CN110441442A - A kind of Poria cocos standard decoction UPLC characteristic spectrum construction method and detection method - Google Patents
A kind of Poria cocos standard decoction UPLC characteristic spectrum construction method and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of Poria cocos standard decoction UPLC characteristic spectrum construction method and detection methods.The construction method of the Poria cocos standard decoction UPLC characteristic spectrum comprises the following steps: (1) precision weighs Poria cocos standard decoction, and Poria cocos standard decoction test solution is prepared；(2) Poria cocos standard decoction test solution is analyzed using Ultra Performance Liquid Chromatography instrument, obtains Poria cocos standard decoction UPLC characteristic spectrum.This method precision, stability and reproducibility are preferable, sufficiently illustrate the Chemical Composition Characteristics of Poria cocos standard decoction, quality control can be carried out to Poria cocos standard decoction comprehensively and accurately, while also the foundation for Poria cocos medicinal material and Poria cocos granule quality standard provides foundation.
Description
Technical field
The invention belongs to Chinese medicinal granule quality standard research fields, and in particular to a kind of Poria cocos standard decoction UPLC is special
Levy map construction method and detection method.
Background technique
Dry sclerotia that Poria cocos is polyporaceae fungus Poria cocos (Schw.) Wolf (version in 2015 " in
State's pharmacopeia " one record), be conventional Chinese medicine, there is the effect of clearing damp and promoting diuresis, invigorating the spleen, calming heart；It is dizzy for edema and little urine, phlegm retention
Throb with fear, the diseases such as spleen eating less, loose stool diarrhea, confused and worried and insomnia of palpitating with fear, clinically has a wide range of applications.Poria cocos nature and flavor
Sweet, light, flat, return heart, lung, spleen, kidney channel are loaded in Shennong's Herbal, are classified as top grade.
Main component contained in Poria cocos is in addition to this pachymaran and triterpenes components have been reported in Poria cocos also
Contain the inorganic elements such as Analysis of Steroids, fatty acid, protein, adenine, amino acid and calcium, magnesium, iron, potassium.Modern pharmacology
Studies have shown that pachymaran and triterpenes components are in anti-aging, anticancer and in terms of immune regulation have a significant pharmacological activity.
2015 version " Chinese Pharmacopoeia " one the qualitative of clear ingredient and fixed is not related to the control of Poria cocos quality
Amount, this haves the defects that the quality control of Poria cocos medicinal material certain.At present about Poria cocos medicinal material index components content measure with
And feature refers to that the method for map has many reports.Zhang Jingqi using UPLC by being established while measuring 6 kinds of ingredients of Poria cocos
Method, its whole spectral peak can have been detected completely in 10min, analysis time is effectively shortened, and analysis efficiency obtains
It significantly improves, for effectively, accurately and rapidly control Poria cocos quality provides methodology foundation.Fourth hilllock is linearly terraced using RP-HPLC
The methods of degree elution filters out optimum controlling condition, carries out Triterpene acids in Poria cocos at 210nm and 242nm
The research of HPLC finger-print, identifies 9 chromatographic peaks, more fully reflects Triterpene acids in Poria cocos, for effectively control
Poria cocos quality of medicinal material provides methodology basis.
According to " control of Chinese medicinal granule quality and the standard formulation technical requirements of Chinese Pharmacopoeia Commission's publication in 2016
(exposure draft) ", Chinese medicine standard decoction system follows traditional Chinese medical theory, is advised using qualified medicine materical crude slice according to clinical decoction decocting mode
Model decocts, and is separated by solid-liquid separation, and is suitably concentrated and is made or is made through proper method is dry, as measure Chinese medicinal granule whether with
The almost the same marker of clinical decoction.
Since the decoction solvent of Poria cocos standard decoction is water, many Triterpenoids not soluble in water cannot in standard decoction
It is embodied very well.Therefore for the present invention on the basis of forefathers study Poria cocos medicinal material, Poria cocos mark can be reflected comprehensively by establishing
The measuring method of the UPLC characteristic spectrum of water-soluble character ingredient in quasi- decoction.Using Poria cocos characteristic spectrum determined pachymic acid A,
4 characteristic peaks such as pachymic acid B, and pointed out 3 peaks therein.
Summary of the invention
It is an object of the invention to deficiency, provide a kind of Poria cocos standard decoction in view of the defects existing in the prior art
The construction method and detection method of UPLC characteristic spectrum, this method precision is high, stability is good, reproducible, by constructed
Poria cocos standard decoction UPLC characteristic spectrum for Poria cocos standard decoction quality control quick, comprehensive detection means is provided.
Above-mentioned technical problem to be solved by this invention, is achieved by following technical solution:
A kind of Poria cocos standard decoction UPLC characteristic spectrum construction method, it includes following steps:
(1) precision weighs Poria cocos standard decoction, and Poria cocos standard decoction test solution is prepared；
(2) Poria cocos standard decoction test solution is analyzed using Ultra Performance Liquid Chromatography instrument, obtains Poria cocos standard decoction 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, using acetonitrile as mobile phase A, using 0.08 ~ 0.12% formic acid as Mobile phase B, carries out gradient elution, flow velocity
For 0.17 ~ 0.22ml/min, column temperature is 28 ~ 32 DEG C, and sample volume is 0.08 ~ 0.12 μ l, and Detection wavelength is 220 ~ 280nm.
