ul, put respectively on same silica G plate, in petroleum ether that volume ratio is 11:1 (60~ 90 DEG C) be unfolded in-ethyl acetate Mixed Expansion agent, take out, dry, spray with 4% vanillin-sulfuric acid ethanol solution, at 105 DEG C plus Heat is clear to spot development.

Embodiment 13

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out thin layer color to schizonepeta volatile component The step of spectrum identifies, specific as follows:

Take originate from Anhui for try schizonepeta crude drug, schizonepeta medicine materical crude slice, according to determination of volatile oil method (Pharmacopoeia of People's Republic of China Four general rules 2204 of version in 2015) measurement, divides and takes volatile oil 0.3ml, add ethyl acetate to be diluted to 4.5ml, obtain 2 parts of test samples Solution.Separately take (-)-menthones, pulegone's reference substance, add ethyl acetate that solution of every 1ml containing 10mg is made, obtain 2 parts it is right According to product solution.Respectively draw above-mentioned solution 5ul, put respectively on same silica G plate, in petroleum ether that volume ratio is 13:1 (60~ 90 DEG C) be unfolded in-ethyl acetate Mixed Expansion agent, take out, dry, spray with 6% vanillin-sulfuric acid ethanol solution, at 105 DEG C plus Heat is clear to spot development.

Embodiment 14

The quality determining method for present embodiments providing a kind of schizonepeta further includes the step for carrying out leaching analyte detection to schizonepeta Suddenly, specific as follows:

The coarse powder 4g for examination schizonepeta crude drug preparation for originating from Anhui is taken, accurately weighed, precision plus volume fraction are 70% Ethyl alcohol 100ml, shake 6 hours, stand 18h, filtration, precision measure subsequent filtrate 20ml, set the evaporating dish dried to constant weight In, it is evaporated, it is 3 hours dry in 105 DEG C, cooling 30 minutes in drier are set, weighed weight calculates the content of extract；It measures Determination of extractives content is 8.03%.

Embodiment 15

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out leaching analyte detection to schizonepeta, specifically It is as follows:

Take originate from Anhui for try schizonepeta medicinal material coarse powder 2g, it is accurately weighed, precision plus volume fraction be 50% ethyl alcohol 100ml shakes 4 hours, stands 16 hours, and filtration, precision measures subsequent filtrate 10ml, sets and has dried into the evaporating dish of constant weight, It is evaporated, it is 3 hours dry in 100 DEG C, cooling 30 minutes in drier are set, weighed weight calculates the content of extract；Measure leaching It is 8.54% that object, which measures content,.

Embodiment 16

The quality determining method for present embodiments providing a kind of schizonepeta further includes carrying out leaching analyte detection to schizonepeta, specifically It is as follows:

The coarse powder 5g for examination schizonepeta medicine materical crude slice preparation for originating from Hebei is taken, accurately weighed, precision plus volume fraction are 95% Ethyl alcohol 100ml shakes 9 hours, stands 36 hours, and filtration, precision measures subsequent filtrate 30ml, sets the evaporating dish dried to constant weight In, it is evaporated, it is 5 hours dry in 120 DEG C, cooling 30 minutes in drier are set, weighed weight calculates the content of extract；It measures Determination of extractives content is 19.20%.

Embodiment 17

The quality determining method for present embodiments providing a kind of schizonepeta further includes the measurement of heavy metal and harmful element, packet Include the assay of heavy metal lead, cadmium, arsenic, mercury, copper, used test sample be originate from Anhui for trying schizonepeta medicinal material (sample 1), originate from Hebei for examination schizonepeta medicinal material (sample 2), originate from Hebei for try schizonepeta medicine materical crude slice (sample 3), step specific as follows:

According to lead, cadmium, arsenic, mercury, copper measuring method (2321 atomic absorption spectrophotometry of " Chinese Pharmacopoeia " general rule or inductive coupling Plasma Mass Spectrometry) it measures, lead must not cross 5mg/kg；Cadmium must not cross 0.3mg/kg；Arsenic must not cross 2mg/kg；Mercury must not mistake 0.2mg/kg；Copper must not cross 20mg/kg.

Sample lead, cadmium, arsenic, mercury, copper content see the table below 6.

Lead, cadmium, arsenic, mercury, copper content testing result in 6 sample of table

Sample name	Lead (mg/kg)	Cadmium (mg/kg)	Arsenic (mg/kg)	Mercury (mg/kg)	Copper (mg/kg)
						Sample 1	0.29	0.022	0.12	0.010	1.48
Sample 2	0.79	0.067	0.26	0.018	5.71
						Sample 3	1.12	0.150	0.16	0.014	5.18

According to persticide residue measuring method (2341 the-the first method of organic chlorine agriculture chemicals determination of residual amount method of " Chinese Pharmacopoeia " general rule) Measurement.

(the sum of α-BHC, β-BHC, γ-BHC, δ-BHC) containing total six six six must not cross 0.2mg/kg；Total DDT (pp'- DDE, pp'-DDD, op'-DDT, the sum of pp'-DDT) 0.2mg/kg must not be crossed；Pentachloronitrobenzene must not cross 0.1mg/kg.

Sample persticide residue result see the table below 7.

7 sample persticide residue measurement result of table

Sample name	Total six six six (mg/kg)	Total DDT (mg/kg)	Pentachloronitrobenzene (mg/kg)
				Sample 1	(< 0.01) is not detected	(< 0.01) is not detected	(< 0.01) is not detected
Sample 2	(< 0.01) is not detected	(< 0.01) is not detected	(< 0.01) is not detected
				Sample 3	(< 0.01) is not detected	(< 0.01) is not detected	(< 0.01) is not detected

Experimental example

1, schizonepeta thin-layered chromatography discrimination method

The optimization of 1.1 methods

Schizonepeta medicinal material is carried out according to the lower thin-layer identification method of schizonepeta in Pharmacopoeia of the People's Republic of China version in 2015 Identify, as follows:

Schizonepeta medicinal material coarse powder 0.8g is taken, petroleum ether (60~90 DEG C) 20ml is added, close plug constantly shakes, stands overnight, filter It crosses, filtrate is waved to 1ml, as test solution.Schizonepeta control medicinal material 0.8g separately is taken, is made in the same way of control medicinal material solution；According to thin Layer chromatography (four general rules 0502 of Pharmacopoeia of the People's Republic of China version in 2015) test, draws each 10 μ of above two solution L puts respectively on same silica gel H lamellae, with n-hexane-ethyl acetate (17:3) for solvent, is unfolded, takes out, dry, spray With 5% ethanol solution of sulfuric acid of 5% vanillic aldehyde, it is clear that spot development is heated at 105 DEG C.In sample chromatogram, with compare On the corresponding position of medicinal material chromatography, the spot of same color is shown.Figure 12 is shown in using the thin-layer chromatogram that official method obtains.In figure " control "-reference medicine chromatography, No. 2 from left to right, No. 8, No. 10, No. 11 and No. 12 successively represent 5 kinds of different sources for examination Schizonepeta medicinal material.It is ineffective using official method identification Rhizoma Atractylodis Macrocephalae by can be seen that in figure, therefore, it is optimized.

