CN110441444A - A kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method and its discrimination method - Google Patents

A kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method and its discrimination method Download PDF

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CN110441444A
CN110441444A CN201910895240.5A CN201910895240A CN110441444A CN 110441444 A CN110441444 A CN 110441444A CN 201910895240 A CN201910895240 A CN 201910895240A CN 110441444 A CN110441444 A CN 110441444A
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mobile phase
volume fraction
folium sauropi
medicinal material
variation
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CN110441444B (en
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李国卫
魏梅
索彩仙
吴文平
曹斯琼
孙冬梅
陈向东
程学仁
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Guangdong Yifang Pharmaceutical Co Ltd
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02Column chromatography
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The present invention relates to a kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method and its discrimination methods.The Folium Sauropi medicinal material UPLC characteristic spectrum construction method comprises the following steps: (1) precision weighs Folium Sauropi medicinal material, and Folium Sauropi test solution is prepared;(2) Folium Sauropi test solution is analyzed using Ultra Performance Liquid Chromatography instrument, obtains Folium Sauropi UPLC characteristic spectrum.The reference data of Folium Sauropi medicinal material characteristic spectrum analysis method is worked out, from the distinctive characteristic spectrum angle of Folium Sauropi medicinal material, selected characteristic peak, calculate the relative retention time and relative peak area of characteristic peak, the overall picture for constituting Folium Sauropi medicinal material characteristic spectrum, make the quality of Folium Sauropi medicinal material control risen to from certain original several component contents measurement in entire drug Quality Detection, the defect that single chemical component is examined is avoided, the quality of Folium Sauropi medicinal material can be preferably evaluated.

Description

A kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method and its discrimination method
Technical field
The invention belongs to TCD identificafion fields, and in particular to a kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method and its Discrimination method.
Background technique
Folium Sauropi is the dried leaf of euphorbia plant Folium Sauropi Sauropus spatulifolius Beille.Summer, autumn two Ji Caishou is dried.Folium Sauropi (Sauropus spatulifolius Beille) also known as leaf of dragon's tongue, imperial taste leaf, Long Liye and The ears of an ox or cow leaf etc. belongs to Euphorbiaceae Sauropus, is unique medicinal plant in Sauropus.It is recorded in for the first time in " south of the Five Ridges gather medicinal herbs record ", Property it is sweet, light, put down, attach to the lung and stomach meridians, have clearing lung-heat, control internal injury cough with lung heat, bronchitis, bronchial asthma, only phlegm-fire cough, Asthma, the effect for controlling internal injury tuberculosis aphonia, laryngalgia etc..It is regional to be mainly distributed on the Guangdong, Guangxi and Hainan in China etc., is evergreen Undershrub, up to 40cm mostly cultivate or are born in mountain valley, hillside, moisten in fertile jungle.
The various bioactive components such as monoterpene, sequiterpene, cumarin, flavonoid glycoside are mainly contained in Folium Sauropi, wherein flavones Glycosides ingredient Kaempferol-O- O-gentibioside has anti-inflammatory, cough-relieving, spasmolysis, antiulcer and other effects, is the main effective of Folium Sauropi Ingredient.It has been reported that studying the leaf of Folium Sauropi using ethyl acetate as solvent, 15 have been demarcated using relative retention time and has been shared Peak obtains 10 batches of Folium Sauropi medicinal materials fingerprint similarities of different sources 0.8 or more;It is with the root of Folium Sauropi, stem respectively Research object has respectively determined 13,14 shared peaks by the statistical analysis of the Folium Sauropi HPLC chromatogram of different sources;Mesh The quality of preceding Folium Sauropi medicinal material is the content from several elements to judge, it is clear that single chemical component is examined to exist and be lacked It falls into, Research Literature majority is conducted a research with HPLC finger-print, perfect to establish for thoroughly evaluating Folium Sauropi quality of medicinal material Folium Sauropi evaluation of medical materials' quality system, accelerates experimental study paces, this experiment grinds 16 batches of Folium Sauropi medicinal materials using UPLC Study carefully, establish its characteristic spectrum, provides the new method of science for the control of Folium Sauropi quality of medicinal material.
Summary of the invention
It is an object of the invention to deficiency, provide a kind of Folium Sauropi medicinal material in view of the defects existing in the prior art UPLC characteristic spectrum construction method and its discrimination method, this method precision is high, stability is good, reproducible, can be more comprehensively Reaction Folium Sauropi medicinal material feature, provide scientific new method for the control of Folium Sauropi quality of medicinal material.
