CN106018577A - Three-Huang preparation component detecting method and fingerprint spectrum establishing method - Google Patents
Three-Huang preparation component detecting method and fingerprint spectrum establishing method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention belongs to the field of medicine analysis, and relates to a determination method of a traditional Chinese medicine fingerprint spectrum, and discloses a three-Huang preparation component detecting method. A test article solution is injected into a UPLC-Q-TOF-MS, determination is conducted by means of gradient eluting through a UPLC-Q-TOF-MS method, and components of a three-Huang preparation are obtained according to the detection result of the UPLC-Q-TOF-MS. Through the three-Huang preparation component detecting method, different batches of three-Huang preparation test article solutions are taken to be detected according to the method, common peaks are calibrated according to the detection result, and the UPLC-Q-TOF-MS fingerprint spectrum of the three-Huang preparation is established. The method and the fingerprint spectrum can be used as one of effective methods for mass control and analysis of the three-Huang preparation.
Description
Technical field
The invention belongs to pharmaceutical analysis field, relate to the assay method that Chinese medicine refers to that Fructus Citri tangerinae green trichoderma bacterium is composed, particularly relate to one
SANHUANG square preparation component detection method and Ultra Performance Liquid Chromatography-high-resolution-flight time mass spectrum combination fingerprint map construction side
Method.
Background technology
Chinese medicine fingerprint is certain class that can reflect Chinese crude drug or Chinese patent medicine or a few class using modern analytical technique to set up
The chromatograph of composition or the collection of illustrative plates of spectral information, can more fully reflect the total chemical characteristics of Chinese medicine, embody in it entirety
Quality, can make up the drawback only controlling Chinese medicine quality with minority composition, it has also become internationally recognized control Chinese medicine or crude drug
The effective means of material amount.But, the multiformity of chemical composition of Chinese materia medica and complexity and the uncertainty of effective substance,
Add the difficulty of Chinese medicine fingerprint foundation and the effective and repeated of finger printing.
At present, what Chinese medicine fingerprint research was more is Chemistry for Chinese Traditional Medicine finger printing, is to use chromatograph, wave spectrum and other points
The finger printing for characterizing chemical composition of Chinese materia medica feature that analysis method is set up.High performance liquid chromatography (HPLC) has separation efficiency
Height, analysis speed are fast, the feature of favorable reproducibility, and different detector can be coordinated to analyze the advantage of all kinds sample,
Become the generally acknowledged predominant methods setting up Chinese medicine fingerprint.The Ultra Performance Liquid Chromatography technology (UPLC) of rising in recent years has
The features such as supertension, hypersensitivity, superelevation separating degree, it, by the theory of HPLC and principle, uses the chromatograph of 1.7 μm particle diameters
Column packing, can make analysis time of a finger printing by within original at least 1 hour, shortening to as about 10min, and chromatograph
Peak capacity and stability also significantly improve, and UPLC has the most excellent in the separation analysis of the complex system such as Chinese medicine and natural product
Gesture, provides new separation platform for Chinese medicine fingerprint research.
Ultraviolet (UV) detector is to carry out finger printing study most widely used detection side with liquid chromatograph combination at present
Method.But the ultraviolet absorptivity of chemical composition has additivity, so that measurement result produces deviation, it is impossible to true reflection Chinese medicine
The content studied point.And uv detection method is only suitable for the chemical composition of Chinese materia medica with uv absorption, for without uv absorption or
Absorb more weak composition, it is difficult to effectively measure.Significant important chemical composition in the monarch drug Radix Astragali in this compound preparation special
Significant important chemical composition former note alkane triterpenes components in triterpene saponin (such as astragaloside IV etc.) and adjuvant drug Rhizoma Alismatis (as
Alisol A etc.) absorb the most weak in ultra-violet (UV) band and disturb more, be not suitable for measuring by UV-detector.
Mass spectrum (MS) detector can be used for the detection of various compounds in Chinese medicine, all can produce preferably response, is a kind of
Highly sensitive all-purpose detector, it is often more important that be provided that molecular weight and the molecular structure information (tandem mass spectrum) of compound.