(1) extracting method is investigated

Official method are as follows: take schizonepeta coarse powder 0.8g, add petroleum ether (60~90 DEG C) 20ml, close plug constantly shakes, placed At night, filtration, filtrate is waved to 1ml, as test solution；In order to shorten the time, pass through Different Extraction Method, different solvents Comparison, determines extracting method are as follows: takes schizonepeta medicine materical crude slice coarse powder 0.8g, adds ethyl acetate 20ml, ultrasound 20 minutes filters, and filtrate is waved To 1ml, as test solution.

(2) coloration method is investigated

Official method are as follows: it is clear to be heated to spot development at 105 DEG C with 5% ethanol solution of sulfuric acid of 5% vanillic aldehyde for spray. The study found that the lamellae after expansion has multiple apparent fluorescence spots, and relatively spray color developing agent effect at ultraviolet lamp 366nm More preferably, more environmentally friendly, therefore selection is inspected at ultraviolet lamp 366nm.

(3) solvent selects

The type and proportion of solvent are groped, select petroleum ether-ethyl acetate, n-hexane-acetic acid second respectively Ester, toluene-ethyl acetate, cyclohexane-ethyl acetate-isopropanol-formic acid, n-hexane-acetone-glacial acetic acid, methylene chloride-acetic acid Ethyl ester-glacial acetic acid, dichloromethane-ethanol-glacial acetic acid, hexamethylene-dichloromethane-ethyl acetate-glacial acetic acid, chloroform-second Acetoacetic ester-glacial acetic acid etc. be used as solvent, final choice chloroform-ethyl acetate-glacial acetic acid be solvent, ratio 10:1: 0.5 is preferred.

(4) lamellae is investigated

It is investigated by silica G plate, H plate, GF254 plate etc., determines that silica G plate separating effect is best.

1.2 methodological study

(1) low-temperature study

It is operated according to the scheme in the embodiment of the present invention 8, point sample, is unfolded in 4 DEG C of refrigerators, taken out, dry, set purple It is inspected under outer smooth lamp (366nm), the results show that being unfolded to work well in the environment at 4 DEG C, sees Figure 13,1- comparison medicine in figure Material；2- schizonepeta medicinal material；3- schizonepeta medicine materical crude slice.

(2) humidity is investigated

It is operated according to the scheme in the embodiment of the present invention 8, point sample, is unfolded in 18%, 72% relative humidity, takes Out, it dries, sets and inspected under ultraviolet lamp (366nm), the results show that expansion effect is preferable in different humidity environments, see Figure 14 (18%), Figure 15 (72%), in Figure 14-15,1- control medicinal material；2- schizonepeta medicinal material；3- schizonepeta medicine materical crude slice.

(3) specificity is investigated

It is operated according to the scheme in the embodiment of the present invention 8, using ethyl acetate reagent as blank solution, point sample, Expansion takes out, dries, set and inspect under ultraviolet lamp (366nm), the results show that ethyl acetate is noiseless, it was demonstrated that this method has Good specificity is shown in Figure 16,1- ethyl acetate in figure；2- control medicinal material；3- schizonepeta medicinal material；4- schizonepeta medicine materical crude slice.

(4) durability is investigated

It is operated according to the scheme in the embodiment of the present invention 8, respectively point sample and Qingdao Haiyang chemical industry subsidiary factory and Yantai City On the silica gel g thin-layer plate of chemical industry research institute, it is unfolded, takes out, dry, set and inspected under ultraviolet lamp (366nm), as a result shown Show there is preferable separating effect on 2 kinds of plates, it was demonstrated that this method has good durability, sees Figure 17, Figure 18, Figure 17-18 In, 1- control medicinal material；2- schizonepeta medicinal material；3- schizonepeta medicine materical crude slice.

(5) reproducibility is investigated

It is operated simultaneously according to the scheme in the embodiment of the present invention 8 by 2 experimenters, as a result proves that this method has Good reproducibility, is shown in Figure 19, Figure 20, in Figure 19-20,1- control medicinal material；2- schizonepeta medicinal material；3- schizonepeta medicine materical crude slice.

2, Herba Schizonepetae volatile oil thin-layered chromatography discrimination method

The optimization of 2.1 methods

(1) solvent selects

The type and proportion of solvent are groped, respectively select n-hexane-ethyl acetate, toluene-ethyl acetate, Petroleum ether-ethyl acetate etc. be used as solvent, final choice petroleum ether (60~90 DEG C)-ethyl acetate be solvent, ratio 12: 1 is preferred.

(2) lamellae is investigated

It is investigated by silica G plate, H plate, GF254 plate etc., determines that silica G plate separating effect is best.

2.2 methodological study

(1) low-temperature study

It is operated according to the scheme in the embodiment of the present invention 11, point sample, is unfolded in 4 DEG C of refrigerators, taken out, dry, sprayed With 5% vanillin-sulfuric acid ethanol solution, it is heated to that spot development is clear at 105 DEG C, the results show that opening up in the environment at 4 DEG C It opens and works well, see Figure 21,1- (-)-menthones in figure；2- pulegone；3- schizonepeta medicinal material；4- schizonepeta medicine materical crude slice.

(2) humidity is investigated

It is operated according to the scheme in the embodiment of the present invention 11, point sample, is unfolded in 18%, 72% relative humidity, It takes out, dries, spray with 5% vanillin-sulfuric acid ethanol solution, it is clear to be heated to spot development at 105 DEG C, the results show that in difference Humidity environment in expansion effect it is preferable, see Figure 22 (18%), Figure 23 (72%), in Figure 22-23,1- (-)-menthones；2- Pulegone；3- schizonepeta medicinal material；4- schizonepeta medicine materical crude slice.