Above-mentioned technical problem to be solved by this invention, is achieved by following technical solution:
A kind of UPLC characteristic spectrum construction method of Folium Sauropi medicinal material, it includes following steps:
(1) precision weighs Folium Sauropi medicinal material, and Folium Sauropi test solution is prepared;
(2) Folium Sauropi test solution is analyzed using Ultra Performance Liquid Chromatography instrument, obtains Folium Sauropi UPLC characteristic spectrum.
As a preferred embodiment, the chromatographic condition of Ultra Performance Liquid Chromatography instrument analysis used are as follows: with octadecylsilane Bonded silica gel is filler, and column temperature is 28 ~ 32 DEG C, water-soluble with the phosphoric acid of volume fraction 0.34 ~ 0.45% using methanol as mobile phase A Liquid is that Mobile phase B carries out gradient elution, and flow velocity is 0.25 ~ 0.36ml/min, and Detection wavelength is 320 ~ 400nm, sample volume 0.5 ~3μl。
As a kind of most preferably scheme, the chromatographic condition of Ultra Performance Liquid Chromatography instrument analysis used are as follows: with octadecyl silicon Alkane bonded silica gel is filler, and column temperature is 30 DEG C, is stream with the phosphate aqueous solution of volume fraction 0.4% using methanol as mobile phase A Dynamic phase B carries out gradient elution, flow velocity 0.3ml/min, Detection wavelength 349nm, and sample volume is 1 μ l.
As a preferred embodiment, the condition of gradient elution are as follows: 0 ~ 10min, the volume fraction of mobile phase A are 6%, stream The volume fraction of dynamic phase B is 94%;The volume fraction variation of 10 ~ 15min, mobile phase A are 6 ~ 7%, and the volume fraction of Mobile phase B becomes Turn to 94 ~ 93%;15 ~ 20min, the volume fraction of mobile phase A are 7%, and the volume fraction of Mobile phase B is 93%;20 ~ 25min, stream The volume fraction variation of dynamic phase A is 7 ~ 18%, and the volume fraction variation of Mobile phase B is 93 ~ 82%;25 ~ 30min, the body of mobile phase A Fraction variation is 18 ~ 23%, and the volume fraction variation of Mobile phase B is 82 ~ 77%;The volume fraction of 30 ~ 40min, mobile phase A become 23 ~ 45% are turned to, the volume fraction variation of Mobile phase B is 77 ~ 55%;40 ~ 50min, the volume fraction variation of mobile phase A for 45 ~ 59%, the volume fraction variation of Mobile phase B is 55 ~ 41%.
As a preferred embodiment, the test solution is prepared by the inclusion of the method for following steps: taking dragon 45 ~ 55% 20 ~ 30ml of methanol are added in Lei leaf 0.4 ~ 0.6g of powder, precision, are ultrasonically treated 25 ~ 35 minutes, let cool, shake up, and filter, Take subsequent filtrate to get.
It is prepared: taking by the inclusion of the method for following steps as one kind most preferably scheme, test solution Folium Sauropi powder 0.5g, sets in stuffed conical flask, and 50% methanol 25ml is added in precision, is ultrasonically treated 30 minutes, lets cool, shake up, and filters Cross, take subsequent filtrate to get.
The present invention also provides a kind of discrimination methods of Folium Sauropi medicinal material to include the following steps:
(1) precision weighs Folium Sauropi medicinal material to be identified, and Folium Sauropi sample solution to be identified is prepared;
(2) accurate to draw Folium Sauropi sample solution to be identified, Ultra Performance Liquid Chromatography instrument is injected, is measured to get dragon Lei to be identified Leaf UPLC characteristic spectrum;
(3) the UPLC characteristic spectrum measured is compared with the Folium Sauropi UPLC characteristic spectrum constructed, and if Folium Sauropi UPLC characteristic spectrum is consistent, then the Folium Sauropi quality of medicinal material is qualified.
As a preferred embodiment, the chromatographic condition of Ultra Performance Liquid Chromatography instrument analysis used are as follows: with octadecylsilane Bonded silica gel is filler, and column temperature is 28 ~ 32 DEG C, water-soluble with the phosphoric acid of volume fraction 0.34 ~ 0.45% using methanol as mobile phase A Liquid is that Mobile phase B carries out gradient elution, and flow velocity is 0.25 ~ 0.36ml/min, and Detection wavelength is 320 ~ 400nm, sample volume 0.5 ~3μl。
As a kind of most preferably scheme, the chromatographic condition of Ultra Performance Liquid Chromatography instrument analysis used are as follows: with octadecyl silicon Alkane bonded silica gel is filler, and column temperature is 30 DEG C, is stream with the phosphate aqueous solution of volume fraction 0.4% using methanol as mobile phase A Dynamic phase B carries out gradient elution, flow velocity 0.3ml/min, Detection wavelength 349nm, and sample volume is 1 μ l.