As a kind of most preferably scheme, the chromatographic condition of high performance liquid chromatograph analysis used are as follows: with octadecylsilane
Bonded silica gel is filler, using acetonitrile as mobile phase A, using 0.1% formic acid as Mobile phase B, carries out gradient elution, flow velocity is
0.2ml/min, column temperature are 30 DEG C, and sample volume is 1 μ l, Detection wavelength 252nm.
As a preferred embodiment, the condition of gradient elution are as follows: 0 ~ 21min, the volume fraction of mobile phase A, which changes, is
40 ~ 99%, the volume fraction variation of Mobile phase B is 60 ~ 1%；The volume fraction variation of 21 ~ 22min, mobile phase A are 99 ~ 40%, stream
The volume fraction variation of dynamic phase B is 1 ~ 60%.
As a preferred embodiment, the Poria cocos standard decoction adds decocting the preparation method comprises the following steps: take Poria cocos 180 ~ 220g of medicine materical crude slice
It boils twice, decocts be added in 8 ~ 10 times of amount water for the first time, impregnate 40 ~ 60 minutes, be cooked by slow fire after boiling by intense fire 26 ~ 32 minutes,
Decocting liquid filters while hot, and filtrate is cooling with cold water rapidly；Second plus 6.4 ~ 7.6 times of amount water, intense fire heating are cooked by slow fire after boiling
22 ~ 28 minutes, for decocting liquid with filtering while hot, filtrate was cooling with cold water rapidly, merged decocting liquid twice；Decocting liquid is transferred to container, is subtracted
Force down the medicinal extract that temperature is concentrated into 120 ~ 140g；Stirring is dispensed into 8 ~ 12ml bottles, and every bottle of packing volume is 1.6 ~ 2.4ml, packing
It is complete after freeze-drying, take out to get.
As a kind of most preferably scheme, the Poria cocos standard decoction sets electroceramics the preparation method comprises the following steps: take Poria cocos medicine materical crude slice 200g
It in pot, adds water to cook twice, decocts be added in 9 times of amount water for the first time, impregnate 50 minutes, 500W boils rear 300W by intense fire and is cooked by slow fire
It boils 30 minutes, decocting liquid filters while hot through 400 mesh screens, and filtrate is cooling with cold water rapidly, second plus 7 times of amount water, intense fire heating
It is cooked by slow fire after boiling 25 minutes, decocting liquid is filtered while hot with 400 mesh screens, and filtrate is cooling with cold water rapidly, merges decocting liquid twice.
Decocting liquid is transferred in round-bottomed flask, in temperature: 62 DEG C；Vacuum degree: dense using Rotary Evaporators decompression low temperature when -0.10MPa
It is reduced to the medicinal extract of 130g；Under magnetic stirring, packing is into 10ml brown cillin bottle, and every bottle of packing volume is 2ml, after having dispensed
It is transferred in vacuum freeze drier and is lyophilized, take out, roll aluminium lid to get Poria cocos vacuum freeze drier eutectic point test knot
Fruit is -32.5 DEG C, and pre-freezing temperature is -45oC, and lyophilization temperature -35oC ~ 0oC, parsing-desiccation temperature is 10oC ~ 20oC.
As a preferred embodiment, the test solution is prepared by the inclusion of the method for following steps: taking Fu
Siberian cocklebur standard decoction is appropriate, finely ground, takes the Poria cocos standard decoction powder equal to 25 ~ 35g Poria cocos medicinal material amount, accurately weighed, and precision adds
Enter 45 ~ 55% 9 ~ 11ml of methanol, weighed weight is ultrasonically treated 28 ~ 32 minutes, lets cool, then weighed weight, is mended with 45 ~ 55% methanol
The weight of sufficient less loss, shakes up, and centrifugation, supernatant crosses 0.18 ~ 0.26 μm of miillpore 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
Poria cocos standard decoction is appropriate, finely ground, takes the Poria cocos standard decoction powder equal to 30g Poria cocos medicinal material amount, accurately weighed, sets tool plug cone
In shape bottle, 50% methanol 10ml is added in precision, and weighed weight, with power 250W, the ultrasonic treatment of frequency 40kHz 30 minutes is put
It is cold, then weighed weight, the weight of less loss is supplied with 50% methanol, is shaken up, is centrifuged, and supernatant crosses 0.22 μm of miillpore filter, takes continuous filter
Liquid to get.
The present invention also provides the detection methods of Poria cocos standard decoction, include the following steps:
(1) precision weighs Poria cocos standard decoction to be detected, and Poria cocos standard decoction sample solution to be detected is prepared；
(2) accurate to draw Poria cocos standard decoction sample solution to be detected, Ultra Performance Liquid Chromatography instrument is injected, is measured to get to be checked
Survey Poria cocos standard decoction UPLC characteristic spectrum；
(3) the UPLC characteristic spectrum measured is compared with the Poria cocos standard decoction UPLC characteristic spectrum constructed, and if Fu
Siberian cocklebur standard decoction UPLC characteristic spectrum is consistent, then up-to-standard.