(3) specificity is investigated

It is operated according to the scheme in the embodiment of the present invention 11, using ethyl acetate reagent as blank solution, point sample, Expansion is taken out, is dried, and with 5% vanillin-sulfuric acid ethanol solution, it is clear to be heated to spot development at 105 DEG C for spray, the results show that Ethyl acetate is noiseless, it was demonstrated that this method has good specificity, sees Figure 24,1- (-)-menthones in figure；2- pulegone； 3- schizonepeta medicinal material；4- schizonepeta medicine materical crude slice；5- ethyl acetate.

(4) durability is investigated

It is operated according to the scheme in the embodiment of the present invention 11, respectively point sample and Qingdao Haiyang chemical industry subsidiary factory and Yantai City On the silica gel g thin-layer plate of chemical industry research institute, it is unfolded, takes out, dry, sprays with 5% vanillin-sulfuric acid ethanol solution, 105 It is clear DEG C to be heated to spot development, the results show that having preferable separating effect on 2 kinds of plates, it was demonstrated that this method has good Durability, see Figure 25, Figure 26, in Figure 25-26,1- (-)-menthones；2- pulegone；3- schizonepeta medicinal material；4- schizonepeta medicine materical crude slice.

(5) reproducibility is investigated

It is operated simultaneously according to the scheme in the embodiment of the present invention 11 by 2 experimenters, as a result proves this method tool There is good reproducibility, sees Figure 27, Figure 28, in Figure 27-28,1- (-)-menthones；2- pulegone；3- schizonepeta medicinal material；4- chaste tree Mustard medicine materical crude slice.

3. schizonepeta heavy metal and harmful element measurement

3.1.1 method

Using lead, cadmium, arsenic, the mercury, copper in atomic absorption spectroscopy determination schizonepeta medicinal material.Specific method is " China People's republic's pharmacopeia " 2015 years version four 2321 lead of general rule, cadmium, arsenic, mercury, copper measuring methods.

3.1.2 result

Lead, cadmium, arsenic, mercury, copper measurement are carried out to 20 batch schizonepeta medicinal materials, measurement result is shown in Table 8.

8 schizonepeta medicinal material heavy metal of table and harmful element measurement result (ppm, mg/kg)

The measurement of 3.2 schizonepeta medicinal material persticide residues

3.2.1 method

Using total six six six (the sum of α-BHC, β-BHC, γ-BHC, δ-BHC) in gas chromatography measurement schizonepeta medicinal material, total DDT (the sum of pp'-DDE, pp'-DDD, op'-DDT, pp'-DDT), pentachloronitrobenzene.Specific method is that " the Chinese people are total With state's pharmacopeia " 2015 years four general rules of version, 2341 organic chlorine agriculture chemicals determination of residual amount methods of method-first.

3.2.2 result

Total six six six (the sum of α-BHC, β-BHC, γ-BHC, δ-BHC), total drop drop have been carried out to 20 batch schizonepeta medicinal materials Tears (the sum of pp'-DDE, pp'-DDD, op'-DDT, pp'-DDT), pentachloronitrobenzene measurement, measurement result are shown in Table 9.

9 schizonepeta medicinal material persticide residue measurement result (ppm, mg/kg) of table

4. schizonepeta medicinal material determination of extractives

4.1 methods are investigated

Take schizonepeta medicinal material 4g, respectively solubilizer (Diluted Alcohol, volume fraction are 70% ethanol solution and ethyl alcohol in table) 100ml shakes 6 hours, impregnates 18 hours, and filtration, filtrate draws 20ml, sets and has dried into the evaporating dish of constant weight, be evaporated, sets It is 3 hours dry at 105 DEG C, weighed weight.The content (%) for calculating extract, the results are shown in Table 10.

10 extract of table investigates result

Solvent	Diluted Alcohol	70% ethyl alcohol	Ethyl alcohol
				Extract (%)	8.09	10.07	8.64

It is best with 70% ethyl alcohol extraction effect in terms of investigation result, therefore select 70% ethyl alcohol as extract solvent.

4.2 result

Each batch schizonepeta medicinal material determination of extractives the results are shown in Table 11.

11 schizonepeta medicinal material determination of extractives result of table

5. schizonepeta medicinal material assay

The investigation of 5.1 chromatographic conditions

Operated according to the scheme in the embodiment of the present invention 5, wherein gradient elution program select respectively according to table 12~ Gradient elution method elution in table 15, chromatogram are successively shown in Figure 29~Figure 32；According to result it is found that gradient elution method 4 obtains The chromatogram aurantiamarin peak shape arrived is preferable, and purity analysis shows that peak purity is good, separating degree 2.11, and USP tailing factor is 1.02, symmetrical factor 1.02, USP theoretical cam curve is 14032.

12 gradient elution method 1 of table

Time (min)	Methanol	5% acetic acid water
			0~45	30%	70%

13 gradient elution method 2 of table

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0~15	10%~14%	90%~86%
15~40	14%~26%	86%~74%
			40~55	26%~60%	74%~40%
55~60	60%~85%	40%~15%
			60~65	85%	15%

14 gradient elution method 3 of table

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0~28	20%	80%
28~29	20%~85%	80%~15%
			29~39	85%	15%

15 gradient elution method 4 of table

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0~28	18%	82%
28~29	18%~85%	82%~15%
			29~39	85%	15%

The investigation of 5.2 extracting methods

5.2.1 the investigation of Extraction solvent

It is operated according to the scheme in the embodiment of the present invention 5, in the preparation step of test solution, selection adds respectively Enter the Extraction solvent 25ml in following table, as a result reflow treatment 30min see the table below.

16 Extraction solvent of table investigates result

Solvent	Methanol	Ethyl alcohol	Water
				Content (mg/g)	0.947	0.573	0.700

From the data in the table, recovery rate highest when methanol is as Extraction solvent, hence it is evident that be better than second alcohol and water, therefore selection mentions Taking solvent is methanol.

5.2.2 the investigation of extracting mode

It is operated according to the scheme in the embodiment of the present invention 5, in the preparation step of test solution, under selecting respectively Extracting mode in table handles 30min, as a result see the table below.

17 extracting mode of table investigates result

Extracting mode	Ultrasound	Reflux	Steeped overnight
				Content (mg/g)	0.947	1.225	0.974

From the data in the table, refluxing extraction recovery rate highest, hence it is evident that be better than ultrasonic extraction and steeped overnight, therefore selection mentions Taking mode is refluxing extraction.

5.2.3 the investigation of sample weighting amount

It is operated according to the scheme in the embodiment of the present invention 5, in the preparation step of test solution, under selecting respectively Sample weighting amount in table weighs test sample, as a result see the table below.