As a preferred embodiment, the condition of gradient elution are as follows: 0 ~ 10min, the volume fraction of mobile phase A are 6%, stream The volume fraction of dynamic phase B is 94%;The volume fraction variation of 10 ~ 15min, mobile phase A are 6 ~ 7%, and the volume fraction of Mobile phase B becomes Turn to 94 ~ 93%;15 ~ 20min, the volume fraction of mobile phase A are 7%, and the volume fraction of Mobile phase B is 93%;20 ~ 25min, stream The volume fraction variation of dynamic phase A is 7 ~ 18%, and the volume fraction variation of Mobile phase B is 93 ~ 82%;25 ~ 30min, the body of mobile phase A Fraction variation is 18 ~ 23%, and the volume fraction variation of Mobile phase B is 82 ~ 77%;The volume fraction of 30 ~ 40min, mobile phase A become 23 ~ 45% are turned to, the volume fraction variation of Mobile phase B is 77 ~ 55%;40 ~ 50min, the volume fraction variation of mobile phase A for 45 ~ 59%, the volume fraction variation of Mobile phase B is 55 ~ 41%.
As a preferred embodiment, the test solution is prepared by the inclusion of the method for following steps: taking dragon 45 ~ 55% 20 ~ 30ml of methanol are added in Lei leaf 0.4 ~ 0.6g of powder, precision, are ultrasonically treated 25 ~ 35 minutes, let cool, shake up, and filter, Take subsequent filtrate to get.
It is prepared: taking by the inclusion of the method for following steps as one kind most preferably scheme, test solution Folium Sauropi powder 0.5g, sets in stuffed conical flask, and 50% methanol 25ml is added in precision, is ultrasonically treated 30 minutes, lets cool, shake up, and filters Cross, take subsequent filtrate to get.
The utility model has the advantages that (1) by being measured to 16 batches of separate sources Folium Sauropi medicinal materials, has determined 8 characteristic peaks altogether, it is right 8 shared peaks have carried out pointing out for chemical component, confirm that No. 6 peaks are 6- Hydroxycoumarin peak, No. 8 peaks are Kaempferol-O- dragon Folium Sauropi medicinal material characteristic spectrum analysis side has been worked out using No. 8 peak as Kaempferol-O- O-gentibioside peak in gallbladder bioside peak The reference data of method;(2) from the distinctive characteristic spectrum angle of Folium Sauropi medicinal material, selected characteristic peak calculates the opposite guarantor of characteristic peak Time and relative peak area are stayed, the overall picture of Folium Sauropi medicinal material characteristic spectrum is constituted, controls the quality of Folium Sauropi medicinal material from original Certain several component contents measurement, rise to in entire drug Quality Detection, avoid single chemical component and examine Defect;(3) this method is quick, stablize and specificity is strong, can more fully react the feature of Folium Sauropi medicinal material, is Folium Sauropi Quality of medicinal material control provides the new method of science.
Detailed description of the invention
Fig. 1 is that Folium Sauropi medicinal material chromatographic peak reference substance points out map, and wherein A is Hydroxycoumarin reference substance solution, and B is mountain Naphthol -3-O- O-gentibioside test solution, C is test solution;Peak 1 is 6- Hydroxycoumarin, and peak 2 is Kaempferol- O- O-gentibioside.
Fig. 2 is Kaempferol-O- O-gentibioside ion flow graph.
Fig. 3 is Kaempferol-O- O-gentibioside mass spectrogram in Folium Sauropi medicinal material, and wherein B is
First mass spectrometric figure, C are second order ms figure.
Fig. 4 is 6- Hydroxycoumarin mass spectrogram in Folium Sauropi medicinal material, and A is ion flow graph, and B is first mass spectrometric figure.
Fig. 5 is that 16 batches of Folium Sauropi medicinal material UPLC characteristic patterns share peak.
Fig. 6 is Folium Sauropi UPLC compare feature map, and wherein peak 8 is Kaempferol-O- O-gentibioside.