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, using acetonitrile as mobile phase A, using 0.08 ~ 0.12% formic acid as Mobile phase B, carries out gradient elution, flow velocity
For 0.17 ~ 0.22ml/min, column temperature is 28 ~ 32 DEG C, and sample volume is 0.08 ~ 0.12 μ l, and Detection wavelength is 220 ~ 280nm.
As a kind of most preferably scheme, the chromatographic condition of high performance liquid chromatograph analysis used are as follows: with octadecylsilane
Bonded silica gel is filler, using acetonitrile as mobile phase A, using 0.1% formic acid as Mobile phase B, carries out gradient elution, flow velocity is
0.2ml/min, column temperature are 30 DEG C, and sample volume is 1 μ l, Detection wavelength 252nm.
As a preferred embodiment, the condition of gradient elution are as follows: 0 ~ 21min, the volume fraction of mobile phase A, which changes, is
40 ~ 99%, the volume fraction variation of Mobile phase B is 60 ~ 1%；The volume fraction variation of 21 ~ 22min, mobile phase A are 99 ~ 40%, stream
The volume fraction variation of dynamic phase B is 1 ~ 60%.
As a preferred embodiment, the Poria cocos standard decoction to be detected is the preparation method comprises the following steps: take Poria cocos 180 ~ 220g of medicine materical crude slice,
It adds water to cook twice, decocts be added in 8 ~ 10 times of amount water for the first time, impregnate 40 ~ 60 minutes, be cooked by slow fire 26 ~ 32 after boiling by intense fire
Minute, decocting liquid filters while hot, and filtrate is cooling with cold water rapidly；Rear mild fire is boiled in second plus 6.4 ~ 7.6 times of amount water, intense fire heating
It decocts 22 ~ 28 minutes, for decocting liquid with filtering while hot, filtrate is cooling with cold water rapidly, merges decocting liquid twice；Decocting liquid is transferred to appearance
Device, decompression low temperature are concentrated into the medicinal extract of 120 ~ 140g；Stirring, packing in 8 ~ 12ml bottle, every bottle of packing volume for 1.6 ~
2.4ml, dispensed after freeze-drying, take out to get.
As a kind of most preferably scheme, the Poria cocos standard decoction to be detected is set the preparation method comprises the following steps: take Poria cocos medicine materical crude slice 200g
It in electroceramics pot, adds water to cook twice, decocts be added in 9 times of amount water for the first time, impregnate 50 minutes, 500W boils rear 300W by intense fire
It is cooked by slow fire 30 minutes, decocting liquid filters while hot through 400 mesh screens, and filtrate is cooling with cold water rapidly, second plus 7 times of amount water, military
Fire heating is cooked by slow fire 25 minutes after boiling, and decocting liquid is filtered while hot with 400 mesh screens, and filtrate is cooling with cold water rapidly, merges two
Secondary decocting liquid.Decocting liquid is transferred in round-bottomed flask, in temperature: 62 DEG C；Vacuum degree: it is depressurized when -0.10MPa using Rotary Evaporators
Low temperature is concentrated into the medicinal extract of 130g；Under magnetic stirring, into 10ml brown cillin bottle, every bottle of packing volume is 2ml for packing, point
It is transferred in vacuum freeze drier and is lyophilized after installing, take out, roll aluminium lid to get Poria cocos vacuum freeze drier eutectic point
Test result is -32.5 DEG C, and pre-freezing temperature is -45oC, and lyophilization temperature -35oC ~ 0oC, parsing-desiccation temperature is 10oC ~ 20
ºC。
As a preferred embodiment, the Poria cocos standard decoction sample solution to be detected by the inclusion of following steps side
Method is prepared: take Poria cocos standard decoction appropriate, it is finely ground, and the Poria cocos standard decoction powder equal to 25 ~ 35g Poria cocos medicinal material amount is taken,
Accurately weighed, 45 ~ 55% 9 ~ 11ml of methanol are added in precision, and weighed weight is ultrasonically treated 28 ~ 32 minutes, lets cool, then weighed weight,
The weight that less loss is supplied with 45 ~ 55% methanol, shakes up, and centrifugation, supernatant crosses 0.18 ~ 0.26 μm of miillpore filter, takes subsequent filtrate, i.e.,
.
As a kind of most preferably scheme, the Poria cocos standard decoction sample solution to be detected is by the inclusion of following steps
Method is prepared: take Poria cocos standard decoction appropriate, it is finely ground, and the Poria cocos standard decoction powder equal to 30g Poria cocos medicinal material amount is taken, essence
It is close weighed, it sets in stuffed conical flask, 50% methanol 10ml, weighed weight, with power 250W, the ultrasound of frequency 40kHz is added in precision
Processing 30 minutes, let cool, then weighed weight, the weight of less loss supplied with 50% methanol, is shaken up, be centrifuged, supernatant cross 0.22 μm it is micro-
Hole filter membrane, take subsequent filtrate to get.