18 sample weighting amount of table investigates result

Sample weighting amount	0.5g	1g	2g
				Content (mg/g)	1.299	1.225	1.198

From the data in the table, recovery rate highest when sample weighting amount is 0.5g, therefore select sample weighting amount for 0.5g.

5.2.4 the investigation of extraction time

It is operated according to the scheme in the embodiment of the present invention 5, in the preparation step of test solution, is flowed back respectively As a result 30min, 60min, 120min see the table below.

19 extraction time of table investigates result

Return time	30min	60min	120min
				Content mg/g	1.299	1.248	1.198

From the data in the table, recovery rate highest when extraction time is 30min, therefore the selective extraction time is 30min.

5.3 methodological study

5.3.1 linear relationship is investigated

Weigh aurantiamarin reference substance 14.2mg (96.1%), set in 25ml measuring bottle, methanol is added to dissolve, then plus methanol dilution extremely Scale shakes up.Precision draws above-mentioned solution 5ml, sets in 10ml measuring bottle, adds methanol dilution to scale, shake up to get 272.924 μ The reference substance solution of g/ml, successively dilutes step by step, and respectively obtaining concentration is 136.462 μ g/ml, 68.231 μ g/ml, 34.116 μ The reference substance solution of g/ml, 17.058 μ g/ml, 8.529 μ g/ml, 4.264 μ g/ml, 2.132 μ g/ml.

Specification Curve of Increasing

Draw respectively above-mentioned concentration be 68.231 μ g/ml, 34.116 μ g/ml, 17.058 μ g/ml, 8.529 μ g/ml, 4.264 μ g/ml, 2.132 μ g/ml reference substance solution, 10 μ l, according in embodiment 7 chromatographic condition and measuring method measure, injection Liquid chromatograph the results are shown in Table 20.

20 linear relationship of table is investigated

Concentration (μ g/ml)	Peak area 1	Peak area 2	Average peak area
				2.132	45689	47099	46394
4.264	94946	94734	94840
				8.529	190110	187972	189041
17.058	375568	347321	361444.5
				34.116	696680	755340	726010
68.231	1541295	1483502	1512399

The results show that aurantiamarin equation of linear regression is Y=21954X, r=0.9997, in 2.132 g/ml~68.231 μ Linear relationship is good within the scope of μ g/ml, and the curve graph of the regression equation as shown in Figure 10, can be counted with one point external standard method It calculates.

5.3.2 precision is investigated

Schizonepeta sample powder (crossing No. two sieves) 0.5g is weighed, is operated according to the scheme in the embodiment of the present invention 5.Continuous sample introduction 6 times, it is 0.47% according to calculated by peak area RSD value, shows that precision is good.

21 precision of table is investigated

5.3.3 study on the stability

Schizonepeta sample powder (crossing No. two sieves) 0.5g is weighed, is operated according to the scheme in the embodiment of the present invention 5.Respectively 0, 2,4,8,12,24 hours sample introductions investigate the stability of test sample according to calculated by peak area RSD value.The results show that test sample is molten Aurantiamarin is basicly stable in 24 hours in liquid.

22 study on the stability of table

5.3.4 repeatability is investigated

Totally 6 parts of 0.5g of schizonepeta sample powder (crossing No. two sieves) is weighed, is operated according to the scheme in the embodiment of the present invention 5.Into Sample, according to calculated by peak area content, the results show that content is 1.30mg/g (0.130%), RSD value is 1.98%, shows we Method repeatability is good.

23 repeatability of table is investigated

5.3.5 accuracy is investigated

Fineleaf Schizonepeta Herb (crossing No. two sieves) 0.25g is taken, the aurantiamarin reference substance that 545.848 μ g/ml of configuration are added in precision is molten Liquid 0.6ml, then accurate addition methanol 24.4ml, close plug, weighed weight, flow back 30min, lets cool, methanol is added to supply the weight of less loss Amount, shakes up, takes subsequent filtrate to get test solution, then operates according to the scheme in the embodiment of the present invention 5.Continuous sample preparation 6 Part, sample introduction calculates sample recovery rate.The result shows that this method sample recovery rate is good.

24 accuracy of table is investigated

5.3.6 durability is investigated

Schizonepeta sample powder (crossing No. two sieves) 0.5g is weighed, is operated according to the scheme in the embodiment of the present invention 5.Not with 3 Same chromatography post detection calculates content and RSD value.The result shows that this method good tolerance.

The different chromatographic columns of table 25 measure sample size

Chromatographic column producer	Waters	Agilent	Kromasil	RSD (%)
					Content (mg/g)	1.30	1.23	1.26	2.78

This research establishes the detection method of content of hesperidin in high effective liquid chromatography for measuring schizonepeta.By to mobile phase The investigation of elution requirement, it is determined that optimal chromatographic condition, and comprehensive investigation has been carried out to methodology；By investigate Extraction solvent, The influence of extracting mode, extraction time, sample weighting amount to aurantiamarin extraction effect in schizonepeta, optimization obtain sample pre-treatments condition. This method is simple, and measurement result is accurate and reliable, reproducible, practical, can be used for the quality evaluation to schizonepeta medicine materical crude slice.

6. schizonepeta medicinal material involatile constituent HPLC characteristic spectrum is studied

6.1.1 the selection of wavelength

Schizonepeta DAD full wavelength scanner map is shown in Figure 33, and as seen from the figure, each chromatographic peak of the sample at 283nm wavelength is opposite Compare balanced, therefore selects 283nm for the Detection wavelength of schizonepeta medicinal material and medicine materical crude slice characteristic spectrum.

6.1.2 the investigation of chromatographic condition

It is operated according to the scheme of the embodiment of the present invention 1, wherein gradient elution method is respectively according in the following table 26-28 Gradient elution method elution, chromatogram is successively shown in Figure 34-36, according to result it is found that the obtained chromatogram of gradient elution method 3 is each Peak shape, resolution are good, therefore, select the method as characteristic spectrum discrimination method.