Fig. 7 is separate sources Folium Sauropi medicinal material clustering figure.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described, it is clear that described embodiments are some of the embodiments of the present invention, rather than Whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise Under every other embodiment obtained, shall fall within the protection scope of the present invention.
Embodiment 1
The building of Folium Sauropi medicinal material characteristic spectrum
Key instrument: Thermo Ultra Performance Liquid Chromatography instrument (Thermo Vanquish, Thermo company);DAD detector, Agilent SB C18Chromatographic column, chameleon work station, ACQUITY UPLC I- Class ultra high efficiency liquid phase flight time high resolution mass spectrum combined system, data processing system are 4.1 work station of MarkerLynx; Hundred a ten thousandth balance of Mettler XP26 (Mettler Toledo company, Switzerland);Ultrapure water purification system (the U.S. Milli-Q Millipore company);KQ5500DE type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
Folium Sauropi medicinal material is derived from national different Chinese Medicinal Materials Markets and pharmacy.
1 Folium Sauropi crude drug source of table
Main agents: methanol, acetonitrile are chromatographically pure (Merk), water is Mili-Q purified water, remaining reagent is that analysis is pure.Kaempferia galanga (lot number: content: 96.2%) 112025-201601 is provided phenol -3-O- O-gentibioside by National Institute for Food and Drugs Control; (lot number: content: 98.0%) Z29D7H27807 is provided 6- Hydroxycoumarin by Shanghai Yuan Ye Biotechnology Co., Ltd.
Test method
The preparation of Folium Sauropi medicinal material test sample
Folium Sauropi powder 0.5g is taken, is set in stuffed conical flask, 50% methanol 25ml is added in precision, and it is ultrasonically treated 30 minutes, lets cool, Shake up, filter, take subsequent filtrate to get.
The preparation of reference substance solution
Accurately weighed Kaempferol-O- O-gentibioside reference substance 3.233mg, sets in 25ml measuring bottle, methanol is added to dissolve and dilute To scale, Kaempferol-O- O-gentibioside reference substance solution stock solution is obtained, concentration is 124.4058 μ g/ml.On precision is drawn It states Kaempferol-O- O-gentibioside reference substance solution stock solution 0.5ml to set in 10ml measuring bottle, adds methanol constant volume to scale, shake It is even, the reference substance solution that concentration is 6.2203 μ g/ml is made.Accurately weighed 6- Hydroxycoumarin reference substance 2.126mg, sets 25ml In measuring bottle, add methanol to dissolve and be diluted to scale, obtain 6- Hydroxycoumarin reference substance solution stock solution, concentration is 83.3392 μ g/ ml.Precision is drawn above-mentioned 6- Hydroxycoumarin reference substance solution stock solution 1ml and is set in 10ml measuring bottle, adds methanol constant volume to scale, It shakes up, the reference substance solution that concentration is 8.3339 μ g/ml is made.
Chromatographic condition
Use specification forAgilent SB C18Chromatographic column, column temperature are 30 DEG C, with Methanol is mobile phase A, carries out gradient elution, flow velocity 0.3ml/ by Mobile phase B of the phosphate aqueous solution of volume fraction 0.4% Min, Detection wavelength 349nm, sample volume are 1 μ l.
Condition of gradient elution are as follows: 0 ~ 10min, the volume fraction of mobile phase A are 6%, and the volume fraction of Mobile phase B is 94%; The volume fraction variation of 10 ~ 15min, mobile phase A are 6 ~ 7%, and the volume fraction variation of Mobile phase B is 94 ~ 93%;15 ~ 20min, The volume fraction of mobile phase A is 7%, and the volume fraction of Mobile phase B is 93%;20 ~ 25min, the volume fraction of mobile phase A, which changes, is 7 ~ 18%, the volume fraction variation of Mobile phase B is 93 ~ 82%;The volume fraction variation of 25 ~ 30min, mobile phase A are 18 ~ 23%, stream The volume fraction variation of dynamic phase B is 82 ~ 77%;The volume fraction variation of 30 ~ 40min, mobile phase A are 23 ~ 45%, Mobile phase B Volume fraction variation is 77 ~ 55%;The volume fraction variation of 40 ~ 50min, mobile phase A are 45 ~ 59%, the volume fraction of Mobile phase B Variation is 55 ~ 41%.
Mass Spectrometry Conditions
Use specification forAgilent SB C18Chromatographic column, column temperature are 30 DEG C, with Methanol is mobile phase A, carries out gradient elution by Mobile phase B of the aqueous formic acid of volume fraction 0.05%;Flow velocity is 0.3ml/ min;Detection wavelength is 349nm, and sample volume is 1 μ l.