Use mass detector, be determined by the mass scan range of ion, it can be made to comprise the information of all target compounds.Should
Pattern is detected with total ion current, can be with the full detail of synthesization compound；And then, believed by the mass spectrum of the total chromatographic peak of examination
Breath, it may be determined that the composition situation of chromatographic peak, it is ensured that chromatographic peak is single compound composition, has preferable science.Due to
The complexity of chemical composition of Chinese materia medica, it is ensured that the chromatographic peak in Chinese medicine fingerprint be single compound peak be very difficult the most several
Impossible, and use mass detector, the ion of specific m/z can be extracted from total ions chromatogram (TIC), foundation carries
Take chromatography of ions figure (EIC), thus ensure that chromatographic peak is single compound composition.It addition, by total peak is carried out mass spectrum
Qualitative, set up the extraction chromatography of ions figure at total peak, and then by the one-to-one relationship of chromatographic peak with compound m/z, it is achieved
Each total peak is accurately positioned and measures, thus the shortcoming that the poor reproducibility between other method, laboratory can be made up.
Flight time tandem mass spectrometer (Q-TOF-MS) is high-resolution tandem mass spectrum, and its distinguishing feature is high sensitivity, height
Selectivity, can obtain high-quality mass spectrum and compound accurate molecular weight (Mass accuracy < 3 × 106), compound can be greatly improved
The accuracy of Structural Identification.UPLC-Q-TOF-MS combines the most excellent chromatogram separating capacity and the sensitiveest, the most intelligent
Power of test, great development prospect and space in Chemistry for Chinese Traditional Medicine finger printing, advantage highlights, and feature is obvious, will become
The development trend of Chinese medicine fingerprint and direction.
Three yellow party are Huashan Hospital Affiliated To Fudan Univ cipher prescription, by the Radix Astragali, Rhizoma Coptidis, Rhizoma Alismatis, Pollen Typhae, Herba Artemisiae Scopariae compatibility and
Becoming, clinical practice for many years, in treatment metabolic syndrome and non-alcoholic fatty liver disease, dissipates for department of Chinese medicine cipher prescription QI invigorating in early days
Side's of simplifying (number of patent application: 201511018056.0) of poly-side's (patent No.: 200810034507.3), has QI invigorating and dissipates poly-merit
Effect, it is possible to improve patient sign and internal insulin-resistant states, the precise and tiny normal transporting of QI and blood can be promoted, correct organism metabolism different
Often, i.e. there is QI invigorating eliminating stagnation, removing heat-phlegm, damp eliminating the turbid descending, the effect of Tiaozhi Huayu.Owing to effective ingredient important in the Radix Astragali is as yellow
The purple of the important effective ingredient such as protostane such as alisol and esters thereof triterpenes in the triterpene saponins such as stilbene saponin I V and Rhizoma Alismatis
Outer absorption is poor, and general UV-detector detection sensitivity is poor；Evaporative light scattering detector sensitivity is the most poor, limits
The finger printing research of the compound preparation containing both Chinese medicine.We report employing 2 compositions so far, 4 compositions contain
Quantity measuring method controls the content of SANHUANG square preparation, but still without its multicomponent assay and utilize UPLC-Q-TOF-
MS carries out the report of quality control.The present invention surveys based on needed for " Chinese Pharmacopoeia " 2015 editions Quality Identification to 5 kinds of composition Chinese medicines
(Radix Astragali is astragaloside IV to 11 kinds of chemical compositions of fixed kind, calycosin glucoside；Rhizoma Coptidis be epiberberine, coptisine,
Palmatine, berberine；Rhizoma Alismatis is Alisol B monoacetate；Pollen Tyjphae is Isorhamnetin-3-O-neohespeidoside and typhaneoside；Mattress
Old for chlorogenic acid and escoparone) and State Food and Drug Administration's standard substance catalogue, and with reference to SANHUANG square preparation blood
Clear chemical research (18 kinds of compositions can enter blood), the 23 kinds of determining fingerprint pattern indexs selected are except containing above chemical composition
And be respectively directed in pharmacopeia the detection less shortcoming of target component needed for 5 kinds of special Pollen Tyjphae of Chinese medicine, Rhizoma Alismatis has been increased newly 12 kinds
Chemical composition, and through the mensuration of 10 batches of three yellow party solution finger printing, primarily determine that total chemical composition, as SANHUANG square preparation
The reference method of quality control.