The utility model has the advantages that (1) present invention constructs the UPLC characteristic spectrum and detection method of Poria cocos standard decoction for the first time；(2)
The UPLC characteristic spectrum for the Poria cocos standard decoction that the present invention constructs, sufficiently illustrates the Chemical Composition Characteristics of Poria cocos standard decoction；
(3) characteristic spectrum that the present invention constructs comprehensively reflects the characteristic peak information of sample, and method is stablized, and precision is high, reproducibility
Preferably；(4) measuring method of Poria cocos standard decoction of the present invention, for Poria cocos standard decoction quality control provide quickly,
Comprehensive detection means；(5) present invention can carry out quality control to Poria cocos standard decoction comprehensively and accurately, while also be Fu
The foundation of Siberian cocklebur medicinal material and Poria cocos granule quality standard provides foundation.
Detailed description of the invention
Fig. 1 is 25 batch Poria cocos standard decoction UPLC characteristic spectrum stacking charts.
Fig. 2 is Poria cocos standard decoction UPLC compare feature map.
Fig. 3 is Poria cocos control medicinal material UPLC compare feature map.
Fig. 4 is Poria cocos standard decoction PDA chromatogram and mass spectrum total ion current figure.
Fig. 5 is Poria cocos standard decoction and mixed reference substance solution UPLC characteristic spectrum.
Fig. 6 is low energy and high-energy mass spectrogram under 2 pachymic acid B negative ion mode of peak.
Fig. 7 is low energy and high-energy mass spectrogram under 2 pachymic acid B positive ion mode of peak.
Fig. 8 is the chemical structural formula figure of pachymic acid B.
Fig. 9 is low energy and high-energy mass spectrogram under 3 pachymic acid A negative ion mode of peak.
Figure 10 is low energy and high-energy mass spectrogram under 3 pachymic acid A negative ion mode of peak.
Figure 11 is the chemical structural formula figure of pachymic acid A.
Figure 12 is low energy and high-energy mass spectrogram under 4 umbellate pore furgus acid C negative ion mode of peak.
Figure 13 is low energy and high-energy mass spectrogram under 4 umbellate pore furgus acid C negative ion mode of peak.
Figure 14 is the chemical structural formula figure of umbellate pore furgus acid C.
Figure 15 is that Poria cocos standard decoction measures UPLC characteristic spectrum.
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 Poria cocos standard decoction characteristic spectrum
Key instrument, reagent, reference substance information, Poria cocos standard decoction source are shown in Table 1, table 2, table 3, table 4 respectively.
1 key instrument of table
2 main agents of table
3 reference substance information of table
4 25 batches of Poria cocos standard decoction place of production information tables of table
Test method
The preparation of reference solution
It is appropriate that precision weighs pachymic acid B reference substance, add methanol be made every 20 μ g solution of 1ml B containing pachymic acid to get.
The preparation of Poria cocos standard decoction
Poria cocos medicine materical crude slice 200g is taken, is set in electroceramics pot, is added water to cook twice, decocts be added in 9 times of amount water for the first time, impregnates 50 points
Clock, 500W boil rear 300W by intense fire and are cooked by slow fire 30 minutes, and decocting liquid filters while hot through 400 mesh screens, and filtrate uses rapidly cold water cold
But, second plus 7 times of amount water, intense fire heating are cooked by slow fire 25 minutes after boiling, and decocting liquid is filtered while hot with 400 mesh screens, filtrate
It is cooling with cold water rapidly, merge decocting liquid twice.Decocting liquid is transferred in round-bottomed flask, in temperature: 62 DEG C；Vacuum degree :-
The medicinal extract of 130g is concentrated into when 0.10MPa using Rotary Evaporators decompression low temperature；Under magnetic stirring, packing is western to 10ml brown
In woods bottle, every bottle of packing volume is 2ml, is transferred in vacuum freeze drier and is lyophilized after having dispensed, and takes out, rolls aluminium lid, i.e.,
.Poria cocos vacuum freeze drier eutectic point test result is -32.5 DEG C, and pre-freezing temperature is -45oC, lyophilization temperature -35o
C ~ 0oC, parsing-desiccation temperature are 10oC ~ 20oC, and drying process total time is 47 hours.
Chromatographic condition
Using octadecylsilane chemically bonded silica as filler, using acetonitrile as mobile phase A, using 0.1% formic acid as Mobile phase B, by table 5
Regulation carries out gradient elution；Flow velocity is 0.2ml per minute；Column temperature is 30 DEG C；Detection wavelength is 252nm.
5 gradient elution table of table
The preparation of Poria cocos standard decoction test solution
Take Poria cocos standard decoction appropriate, it is finely ground, the Poria cocos standard decoction powder equal to Poria cocos medicinal material amount 30g is taken, it is accurately weighed, it sets
In stuffed conical flask, 50% methanol 10ml is added in precision, and weighed weight is divided with the ultrasonic treatment 30 of power 250W, frequency 40kHz
Clock is let cool, then weighed weight, and the weight of less loss is supplied with 50% methanol, is shaken up, and centrifugation, supernatant crosses 0.22 μm of miillpore filter,
Take subsequent filtrate to get.