Condition of gradient elution are as follows: 0 ~ 10min, the volume fraction of mobile phase A are 6%, and the volume fraction of Mobile phase B is 94%; The volume fraction variation of 10 ~ 15min, mobile phase A are 6 ~ 7%, and the volume fraction variation of Mobile phase B is 94 ~ 93%;15 ~ 20min, The volume fraction of mobile phase A is 7%, and the volume fraction of Mobile phase B is 93%;20 ~ 25min, the volume fraction of mobile phase A, which changes, is 7 ~ 18%, the volume fraction variation of Mobile phase B is 93 ~ 82%;The volume fraction variation of 25 ~ 30min, mobile phase A are 18 ~ 23%, stream The volume fraction variation of dynamic phase B is 82 ~ 77%;The volume fraction variation of 30 ~ 40min, mobile phase A are 23 ~ 45%, Mobile phase B Volume fraction variation is 77 ~ 55%;The volume fraction variation of 40 ~ 50min, mobile phase A are 45 ~ 59%, the volume fraction of Mobile phase B Variation is 55 ~ 41%.
Atomization and dry gas of the nitrogen as mass ion source, electrospray ionisation (positive and negative) ion mode, spray voltage 3.0KV, Lens voltage 50V, 400 °C of desolvation temperature, sheath atmospheric pressure 40Arb, assist gas pressure power 12Arb, mass charge ratio range 100~1000。
Characteristic spectrum methodological study
Precision test: taking Folium Sauropi medicinal material S1 sample, be prepared into test solution, under defined chromatographic condition continuously into Sample 6 times, investigate the consistency of shared peak relative retention time and relative peak area, gained shares the relative peak area at peak and opposite Retention time RSD < 2% shows that instrument precision is good.
Stability test: taking Folium Sauropi medicinal material S1 sample, be prepared into test solution, in the condition of regulation chromatography Under, it is measured respectively at 0,2,4,6,8,12,20 sample introductions, investigates the consistency of shared peak relative retention time and relative peak area, And calculate RSD value.Gained shares the relative peak area and relative retention time RSD < 2% at peak, shows test solution in 20h It has good stability.
Repetitive test: taking Folium Sauropi medicinal material S1 sample, prepare 6 parts of test solution, in the case where providing chromatographic condition, into Sample measurement, investigates the consistency of shared peak relative retention time and relative peak area, and calculates RSD value.Gained shares the phase at peak To peak area RSD < 3%, relative retention time RSD < 2% shows that this method repeatability is good.
The analysis of Folium Sauropi medicinal material characteristic spectrum and evaluation
Sample measurement
16 batches of Folium Sauropi medicinal material samples are taken, test solution is prepared, under defined chromatography and Mass Spectrometry Conditions, records UPLC respectively Map and mass spectrogram.Folium Sauropi medicinal material chromatographic peak reference substance points out map and sees Fig. 1.
Folium Sauropi medicinal material characteristic spectrum shares the determination at peak
" similarity evaluation (2012.0 version) " recommended using Chinese Pharmacopoeia Commission is to 16 It criticizes Folium Sauropi medicinal material UPLC map and carries out interpretation of result, determine 8 shared peaks altogether.In 8 shared peaks, No. 8 peak (Kaempferol- 3-O- O-gentibioside) separating degree is good, and peak shape is symmetrical, and is one of anti-inflammatory, cough-relieving, main component of antiulcer in Folium Sauropi, It therefore is to calculate separately the opposite guarantor at each shared peak referring to peak (the as peak S) with No. 8 peak (Kaempferol-O- O-gentibioside) Time and relative peak area are stayed, each sample shares the relative retention time RSD at peak 0.05%~1.04%, shows shared peak selection Accuracy;Peak area difference without each shared peak of same sample is bigger, shows that the Folium Sauropi medicinal material of different sources shares peak Content has a certain difference, and is shown in Table 2.
2 16 batches of Folium Sauropi medicinal material characteristic spectrum relative retention times of table and relative peak area
The mass spectrum of Folium Sauropi medicinal material characteristic spectrum is pointed out
According to above-mentioned Mass Spectrometry Conditions, characteristic peak 8 is analyzed, the first mass spectrometric of sample and the mass spectrum of second order ms respective peaks Information is shown in Fig. 2, and wherein the accurate molecular weight at peak 8 is 287.055, is judged according to document and database, with Kaempferol-O- rough gentian Bioside is almost the same, thus may determine that the chromatographic peak is Kaempferol-O- O-gentibioside, sees Fig. 3.By in test sample First mass spectrometric figure and leading ion mass-to-charge ratio (163.039), according to the literature and first mass spectrometric information calculate it is corresponding Molecular formula is C9H6O3, thus it is speculated that it should be Hydroxycoumarin or corresponding isomers, see Fig. 4.