26 gradient elution method 1 of table

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0~20	20%	80%
20~30	25%	75%
			30~50	30%	70%
50~60	50%	50%
			60~70	80%	20%

27 gradient elution method 2 of table

Time (min)	Acetonitrile	0.05% trifluoroacetic acid water
			0~40	5%~40%	95%~60%
40~60	40%~60%	60%~40%
			60~65	60%~5%	40%~95%
65~90	5%	95%

28 gradient elution method 3 of table

Time (min)	Acetonitrile	0.2% phosphoric acid water
			0~15	10%~14%	90%~86%
15~40	14%~26%	86%~74%
			40~55	26%~60%	74%~40%
55~60	60%~85%	40%~15%
			60~65	85%	15%

6.2 methodological study

6.2.1 precision is investigated

According to the scheme operation in the embodiment of the present invention 1, the accurate same schizonepeta test solution of absorption, continuous sample introduction 7 times, It is referring to peak (S), the RSD < 3.0% of each characteristic peak relative retention time, the RSD < 5.0% of relative peak area, table with No. 1 peak Bright instrument precision is good.

29 precision relative retention time calculated result of table

	S1	S2	S3	S4	S5	S6
							1	1.00	1.07	1.09	1.20	1.25	1.64
2	1.00	1.07	1.09	1.21	1.25	1.64
							3	1.00	1.07	1.09	1.21	1.26	1.64
4	1.00	1.07	1.09	1.21	1.26	1.63
							5	1.00	1.07	1.09	1.21	1.26	1.63
6	1.00	1.07	1.09	1.21	1.26	1.63
							7	1.00	1.07	1.09	1.21	1.26	1.63
RSD%	0.00	0.25	0.17	0.19	0.10	0.18

30 precision relative peak area calculated result of table

	S1	S2	S3	S4	S5	S6
							1	1.00	0.39	0.64	0.23	0.50	0.39
2	1.00	0.39	0.64	0.23	0.50	0.39
							3	1.00	0.39	0.64	0.23	0.49	0.38
4	1.00	0.39	0.64	0.23	0.49	0.38
							5	1.00	0.39	0.64	0.23	0.49	0.38
6	1.00	0.38	0.62	0.22	0.53	0.37
							7	1.00	0.39	0.63	0.23	0.54	0.38
RSD%	0.00	1.10	0.93	1.67	3.36	1.68

6.2.2 study on the stability

According to the scheme operation in the embodiment of the present invention 1, precision draws same schizonepeta test solution, respectively at test sample 0h, 1h, 3h, 6h, 9h, 12h after solution preparation, sample introduction, is referring to peak (S), when each characteristic peak retains relatively with No. 1 peak for 24 hours Between RSD < 3.0%, the RSD < 6.0% of relative peak area, show test solution after preparation for 24 hours in stablize.

31 stability relative retention time calculated result of table

	S1	S2	S3	S4	S5	S6
							0h	1.00	1.07	1.09	1.20	1.25	1.64
1h	1.00	1.07	1.09	1.21	1.25	1.64
							3h	1.00	1.07	1.09	1.21	1.26	1.64
6h	1.00	1.07	1.09	1.21	1.26	1.63
							9h	1.00	1.07	1.09	1.21	1.26	1.63
12h	1.00	1.08	1.10	1.21	1.26	1.63
							24h	1.00	1.07	1.09	1.21	1.26	1.64
RSD%	0.00	0.31	0.22	0.24	0.13	0.24

32 stability relative peak area calculated result of table

	S1	S2	S3	S4	S5	S6
							0h	1.00	0.39	0.64	0.23	0.50	0.39
1h	1.00	0.39	0.64	0.23	0.50	0.39
							3h	1.00	0.39	0.64	0.23	0.49	0.38
6h	1.00	0.39	0.64	0.23	0.49	0.38
							9h	1.00	0.39	0.63	0.23	0.54	0.38
12h	1.00	0.39	0.64	0.24	0.56	0.38
							24h	1.00	0.40	0.65	0.22	0.52	0.39
RSD%	0.00	0.96	0.90	2.40	5.20	1.36

6.2.3 repeatability is investigated

According to the scheme operation in the embodiment of the present invention 1, take with a collection of Fineleaf Schizonepeta Herb about 0.5g, parallel 6 parts, precision claims Fixed, sample introduction, records characteristic spectrum respectively, with No. 1 peak be referring to peak (S), the RSD < 3.0% of each characteristic peak relative retention time, The RSD < 6.0% of relative peak area shows that the repeatability of this method is good.

The repeated relative retention time calculated result of table 33

The repeated relative peak area calculated result of table 34

	S1	S2	S3	S4	S5	S6
							1	1.00	0.39	0.64	0.23	0.50	0.39
2	1.00	0.38	0.60	0.21	0.52	0.35
							3	1.00	0.38	0.63	0.23	0.52	0.36
4	1.00	0.39	0.62	0.23	0.50	0.39
							5	1.00	0.38	0.61	0.20	0.48	0.36
6	1.00	0.39	0.60	0.21	0.51	0.40
							RSD%	0.00	1.19	2.26	5.93	3.08	5.19

6.2.4 durability is investigated

According to the scheme operation in the embodiment of the present invention 1, detection checks each peak point using the chromatogram under different chromatographic columns From degree.Each peak separating degree of Waters column, AlchromBond-AQ column, Phenomex-Luna column is good.

The different chromatographic columns of table 35 measure sample relative retention time

Chromatographic column producer	Waters	AlchromBond-AQ	Phenomex-Luna
				S1	1.00	1.00	1.00
S2	1.07	1.07	1.07
				S3	1.09	1.14	1.09
S4	1.20	1.20	1.20
				S5	1.25	1.23	1.25
S6	1.64	1.49	1.59

The different chromatographic columns of table 36 measure sample relative peak area

The acquisition of 6.3 characteristic spectrums

" similarity evaluation " (V2.0) worked out using National Pharmacopeia committee member, it is each to schizonepeta The testing result of batch sample is analyzed.Using median method, time window parameter is 0.30, by Software Create compare feature Map (R finger-print).

6.3.1 characteristic peak is demarcated

According to the scheme constructs schizonepeta medicinal material representativeness test sample in the embodiment of the present invention 1 characteristic spectrum and divided Analysis, takes the typical chromatographic peak all occurred in every batch of test sample characteristic spectrum as characteristic peak.

In 283nm, totally 6 characteristic peaks, No. 1 peak are aurantiamarin, and No. 2 peaks are Rosmarinic acid, and No. 3 peaks, No. 4 peaks, No. 5 peaks are Common characteristic peaks, No. 6 peaks are pulegone, and each chromatographic peak is indicated respectively with 1~6 serial number.

6.3.2 the measurement of each batch sample and the foundation of compare feature map

The relative retention time and relative peak area of each batch sample are shown in Table 37~table 38, the compare feature map of generation, See Figure 37,6 characteristic peaks should be presented in the characteristic spectrum of each batch sample, is label with S1, S2, S3, S4, S5, S6.