Control medicinal material solution preparation
Poria cocos control medicinal material 3g is taken, it is accurately weighed, it sets in stuffed conical flask, 50% methanol 25ml is added in precision, and weighed weight is set
It is shaken 2 hours in constant temperature oscillator, is ultrasonically treated 60 minutes, is let cool, then weighed weight with power 250W, frequency 40kHz, used
50% methanol supplies the weight of less loss, shakes up, and centrifugation, supernatant crosses 0.22 μm of miillpore filter, take subsequent filtrate to get.
Poria cocos standard decoction characteristic spectrum shares the determination at peak
(1) 25 batches of Poria cocos standard decoctions are taken, by test solution in the case where providing chromatographic condition sample introduction, obtain 25 batch Poria cocos standards
Decoction UPLC characteristic spectrum (see figure 1), and shared peak mark is carried out, establish Poria cocos standard decoction UPLC compare feature map (figure
2).
(3) experimental result: selection separating degree is preferable, and the purer shared peak of chromatographic peak is Poria cocos standard decoction characteristic spectrum
Characteristic peak, the 25 batches of Poria cocos standard decoctions have 4 characteristic peaks (see Fig. 1), and 4 characteristic peaks can from Poria cocos control medicinal material (see
It Fig. 3) is found in characteristic spectrum, illustrates that 4 characteristic peaks can be transferred in Poria cocos standard decoction from Poria cocos medicinal material stabilization；Therefore it selects
Characteristic peak of the above-mentioned 4 shared peaks as Poria cocos standard decoction UPLC characteristic spectrum is selected, and with peak 2(pachymic acid B) it is referring to peak
Research on standard is carried out, and attempts to point out its characteristic peak progress chemical component using UPLC/MS Q-TOF technology, it is determined that its
In peak 2 be pachymic acid B, peak 3 is pachymic acid A, peak 4 is umbellate pore furgus acid C.
Poria cocos standard decoction test solution study on the stability
Poria cocos standard decoction is taken to prepare test solution, it is small 0,2,4,8,12,16,20,24 respectively in the case where providing chromatographic condition
When sample introduction, 1 μ l of sampling volume.It is that the relative retention time of each characteristic peak and opposite is calculated referring to peak S with pachymic acid B chromatographic peak
Peak area is shown in Table 6 and table 7, the experimental results showed that, the relative retention time of each characteristic peak and relative peak area RSD in 24 hours
Value is respectively less than 2.0%, and the test solution is moderately good in 24 hours.
6 Poria cocos standard decoction characteristic spectrum study on the stability result table (relative retention time) of table
7 Poria cocos standard decoction characteristic spectrum study on the stability result table of table (opposite to retain peak area)
The research of Poria cocos standard decoction characteristic spectrum
For the quality of strict control Poria cocos granule, more comprehensive quality control is provided for the production technology of Poria cocos granule
Parameter establishes Poria cocos standard decoction characteristic spectrum, and sets to the characteristic peak relative peak area of Poria cocos standard decoction characteristic spectrum
Threshold quantity standard.Adequate representation of this research based on 25 batch Poria cocos standard decoctions takes its maximum, minimum value for according to phase
Limitation regulation is carried out to peak area；Selection peak area is of moderate size, and the preferable peak 2 of separating degree is calculated and provided as referring to peak
The relative peak area ranges of other each characteristic peaks, therefore the relative peak area ranges for each characteristic peak established are with quantitative.Institute
The peak area upper and lower limits of foundation have breediness, are avoided that the erroneous judgement of mass production part qualification batch, and can be to big raw
Production. art provides important quality control parameter, especially controls the quality of unstable characteristic peak ingredient, to improve product
Quality and quality control level.
In research, it is determined that 4 shared peaks of Poria cocos standard decoction, and attempt using PLC/MS Q-TOF technology to its spy
Sign peak carries out pointing out for chemical component, as a result 4 characteristic peak initial guess are as follows: peak 1 is 3- ketone group -6,16 alpha-dihydroxy-wool
Steroid -7, -9 (11), -24 (31)-triolefin -21- acid；Peak 2 is pachymic acid B；Peak 3 is 16 Alpha-hydroxy -3, -4 open loops-wool steroid -4
(28), -7,9 (11), 4 (31)-tetraene -3,21- diacid (i.e. pachymic acid A)；Peak 4 is umbellate pore furgus acid C.Through being compared really with reference substance
Recognize, it is determined that peak 2 therein, peak 3, peak 4 qualification result.
The measuring method of characteristic spectrum is established by high performance liquid chromatography and carries out methodological study, to 25 batches of Poria cocos marks
Quasi- decoction characteristic spectrum measurement, has finally determined Poria cocos standard decoction characteristic spectrum standard: 4 spies should be presented in sample chromatogram
Peak is levied, with peak 2 for the peak S, the relative retention time and relative peak area for calculating each characteristic peak and the peak S are, it is specified that each characteristic peak model
It encloses, as a result as follows:
8 25 batches of Poria cocos standard decoction characteristic spectrum relative retention times of table
9 25 batches of Poria cocos standard decoction characteristic spectrum relative peak areas of table
Each characteristic peak relative retention time of 10 Poria cocos standard decoction of table and relative peak area ranges
Poria cocos standard decoction characteristic spectrum characteristic peak is pointed out
Experiment condition
Instrument: Waters ACQUITY UPLCTMI-Class liquid phase systems, Xevo G2-XS Q-TOF mass spectrometer system (water
Generation Science and Technology Ltd.)；Waters Hss T3 C18(2.1mm × 100mm, 1.8 μm) chromatographic column.