Folium Sauropi medicinal material characteristic spectrum similarity evaluation
" similarity evaluation ((2012.0 version) " recommended using Chinese Pharmacopoeia Commission is to S1 ~S16 Folium Sauropi medicinal material sample characteristic map is overlapped, and is carried out shared peak mark, and generate control map, is investigated The consistency of chromatographic peak similarity carries out similarity evaluation, as a result sees Fig. 5, Fig. 6, table 3.The result shows that 16 batches of different sources dragons The similarity of Lei leaf medicinal material sample illustrates that sample characteristic map and the similarity for compareing map are higher 0.9 or more, and 8 altogether There is peak can stablize appearance in each sample, and the goodness of fit is preferable, therefore the map can be used as the characteristic spectrum of Folium Sauropi medicinal material.
The similarity of the different Folium Sauropi samples of table 3
Separate sources Folium Sauropi medicinal material clustering
Characteristic spectrum analysis is carried out by the Folium Sauropi medicinal material to 16 batches of separate sources, has obtained including 6- Hydroxycoumarin (peak 7), 8 shared peaks including Kaempferol-O- O-gentibioside (peak 8), the relative peak area at each shared peak is quantified, Hierarchial-cluster analysis is carried out to it using 22.0 software of SPSS, is to estimate with Euclidean distance, to 16 batches of Folium Sauropi medicinal materials with respect to peak Area is analyzed, and as a result sees Fig. 7.When judging distance for 5,16 batches of Folium Sauropi medicinal materials are divided into 3 classes.S1~S3,S5~S6, S11 ~ 12, S14, S16 are gathered for one kind, such sample crude drug source is Gaoyao City, Qingyuan City, Zhaoqing;S4, S8, S10, S13 are poly- For one kind, such sample medicinal material main source is the peaceful medicinal material market in Guangzhou;S7, S9, S15 gather for one kind, and main source is wide The peaceful medicinal material market in state and Zhaoqing, wherein S4, S13, S15 sample and its crude drug source have differences, and are originated from height respectively City, Qingyuan City and Zhaoqing are wanted, clustering is not returned in corresponding one kind, come from the relative peak area of S4 sample It sees, there is some difference for the relative peak area of peak 2, peak 3 and peak 6 and other samples, the phase at 8 shared peaks of S13 sample The sample in the same place of production is all larger than to peak area, and S15 sample is then in reversed trend, relative peak area is respectively less than the sample with the place of production Product.By clustering the study found that the Folium Sauropi medicinal material chemical component difference in different sources source is larger, between provenance There were significant differences, and the Folium Sauropi quality of medicinal material in the same place of production is also and disunity, illustrates to influence the factor of quality of medicinal material not only It is provenance, the links such as collecting season, Habitat producing drying also generate certain influence to Folium Sauropi quality of medicinal material.It is wherein imperial Lei leaf processing of crude drugs drying mode are as follows: take Folium Sauropi medicinal material, each 3-5 piece dries in the shade, or dries in the shade to after very likely, and blade is folded It is bundled into small bundle together, is covered with cattail mat, dries.It is analyzed from sample property, compares other samples, S4, S13 and S15 sample medicinal material Quality is softer, and sample moisture content is higher, and consideration may be to cause sample to contain because medicinal material Habitat producing degree of drying is inadequate It is had differences between amount, certain influence is produced to Folium Sauropi quality of medicinal material.
Embodiment 2
The discrimination method of Folium Sauropi medicinal material, steps are as follows for identification:
(1) precision weighs Folium Sauropi medicinal material to be identified, and Folium Sauropi sample solution to be identified is prepared;
(2) accurate to draw Folium Sauropi sample solution to be identified, Ultra Performance Liquid Chromatography instrument is injected, is measured to get dragon Lei to be identified Leaf UPLC characteristic spectrum;
(3) the UPLC characteristic spectrum measured is compared with the Folium Sauropi UPLC characteristic spectrum constructed, and if Folium Sauropi UPLC characteristic spectrum is consistent, then the Folium Sauropi quality of medicinal material is qualified.