37 6 characteristic peak relative retention times of schizonepeta medicinal material each batch of table

Number	S1	S2	S3	S4	S5	S6
							J1	1.00	1.07	1.09	1.21	1.26	1.66
J2	1.00	1.07	1.09	1.21	1.26	1.63
							J3	1.00	1.08	1.10	1.20	1.25	1.63
J4	1.00	1.09	1.11	1.22	1.27	1.65
							J5	1.00	1.07	1.09	1.21	1.26	1.64
J6	1.00	1.06	1.09	1.20	1.25	1.65
							J7	1.00	1.07	1.10	1.21	1.26	1.63
J8	1.00	1.08	1.10	1.21	1.26	1.63

38 6 characteristic peak relative peak areas of schizonepeta medicinal material each batch of table

Number	S1	S2	S3	S4	S5	S6
							J1	1.00	0.41	0.25	0.64	0.46	0.42
J2	1.00	0.26	0.18	0.18	0.26	0.19
							J3	1.00	2.71	0.62	0.02	0.02	0.42
J4	1.00	0.60	0.01	0.05	0.03	0.62
							J5	1.00	4.82	0.85	0.40	1.64	1.22
J6	1.00	0.75	0.21	0.17	0.38	0.23
							J7	1.00	0.70	0.18	0.27	0.77	0.16
J8	1.00	1.25	0.25	0.23	0.47	0.26

7. the research of schizonepeta medicinal material volatile oil GC characteristic spectrum

7.1.1 chromatography condition

According to the embodiment of the present invention 4 scheme operate, column temperature temperature program therein according to 9~table of the following table 3 43 heating Program；The characteristic spectrum obtained using temperature program 1-5 is as shown in Figure 38-42, the results showed that, the chromatography that temperature program 3 obtains Figure peak shape is preferable, therefore selects temperature program 3.

39 temperature program 1 of table

Rate DEG C/min	Temperature DEG C	Retention time min
			——	60	2
3	120	5
			3	130	10
3	160	0
			5	230	0

40 temperature program 2 of table

41 temperature program 3 of table

Rate DEG C/min	Temperature DEG C	Temperature DEG C
			——	60	2
2	120	5
			3	160	0
5	220	0

42 temperature program 4 of table

Rate DEG C/min	Temperature DEG C	Rate DEG C/min
			——	60	2
2	140	5
			5	210	0
10	220	0

43 temperature program 5 of table

Rate (DEG C/min)	Temperature (DEG C)	Temperature (DEG C)
			——	60	2
2	120	5
			5	170	5
10	220	0

7.2 methodological study

7.2.1 precision is investigated

According to the method operation in the embodiment of the present invention 4, the accurate same schizonepeta test solution of absorption, continuous sample introduction 6 times, It is referring to peak (S), the RSD < 3.0% of each characteristic peak relative retention time, the RSD < 3.0% of relative peak area, table with No. 2 peaks Bright instrument precision is good.

44 precision relative retention time calculated result of table

45 precision relative peak area calculated result of table

	S1	S2	S3
				1	1.05	1.00	0.03
2	1.02	1.00	0.03
				3	1.05	1.00	0.03
4	1.04	1.00	0.03
				5	1.05	1.00	0.03
6	1.05	1.00	0.03
				7	1.03	1.00	0.03
RSD%	1.09	0.00	1.61

7.2.2 study on the stability

According to the method operation in the embodiment of the present invention 4, precision draws same schizonepeta test solution, respectively at test sample 0h, 1h, 3h, 6h, 9h, 12h after solution preparation, sample introduction, is referring to peak (S), when each characteristic peak retains relatively with No. 2 peaks for 24 hours Between RSD < 3.0%, the RSD < 5.0% of relative peak area, show test solution after preparation for 24 hours in stablize.

46 stability relative retention time calculated result of table

47 stability relative peak area calculated result of table

	S1	S2	S3
				0h	1.05	1.00	0.03
1h	1.02	1.00	0.03
				3h	1.04	1.00	0.03
6h	1.03	1.00	0.03
				9h	1.04	1.00	0.03
12h	1.04	1.00	0.03
				24h	1.07	1.00	0.03
RSD%	1.39	0.00	3.12

7.2.3 repeatability is investigated

According to the method operation in the embodiment of the present invention 4, take with a collection of Fineleaf Schizonepeta Herb, parallel 6 parts, sample introduction, records respectively Gas chromatogram is the RSD < 3.0% of each characteristic peak relative retention time referring to peak (S) with No. 2 peaks, relative peak area RSD < 3.0% shows that the repeatability of this method is good.

The repeated relative retention time calculated result of table 48

	S1	S2	S3
				1	0.67	1.00	1.51
2	0.67	1.00	1.51
				3	0.67	1.00	1.51
4	0.67	1.00	1.51
				5	0.67	1.00	1.50
6	0.67	1.00	1.50
				RSD%	0.08	0.00	0.10

The repeated relative peak area calculated result of table 49

	S1	S2	S3
				1	1.04	1.00	0.03
2	1.04	1.00	0.03
				3	1.04	1.00	0.03
4	1.04	1.00	0.03
				5	1.06	1.00	0.03
6	1.04	1.00	0.03
				RSD%	0.88	0.00	2.65

The acquisition of 7.3 characteristic spectrums

" similarity evaluation " (V2.0) worked out using National Pharmacopeia committee member, it is each to schizonepeta The testing result of batch gas phase map is analyzed.Using median method, time window parameter is 0.30, is compareed by Software Create Characteristic spectrum (R finger-print).

7.3.1 characteristic peak is demarcated

According to the scheme constructs schizonepeta medicinal material representativeness test sample in the embodiment of the present invention 4 characteristic spectrum and divided Analysis, takes the typical chromatographic peak all occurred in every batch of test sample characteristic spectrum as characteristic peak.

Totally 3 characteristic peaks, common characteristic peaks are as follows: No. 1 peak is menthones, and No. 2 peaks are pulegone, No. 3 peaks, each chromatographic peak It is indicated respectively with 1~3 serial number.

7.3.2 the measurement of each batch sample and the foundation of compare feature map

The relative retention time and relative peak area of each batch sample are shown in Table 50~table 51, and the compare feature map of generation is shown in 3 characteristic peaks should be presented in the characteristic spectrum of each batch sample in Figure 43, be label with S1, S2, S3.