Reagent: methanol (Guangzhou chemical reagent factory) analysis is pure；Liquid phase formic acid (Merck S. A.), acetonitrile
(match Mo Feishier limited liability company), methanol (match Mo Feishier limited liability company) are HPLC chromatogram grade, and water is laboratory
Ultrapure water system (Merck S. A., Milli-Q Direct).
Mass Spectrometry Conditions: acquisition mode: double spray ESI ion sources acquire MSE data under ESI (+/-)；Acquisition quality model
It encloses: 50-1200Da；Correcting fluid: sodium formate, leucine enkephalin；Spray voltage: 2.0/2.0kv；Orifice potential: 40V；Source temperature:
120 degree；Desolvation temperature: 450 DEG C；Dissolve gas flow: 800L/hr taper hole gas flow: 50L/hr.
Chromatographic condition: chromatographic column: Waters Hss T3 C18(2.1mm × 100mm, 1.8 μm)
Flow velocity: 0.3 ml/min column temperature: 40 DEG C of Detection wavelengths: 252nm, sample volume: 1 μ l, mobile phase: using acetonitrile as mobile phase
A, using 0.1% formic acid as Mobile phase B, the regulation according to the form below 11 carries out gradient elution:
11 characteristic peak of table points out eluent gradient
The preparation of reference substance solution
Take pachymic acid A, pachymic acid B, umbellate pore furgus acid C reference substance appropriate, it is accurately weighed, add methanol that every 1ml A containing pachymic acid, Fu is made
The mixing contrast solution of each 50 μ g of Siberian cocklebur acid B, umbellate pore furgus acid C.
The preparation of test solution
Take Poria cocos standard decoction appropriate, it is finely ground, the Poria cocos standard decoction powder equal to Poria cocos medicinal material amount 30g is taken, tool plug taper is set
In bottle, 50% methanol 10ml is added in precision, and weighed weight was let cool with power 250W, ultrasonic treatment 30 minutes of frequency 40kHz,
Weighed weight again is supplied the weight of less loss with 50% methanol, is shaken up, and centrifugation, supernatant crosses 0.22 μm of miillpore filter, takes subsequent filtrate,
To obtain the final product.
Sample introduction is analyzed
It is accurate respectively to draw above-mentioned reference substance solution and each 1 μ l of test solution, LC-MS system is injected, it is soft using UNIFI
Part analyzes collected data, obtains accurate mass number from high-resolution system, and Elemental Composition analysis is carried out to it simultaneously
The chemical formula of compound is obtained, is retained in conjunction with the search of debris analysis and online database, and by the chromatographic peak of compound
Row is compared with reference substance.
As a result
It can be with reference substance pachymic acid B, pachymic acid A, umbellate pore furgus acid by the retention time peak 2, peak 3, peak 4 of reference substance mixing reference substance
The retention time of C corresponds (see figure 5).
Peak 2: under negative ion mode, m/z 483.3115 is [M-H]-peak；Under positive ion mode, there are m/z
485.3262, it is [M+H]+peak, base peak m/z 467.3159 is [M+H-H2O]+peak (see Fig. 6, Fig. 7).Through Elemental Composition
Analysis, neutral formula are as follows: C30H44O5, in conjunction with document (Zhu L, Xu J, Zhang S, et al. Qualitatively and
quantitatively comparing secondary metabolites in three medicinal parts
derived from Poria cocos (Schw.) Wolf using UHPLC-QTOF-MS/MS-based chemical
Profiling. [J] J Pharm Biomed Anal, 2017,150:278-286.).As a result meet following 3 points: 1. it is negative from
Son without significantly -98 neutral loss；2. there are apparent -74 neutral loss in anion；3. there are m/z325 for cation, comprehensive
Upper analysis is determined as pachymic acid B(Fig. 8)
Peak 3: under negative ion mode, m/z 497.3278 is [M-H]-peak；Under positive ion mode, there are m/z 499, are [M+H]
+ peak, base peak m/z 481.3325 are [M+H-H2O]+peak (see Fig. 9, Figure 10).It is analysed through Elemental Composition, neutral formula
Are as follows: C31H46O5, in conjunction with document (the isolating and purifying of triterpenes chemical component, Structural Identification and Poria cocos medicinal material in Li Ke fuling peel
Research [D] Hubei University of Chinese Medicine of finger-print, 2013.).As a result meet following 2 points: 1. anions have it is apparent-
74, explanation is C3, C4 open ring type triterpene；2. cation is to sum up analyzed there are m/z 325, it is determined as pachymic acid A(Figure 11).