Chromatographic condition
Use specification forAgilent SB C18Chromatographic column, column temperature are 30 DEG C, with Methanol is mobile phase A, carries out gradient elution by Mobile phase B of the phosphate aqueous solution of volume fraction 0.4%;Flow velocity is 0.3ml/ min;Detection wavelength is 349nm, and sample volume is 1 μ l.
Condition of gradient elution
0 ~ 10min, the volume fraction of mobile phase A are 6%, and the volume fraction of Mobile phase B is 94%;10 ~ 15min, the body of mobile phase A Fraction variation is 6 ~ 7%, and the volume fraction variation of Mobile phase B is 94 ~ 93%;The volume fraction of 15 ~ 20min, mobile phase A is 7%, the volume fraction of Mobile phase B is 93%;The volume fraction variation of 20 ~ 25min, mobile phase A are 7 ~ 18%, the volume of Mobile phase B Score variation is 93 ~ 82%;The volume fraction variation of 25 ~ 30min, mobile phase A are 18 ~ 23%, and the volume fraction of Mobile phase B changes It is 82 ~ 77%;The volume fraction variation of 30 ~ 40min, mobile phase A are 23 ~ 45%, the variation of the volume fraction of Mobile phase B for 77 ~ 55%;The volume fraction variation of 40 ~ 50min, mobile phase A are 45 ~ 59%, and the volume fraction variation of Mobile phase B is 55 ~ 41%.
The preparation of Folium Sauropi sample solution to be identified
Folium Sauropi powder 0.5g is taken, 50% methanol 25ml of accurate addition in stuffed conical flask is set, is ultrasonically treated 30 minutes, lets cool, shake It is even, filtration, take subsequent filtrate to get.
Sample introduction measurement
Folium Sauropi sample solution to be identified is prepared by the above method, sample introduction measurement is in the case where providing chromatographic condition to get to be identified Folium Sauropi medicinal material UPLC characteristic spectrum.
Folium Sauropi medicinal material characteristic spectrum to be identified is compared with the Folium Sauropi medicinal material characteristic spectrum constructed, the two one It causes, then the Folium Sauropi sample to be identified is qualified Folium Sauropi medicinal material.
This research establishes the UPLC characteristic spectrum of Folium Sauropi medicinal material, establishes 8 shared peaks.The UPLC that this experiment is established Characteristic spectrum can be good at the overall picture for reflecting Folium Sauropi flavonoids, and this method is accurate as the result is shown for methodological study Degree, stability and reproducibility are good, show that the method is accurate and reliable, can be used as the foundation of Folium Sauropi quality of medicinal material control.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and what is described in the above embodiment and the description is only the present invention Principle, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these variation and Improvement is both fallen in the range of claimed invention.The present invention claims protection scope by appended claims and its Equivalent defines.

Claims (10)

1. a kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method, which is characterized in that comprise the following steps:
(1) precision weighs Folium Sauropi medicinal material, and Folium Sauropi test solution is prepared;
(2) Folium Sauropi test solution is analyzed using Ultra Performance Liquid Chromatography instrument, obtains Folium Sauropi UPLC characteristic spectrum.
2. Folium Sauropi medicinal material UPLC characteristic spectrum construction method according to claim 1, which is characterized in that the ultra high efficiency The chromatographic condition of liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature is 28 ~ 32 DEG C, with first Alcohol is mobile phase A, using the phosphate aqueous solution of volume fraction 0.34 ~ 0.45% as Mobile phase B carry out gradient elution, flow velocity be 0.25 ~ 0.36ml/min, Detection wavelength are 320 ~ 400nm, and sample volume is 0.5 ~ 3 μ l.
3. Folium Sauropi medicinal material UPLC characteristic spectrum construction method according to claim 2, which is characterized in that the ultra high efficiency The chromatographic condition of liquid chromatograph analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature is 30 DEG C, is with methanol Mobile phase A carries out gradient elution, flow velocity 0.3ml/min, detection by Mobile phase B of the phosphate aqueous solution of volume fraction 0.4% Wavelength is 349nm, and sample volume is 1 μ l.