50 3 characteristic peak relative retention times of schizonepeta medicinal material each batch of table

51 3 characteristic peak relative peak areas of schizonepeta medicinal material each batch of table

Number	S1	S2	S3
				J1	0.44	1.00	0.05
J2	0.20	1.00	0.04
				J3	0.59	1.00	0.02
J4	0.71	1.00	0.03
				J5	0.27	1.00	0.02
J6	0.39	1.00	0.01
				J7	0.20	1.00	0.02
J8	0.46	1.00	0.01

Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or It changes still within the protection scope of the invention.

Claims (11)

1. a kind of construction method of schizonepeta characteristic spectrum, which is characterized in that the building side including involatile constituent characteristic spectrum The construction method of method and volatile component characteristic spectrum:

A, the construction method of the involatile constituent characteristic spectrum, includes the following steps:

The preparation of test solution I: schizonepeta test sample is taken to prepare test solution I；

The preparation of reference solution I: schizonepeta control medicinal material is taken to prepare control medicinal material reference solution I；Take aurantiamarin reference substance system Standby reference substance reference solution I；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as chromatographic column Filler；Using acetonitrile as mobile phase A, eluted using the phosphate aqueous solution that volume fraction is 0.2% as Mobile phase B；

Measuring method: it is accurate respectively to draw reference solution I and test solution I, inject liquid chromatograph, measurement to get；With

B, the construction method of the volatile component characteristic spectrum, includes the following steps:

The preparation of test solution II: taking the volatile oil prepared by schizonepeta test sample, prepares test solution II；

The preparation of reference solution II: pulegone and menthones is taken to prepare the reference solution II of reference substance respectively；

Chromatographic condition and system suitability: it is measured according to gas chromatography, with N₂For carrier gas, using PEG as stationary phase, control into Sample mouth temperature, detector temperature, column temperature and flow velocity；

Measuring method: it is accurate respectively to draw reference solution II and test solution II, inject gas chromatograph, measurement to get.

2. the construction method of schizonepeta characteristic spectrum according to claim 1, which is characterized in that further include compare feature map Building, at least 15 batches confessions using the similarity evaluation of Chinese Pharmacopoeia Commission, to obtaining Test sample solution I characteristic spectrum is analyzed, and involatile constituent compare feature map is generated；And/or

Using the similarity evaluation of Chinese Pharmacopoeia Commission, to obtained at least 15 batches of test samples The gas phase characteristic map of solution II is analyzed, and volatile component compare feature map is generated.

3. construction method according to claim 1 or 2, which is characterized in that the involatile constituent characteristic spectrum includes 6 A characteristic peak, characteristic peak are as follows: No. 1 peak is aurantiamarin, and No. 2 peaks are Rosmarinic acid, and No. 3 peaks, No. 4 peaks, No. 5 peaks are common characteristic Peak, No. 6 peaks are pulegone；And/or

The volatile component characteristic spectrum includes 3 characteristic peaks, characteristic peak are as follows: No. 1 peak is menthones, and No. 2 peaks are pennyroyal mint Ketone, No. 3 peaks are common characteristic peaks.

4. the construction method of schizonepeta characteristic spectrum according to claim 1-3, which is characterized in that non-waved described In the construction method of hair property composition characteristics map, type of elution selects gradient elution mode, gradient elution program are as follows: 0-15min, Mobile phase A: the volume ratio of Mobile phase B is 8-12%:92-88% → 12-16%:88-84%；15-40min, mobile phase A: stream The volume ratio of dynamic phase B is 12-16%:88-84% → 24-28%:76-72%；40-55min, mobile phase A: the body of Mobile phase B Product is than being 24-28%:76-72% → 58-62%:42-38%；55-60min, mobile phase A: the volume ratio of Mobile phase B is 58- 62%:42-38% → 83-87%:17-13%；60-65min, mobile phase A: the volume ratio of Mobile phase B is 83-87%:17- 13% → 83-87%:17-13%；

Preferably, the gradient elution program are as follows: 0-15min, mobile phase A: the volume ratio of Mobile phase B be 10%:90% → 14%:86%；15-40min, mobile phase A: the volume ratio of Mobile phase B is 14%:86% → 26%:74%；40-55min, stream Dynamic phase A: the volume ratio of Mobile phase B is 26%:74% → 60%:40%；55-60min, mobile phase A: the volume ratio of Mobile phase B For 60%:40% → 85%:15%；60-65min, mobile phase A: the volume ratio of Mobile phase B is 85%:15% → 85%: 15%；

Preferably, chromatographic condition are as follows: column temperature is 25-40 DEG C；Flow velocity is 0.8-1.2ml/min；Detection wavelength is 278-288nm；

Preferably, chromatographic condition are as follows: column temperature is 40 DEG C；Flow velocity is 1ml/min；Detection wavelength is 283nm.

5. the construction method of schizonepeta characteristic spectrum according to claim 1-4, which is characterized in that in the volatilization Property composition characteristics map construction method in, control column temperature heats up in gradient, the gradient increased temperature program are as follows: initial temperature is 55-65 DEG C, keep 1-3min；Temperature rises to 115-125 DEG C from 55-65 DEG C, then keeps 4-6min, heating rate 1.5- 2.5℃/min；Temperature rises to 155-165 DEG C from 115-125 DEG C, and heating rate is 2.5-3.5 DEG C/min；Temperature is from 155-165 235-245 DEG C DEG C is risen to, heating rate is 4.5-5.5 DEG C/min；

Preferably, the gradient increased temperature program are as follows: initial temperature is 60 DEG C, keeps 2min；Temperature rises to 120 DEG C from 60 DEG C, protects 5min is held, heating rate is 2 DEG C/min；Temperature rises to 160 DEG C from 120 DEG C, and heating rate is 3 DEG C/min；Temperature rises from 160 DEG C To 240 DEG C, heating rate is 5 DEG C/min；

Preferably, injector temperature is 245-255 DEG C, and detector temperature is 245-255 DEG C, total flow 3.5-3.9ml/min；

Preferably, injector temperature is 250 DEG C, and detector temperature is 250 DEG C, total flow 3.7ml/min.