Peak 4: under negative ion mode, m/z 481.3321 is [M-H]-peak；Under positive ion mode, there are m/z
483.3467, it is [M+H]+peak, base peak m/z 465.3363 is [M+H-H2O]+peak (see Figure 12, Figure 13).Through Elemental Composition
Analysis, neutral formula are as follows: C31H46O4, in conjunction with document (the isolating and purifying of triterpenes chemical component, structure in Li Ke fuling peel
Research [D] Hubei University of Chinese Medicine of identification and Poria cocos medicinal materials fingerprint, 2013.).As a result meet following 2 points: 1. it is negative
Ion is without apparent -74；2. there are m/z 309 for cation.It to sum up analyzes, is determined as -16 Alpha-hydroxies of 3- ketone group-wool steroid -7,
9 (11), 24 (31)-triolefin -21- acid, i.e. umbellate pore furgus acid C(Figure 14).
Embodiment 2
Poria cocos standard decoction detection method, detecting step are as follows:
(1) precision weighs Poria cocos standard decoction to be detected, and Poria cocos standard decoction sample solution to be detected is prepared；
(2) accurate to draw Poria cocos standard decoction sample solution to be detected, Ultra Performance Liquid Chromatography instrument is injected, is measured to get to be checked
Survey Poria cocos standard decoction UPLC characteristic spectrum；
(3) the UPLC characteristic spectrum measured is compared with the Poria cocos standard decoction UPLC characteristic spectrum constructed, and if Fu
Siberian cocklebur standard decoction UPLC characteristic spectrum is consistent, then up-to-standard.
Chromatographic condition
Using octadecylsilane chemically bonded silica as filler (column length 150mm, internal diameter 2.1mm, partial size are 1.6 μm)；With second
Nitrile is mobile phase A, using 0.1% formic acid as Mobile phase B, provides to carry out gradient elution by table 12；Flow velocity is 0.2ml per minute；Column temperature
It is 30 DEG C；Detection wavelength is 252nm.
12 gradient elution table of table
The preparation of reference solution
It is appropriate that precision weighs pachymic acid B reference substance, add methanol be made every 20 μ g solution of 1ml B containing pachymic acid to get.
The preparation of control medicinal material solution
Poria cocos control medicinal material 3g is taken, it is accurately weighed, it sets in stuffed conical flask, 50% methanol 25ml is added in precision, and weighed weight is set
It is shaken 2 hours in constant temperature oscillator, is ultrasonically treated 60 minutes, is let cool, then weighed weight with power 250W, frequency 40kHz, used
50% methanol supplies the weight of less loss, shakes up, and centrifugation, supernatant crosses 0.22 μm of miillpore filter, take subsequent filtrate to get.
The preparation of test solution
It takes Poria cocos standard decoction finely ground in right amount, takes the Poria cocos standard decoction powder equal to Poria cocos medicinal material amount 30g, it is accurately weighed, it sets
In stuffed conical flask, 50% methanol 10ml is added in precision, and weighed weight is ultrasonically treated 30 minutes with power 250W, frequency 40kHz,
It lets cool, then weighed weight, the weight of less loss is supplied with 50% methanol, is shaken up, be centrifuged, supernatant is crossed 0.22 μm of miillpore filter, taken continuous
Filtrate to get.
Measurement: precision draws 10 μ l of control medicinal material solution, reference solution and each 2 μ l of test solution, injects liquid phase color
Spectrometer, measurement to get.
Measurement result
Each characteristic peak relative retention time of 13 Poria cocos standard decoction of table and relative peak area prescribed limit
The Poria cocos standard decoction characteristic spectrum of measurement and Poria cocos control medicinal material characteristic spectrum (Figure 15) are compared: can detect with
Identical 4 characteristic peaks of Poria cocos control medicinal material；Data result shows (table 13), except reference peak (peak 2) of the sample remaining 3
A peak relative retention time and relative peak area ranges in the range of standard provides, thus illustrate, the Poria cocos standard decoction
Characteristic spectrum method stablize, favorable reproducibility can accurately reflect Poria cocos standard decoction quality requirement.
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 Poria cocos standard decoction UPLC characteristic spectrum construction method, which is characterized in that comprise the following steps:
(1) precision weighs Poria cocos standard decoction, and Poria cocos standard decoction test solution is prepared；
(2) Poria cocos standard decoction test solution is analyzed using Ultra Performance Liquid Chromatography instrument, obtains Poria cocos standard decoction UPLC
Characteristic spectrum.
2. Poria cocos standard decoction UPLC characteristic spectrum construction method according to claim 1, which is characterized in that the superelevation
The chromatographic condition of effect liquid phase chromatogram instrument analysis are as follows: using octadecylsilane chemically bonded silica as filler, using acetonitrile as mobile phase A,
Using 0.08 ~ 0.12% formic acid as Mobile phase B, gradient elution is carried out, flow velocity is 0.17 ~ 0.22ml/min, and column temperature is 28 ~ 32 DEG C, into
Sample amount is 0.08 ~ 0.12 μ l, and Detection wavelength is 220 ~ 280nm.