4. Folium Sauropi medicinal material UPLC characteristic spectrum construction method according to claim 1, which is characterized in that gradient elution item Part are as follows: 0 ~ 10min, the volume fraction of mobile phase A are 6%, and the volume fraction of Mobile phase B is 94%;10 ~ 15min, mobile phase A Volume fraction variation is 6 ~ 7%, and the volume fraction variation of Mobile phase B is 94 ~ 93%;The volume fraction of 15 ~ 20min, mobile phase A is 7%, the volume fraction of Mobile phase B is 93%;The volume fraction variation of 20 ~ 25min, mobile phase A are 7 ~ 18%, the volume of Mobile phase B Score variation is 93 ~ 82%;The volume fraction variation of 25 ~ 30min, mobile phase A are 18 ~ 23%, and the volume fraction of Mobile phase B changes It is 82 ~ 77%;The volume fraction variation of 30 ~ 40min, mobile phase A are 23 ~ 45%, the variation of the volume fraction of Mobile phase B for 77 ~ 55%;The volume fraction variation of 40 ~ 50min, mobile phase A are 45 ~ 59%, and the volume fraction variation of Mobile phase B is 55 ~ 41%.
5. Folium Sauropi medicinal material UPLC characteristic spectrum construction method according to claim 1, which is characterized in that described for examination Product solution is prepared by the inclusion of the method for following steps: taking Folium Sauropi 0.4 ~ 0.6g of powder, 45 ~ 55% methanol are added in precision 20 ~ 30ml, be ultrasonically treated 25 ~ 35 minutes, let cool, shake up, filter, take subsequent filtrate to get.
6. a kind of discrimination method of Folium Sauropi medicinal material, which comprises the steps of:
(1) precision weighs Folium Sauropi medicinal material to be identified, and Folium Sauropi sample solution to be identified is prepared;
(2) accurate to draw Folium Sauropi sample solution to be identified, Ultra Performance Liquid Chromatography instrument is injected, is measured to get dragon Lei to be identified Leaf UPLC characteristic spectrum;
(3) the Folium Sauropi UPLC characteristic spectrum for constructing any one of the UPLC characteristic spectrum measured and claim 1 ~ 5 method It is compared, if consistent with Folium Sauropi UPLC characteristic spectrum, the Folium Sauropi quality of medicinal material is qualified.
7. the discrimination method of Folium Sauropi medicinal material according to claim 6, which is characterized in that the Ultra Performance Liquid Chromatography instrument The chromatographic condition of analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature is 28 ~ 32 DEG C, using methanol as mobile phase A carries out gradient elution by Mobile phase B of the phosphate aqueous solution of volume fraction 0.34 ~ 0.45%, and flow velocity is 0.25 ~ 0.36ml/ Min, Detection wavelength are 320 ~ 400nm, and sample volume is 0.5 ~ 3 μ l.
8. the discrimination method of Folium Sauropi medicinal material according to claim 6, which is characterized in that the Ultra Performance Liquid Chromatography instrument The chromatographic condition of analysis are as follows: using octadecylsilane chemically bonded silica as filler, column temperature is 30 DEG C, using methanol as mobile phase A, with The phosphate aqueous solution of volume fraction 0.4% is that Mobile phase B carries out gradient elution, flow velocity 0.3ml/min, and Detection wavelength is 349nm, sample volume are 1 μ l.
9. the discrimination method of Folium Sauropi medicinal material according to claim 6, which is characterized in that condition of gradient elution are as follows: 0 ~ 10min, the volume fraction of mobile phase A are 6%, and the volume fraction of Mobile phase B is 94%;10 ~ 15min, the volume fraction of mobile phase A Variation is 6 ~ 7%, and the volume fraction variation of Mobile phase B is 94 ~ 93%;15 ~ 20min, the volume fraction of mobile phase A are 7%, flowing The volume fraction of phase B is 93%;The volume fraction variation of 20 ~ 25min, mobile phase A are 7 ~ 18%, and the volume fraction of Mobile phase B becomes Turn to 93 ~ 82%;The volume fraction variation of 25 ~ 30min, mobile phase A are 18 ~ 23%, the variation of the volume fraction of Mobile phase B for 82 ~ 77%;The volume fraction variation of 30 ~ 40min, mobile phase A are 23 ~ 45%, and the volume fraction variation of Mobile phase B is 77 ~ 55%;40~ The volume fraction variation of 50min, mobile phase A are 45 ~ 59%, and the volume fraction variation of Mobile phase B is 55 ~ 41%.
10. the discrimination method of Folium Sauropi medicinal material according to claim 6, which is characterized in that the test solution is logical It crossing the method comprised the following steps to be prepared: taking Folium Sauropi 0.4 ~ 0.6g of powder, 45 ~ 55% 20 ~ 30ml of methanol are added in precision, Ultrasonic treatment 25 ~ 35 minutes, let cool, shake up, filter, take subsequent filtrate to get.
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