6. the construction method of schizonepeta characteristic spectrum according to claim 1-5, which is characterized in that

The preparation method of the test solution I or control medicinal material reference solution I include: to take schizonepeta test sample or schizonepeta pair Powder is made according to medicinal material, precision weighs 0.5-1g, solubilizer 15-30ml, and close plug extracts processing, lets cool, with the solvent The weight for supplying less loss, shakes up, and supernatant is taken to filter, and takes subsequent filtrate to get test solution I or control medicinal material reference solution I；

Preferably, the preparation method of the reference substance reference solution I includes: that aurantiamarin reference substance is taken to add the solvent dissolution system At the reference substance reference solution I of the g/ml aurantiamarin of μ containing 55-65；

Preferably, the test sample is selected from schizonepeta crude drug or schizonepeta medicine materical crude slice；

Preferably, the solvent is methanol, ethyl alcohol or water；

Preferably, the extraction process is using ultrasound 30-90min, reflux 30-120min or dipping 12-24h；

Preferably, the reference substance reference solution I is the reference substance reference solution I containing 60 μ g/ml aurantiamarins.

7. the construction method of schizonepeta characteristic spectrum according to claim 1-6, which is characterized in that the object of reference Solution II is the reference substance solution containing 3-6mg/ml pulegone and 3-6mg/ml menthones, and solvent is ethyl acetate；

Described for II preparation method of reagent material solution includes: to take schizonepeta test sample, using the Pharmacopoeia of the People's Republic of China The first legal system of four 2204 determination of volatile oil of general rule of version in 2015 obtains volatile oil, and precision draws 25-50ul, adds ethyl acetate constant volume To 2ml, water removal is drying to obtain.

8. a kind of schizonepeta quality determining method, which is characterized in that including using the described in any item schizonepeta features of claim 1-7 Map construction method carries out quality testing to schizonepeta product.

9. schizonepeta quality determining method according to claim 8, which is characterized in that according to any one of claim 1-7 institute The schizonepeta characteristic spectrum construction method stated detects schizonepeta product to be measured, obtained schizonepeta involatile constituent characteristic pattern with method operation Spectrum and schizonepeta volatile component characteristic spectrum, 6 characteristic peaks are presented in the non-volatile characteristic spectrum, and should join with control medicinal material Corresponding according to 6 characteristic peaks in object characteristic spectrum, wherein peak 1, peak 6 should be consistent with reference substance object of reference peak retention time； 3 characteristic peaks are presented in the volatile component characteristic spectrum, and wherein peak 1, peak 2 should be with reference substance object of reference peak retention time phases one It causes.

10. schizonepeta quality determining method according to claim 8 or claim 9, which is characterized in that further include carrying out content to schizonepeta The step of measurement, includes the following steps:

Test solution III: schizonepeta test sample is taken to prepare test solution III；

Reference substance solution: taking aurantiamarin reference substance to be dissolved in solvent, prepares reference substance solution；

Chromatographic condition and system suitability: high effective liquid chromatography for measuring is shone, using octadecyl silane as chromatographic column Filler；Using acetonitrile as mobile phase A, gradient elution is carried out by Mobile phase B of the phosphate aqueous solution that volume fraction is 0.2%, is pressed Carry out gradient elution: 0-28min according to following procedure, mobile phase A: the volume ratio of Mobile phase B is 17-19%:83-81% → 17- 19%:83-81%；28-29min, mobile phase A: the volume ratio of Mobile phase B is 17-19%:83-81% → 84-86%:16- 14%；29-39min, mobile phase A: the volume ratio of Mobile phase B is 84-86%:16-14% → 84-86%:16-14%；Column temperature It is 25-35 DEG C；Flow velocity is 0.8-1.2ml/min；Detection wavelength is 278-288nm；Number of theoretical plate is not less than based on aurantiamarin 3000；

Measuring method: it is accurate respectively to draw reference substance solution and test solution III, inject liquid chromatograph, measurement to get.

11. according to the described in any item schizonepeta quality determining methods of claim 8-10, which is characterized in that further include thin-layer chromatography The step of the step of the step of method identifies, heavy metal and harmful element measure and/or determination of extractives:

C, the involatile constituent thin-layered chromatography identifies step, includes the following steps:

The coarse powder and schizonepeta control medicinal material coarse powder 0.5-1.0g for taking schizonepeta test sample respectively, add ethyl acetate 15-25ml, ultrasound Processing 15-25 minutes, filtration, filtrate is waved to 1ml, as test solution IV and control medicinal material solution, then respectively draws 1- 5ul puts respectively on same lamellae, is unfolded in solvent, takes out, dries, and sets the ultraviolet light that wavelength is 361nm-371nm Under inspect；

Preferably, the lamellae selects silica G plate or H plate；

Preferably, the solvent selects chloroform-ethyl acetate-glacial acetic acid mixed solution, and volume ratio is (9-11): 1: (0.3-0.8)；And/or

D, the volatile component thin-layered chromatography identifies step, includes the following steps:

Take schizonepeta test sample coarse powder appropriate, according to determination of volatile oil method " Pharmacopoeia of People's Republic of China four general rules of version in 2015 2204 " it measures, divides and take 0.1~0.3ml of volatile oil, add ethyl acetate to be diluted to 1.5~4.5ml, as test solution V.Separately (-)-menthones, pulegone's reference substance are taken, adds ethyl acetate that the solution that every 1ml contains 5~30mg is made, it is molten as reference substance Liquid.It is each to draw 1-5ul, it is put respectively on same lamellae, is unfolded in solvent, taken out, dry, sprayed with 2%~8% vanilla It is clear to be heated to spot development at 105 DEG C for aldehyde ethanol solution of sulfuric acid；

Preferably, the lamellae selects silica G plate or H plate；

Preferably, the solvent selects petroleum ether (60~90 DEG C)-ethyl acetate, and volume ratio is (11-13): 1；And/or；

E, the measurement of the heavy metal and harmful element includes the assay of heavy metal lead, cadmium, arsenic, mercury, copper, including walks as follows It is rapid:

The persticide residue measurement includes the assay of total six six six, total DDT and pentachloronitrobenzene；And/or

F, the step of determination of extractives includes:

The determination of extractives operating procedure are as follows: take schizonepeta for reagent material coarse powder 2-5g, it is accurately weighed, set the cone of 250~300ml In shape bottle, accurate solubilizer 100ml, close plug, cold soaking constantly shakes in first 6 hours, then stands 18 hours, fast with dry filter Speed filtration, precision measure subsequent filtrate 20ml, set and dried into the evaporating dish of constant weight, after being evaporated in water-bath, in 105 DEG C of dryings 3 hours, 30 minutes cooling in drier, rapid accurately weighed weight is set, with the content of extract in dry product calculating test sample (%)；Wherein, solvent is ethyl alcohol, and volume ratio score is 50-90%.