3. Poria cocos standard decoction UPLC characteristic spectrum construction method according to claim 1, which is characterized in that the Poria cocos
Standard decoction adds water to cook twice the preparation method comprises the following steps: take Poria cocos 180 ~ 220g of medicine materical crude slice, decocts 8 ~ 10 times of amount water are added for the first time
In, it impregnates 40 ~ 60 minutes, is cooked by slow fire after boiling by intense fire 26 ~ 32 minutes, decocting liquid filters while hot, and filtrate is cooling with cold water rapidly；
Second plus 6.4 ~ 7.6 times of amount water, intense fire heating are cooked by slow fire 22 ~ 28 minutes after boiling, and for decocting liquid with filtering while hot, filtrate is rapid
It is cooling with cold water, merge decocting liquid twice；Decocting liquid is transferred to container, decompression low temperature is concentrated into the medicinal extract of 120 ~ 140g；Stirring, point
Be filled in 8 ~ 12ml bottles, every bottle of packing volume is 1.6 ~ 2.4ml, freeze-drying after dispense, taking-up to get.
4. Poria cocos standard decoction UPLC characteristic spectrum construction method according to claim 1, which is characterized in that gradient elution
Condition are as follows: the volume fraction variation of 0 ~ 21min, mobile phase A are 40 ~ 99%, and the volume fraction variation of Mobile phase B is 60 ~ 1%；21~
The volume fraction variation of 22min, mobile phase A are 99 ~ 40%, and the volume fraction variation of Mobile phase B is 1 ~ 60%.
5. Poria cocos standard decoction UPLC characteristic spectrum construction method according to claim 1, which is characterized in that the confession
The method of test sample solution as follows is prepared:
Take Poria cocos standard decoction appropriate, it is finely ground, the Poria cocos standard decoction powder equal to 25 ~ 35g Poria cocos medicinal material amount is taken, precision claims
Fixed, 45 ~ 55% 9 ~ 11ml of methanol are added in precision, and weighed weight is ultrasonically treated 28 ~ 32 minutes, lets cool, then weighed weight, with 45 ~
55% methanol supplies the weight of less loss, shakes up, and centrifugation, supernatant crosses 0.18 ~ 0.26 μm of miillpore filter, take subsequent filtrate to get.
6. a kind of detection method of Poria cocos standard decoction, which comprises the steps of:
(1) precision weighs Poria cocos standard decoction to be detected, and Poria cocos standard decoction sample solution to be detected is prepared；
(2) accurate to draw Poria cocos standard decoction sample solution to be detected, Ultra Performance Liquid Chromatography instrument is injected, is measured to get to be checked
Survey Poria cocos standard decoction UPLC characteristic spectrum；
(3) the Poria cocos standard decoction UPLC constructed any one of the UPLC characteristic spectrum measured and claim 1 ~ 5 method is special
Sign map is compared, up-to-standard if consistent with Poria cocos standard decoction UPLC characteristic spectrum.
7. the detection method of Poria cocos standard decoction according to claim 6, which is characterized in that
The chromatographic condition of the Ultra Performance Liquid Chromatography instrument analysis are as follows: using octadecylsilane chemically bonded silica as filler, with second
Nitrile is mobile phase A, using 0.08 ~ 0.12% formic acid as Mobile phase B, carries out gradient elution, flow velocity is 0.17 ~ 0.22ml/min, column temperature
It is 28 ~ 32 DEG C, sample volume is 0.8 ~ 0.12 μ l, and Detection wavelength is 220 ~ 280nm.
8. the detection method of Poria cocos standard decoction according to claim 6, which is characterized in that the Poria cocos standard decoction system
Preparation Method are as follows: take Poria cocos 180 ~ 220g of medicine materical crude slice, add water to cook twice, decoct be added in 8 ~ 10 times of amount water for the first time, impregnate 40 ~ 60
Minute, it is cooked by slow fire after boiling by intense fire 26 ~ 32 minutes, decocting liquid filters while hot, and filtrate is cooling with cold water rapidly；Second plus 6.4 ~
7.6 times of amount water, intense fire heating are cooked by slow fire 22 ~ 28 minutes after boiling, and for decocting liquid with filtering while hot, filtrate is cooling with cold water rapidly,
Merge decocting liquid twice；Decocting liquid is transferred to container, decompression low temperature is concentrated into the medicinal extract of 120 ~ 140g；Stirring, packing to 8 ~ 12ml
In bottle, every bottle of packing volume is 1.6 ~ 2.4ml, is lyophilized after dispense, taking-up to get.
9. the detection method of Poria cocos standard decoction according to claim 6, which is characterized in that condition of gradient elution are as follows: 0 ~
The volume fraction variation of 21min, mobile phase A are 40 ~ 99%, and the volume fraction variation of Mobile phase B is 60 ~ 1%；21 ~ 22min, stream
The volume fraction variation of dynamic phase A is 99 ~ 40%, and the volume fraction variation of Mobile phase B is 1 ~ 60%.
10. the detection method of Poria cocos standard decoction according to claim 6, which is characterized in that the test solution
Method as follows is prepared: take Poria cocos standard decoction appropriate, it is finely ground, and take the Fu equal to 25 ~ 35g Poria cocos medicinal material amount
Siberian cocklebur standard decoction powder, it is accurately weighed, it is ultrasonically treated 28 ~ 32 minutes, lets cool, then weighed weight, supplied and subtracted with 45 ~ 55% methanol
The weight of mistake, shakes up, and centrifugation, supernatant crosses 0.18 ~ 0.26 μm of miillpore filter, take subsequent filtrate to get.
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