This technology is popularized the most in a large number based on high-end liquid phase determination techniques, equipment needed thereby, but for the most comprehensive
Property university and large-scale Chinese medicine to produce medicine enterprise be all indispensable outfit.SANHUANG square preparation has been basically completed preclinical study the most, will just
Walk as developing preparation, the not yet large-scale production of its preparation, current finger printing master in Huashan Hospital Affiliated To Fudan Univ institute
Will be as with reference to collection of illustrative plates, but this assay method can be as the method for quality control of SANHUANG square preparation.
Summary of the invention
It is an object of the invention to overcome the shortcoming and defect of prior art, it is provided that one utilizes UPLC-Q-TOF-MS letter
Single method quickly carrying out SANHUANG square preparation quality control, and thus SANHUANG square preparation finger printing constructed by method.
First purpose of the present invention is to provide a kind of SANHUANG square preparation component detection method, comprises the following steps:
Step 1: the preparation of need testing solution
Take the Radix Astragali, Rhizoma Coptidis, Rhizoma Alismatis, Pollen Tyjphae and Herba Artemisiae Scopariae, with water and ethanol extraction, constant volume, obtain need testing solution；
Step 2: the mensuration of need testing solution
Draw need testing solution and inject UPLC-Q-TOF-MS combined instrument, use gradient elution to enter with UPLC-Q-TOF-MS method
Row measures, according to UPLC-Q-TOF-MS combined instrument testing result, it is thus achieved that the composition of SANHUANG square preparation.
Preferably, above-mentioned step 1 specifically includes following steps:
Step a: after the Radix Astragali, Rhizoma Coptidis, Rhizoma Alismatis, Pollen Tyjphae and Herba Artemisiae Scopariae being soaked by purified water, adds purified water with boiling, so
Rear leaching medicinal liquid concentration obtain concentrated medicament；
Step b: after addition ethanol stirs in the concentrated medicament that step a obtains, room temperature precipitates 24 hours, then takes
Supernatant reclaims ethanol and carries out concentration, again takes supernatant and reclaims ethanol and carry out concentration, obtains concentrated solution；
Step c: after addition water stirs in the concentrated solution that step b obtains, precipitate 24 hours, then supernatant is entered
Row concentration, constant volume obtains need testing solution.
Preferably, by weight calculating in above-mentioned step 1, each constituent content is respectively as follows: Radix Astragali 1-4 part；Rhizoma Coptidis 1 part；
Rhizoma Alismatis 1-3 part；Pollen Tyjphae 1-3 part；Herba Artemisiae Scopariae 1-3 part.
Preferably, in above-mentioned steps 2, UPLC chromatographic condition is: flowing is the formic acid second of the formic acid water-0.1% of 0.1% mutually
Nitrile；Flow velocity 0.3-0.5mL/min；Column temperature 35-50 DEG C；Sample size 1-5 μ L.
Preferably, in above-mentioned steps 2, UPLC chromatographic determination mode is negative ions full scan, electron spray (ESI) ion source
Detection pattern；Quality of scanning scope is 100-1000Da.
Preferably, in above-mentioned steps 2, the chromatographic column of UPLC chromatograph is ACQUITYUPLC BEH C18 chromatographic column.
Preferably, the chromatographic condition of above-mentioned ACQUITY UPLC BEH C18 chromatographic column is: flow velocity 0.4mL/min, post
Temperature 45-50 DEG C, sample size 1-5 μ L.
Second object of the present invention provides a kind of SANHUANG square preparation fingerprint map construction method, takes the SANHUANG of different batches
Square preparation need testing solution is measured by above-mentioned SANHUANG square preparation component detection method, according to testing result, demarcates total
Peak, sets up SANHUANG square preparation finger printing.
By 10 batches of SANHUANG square preparation need testing solution comparisons, it is determined that 23 kinds of total compositions, and mark total peak.This
Sample does not contains under bright testing conditions in addition to purpose composition other interference component, the detection method that the result display present invention provides
Precision is high, more stable, reproducible, good tolerance, the collection of illustrative plates of the present invention can be used for the quality control of SANHUANG square preparation with
Analyze.
The present invention utilizes finger printing to characterize the chemical composition of SANHUANG square preparation on the whole, and by serum drug chemistry
Method proves that the method can be used for measuring 18 total peaks and is the composition being absorbed into blood, it may be possible to SANHUANG square preparation plays medicine
The potential effective ingredient of effect.
Compared with prior art, the invention have the advantages that
(1) present invention uses UPLC-Q-TOF-MS as analysis means first, utilizes 23 kinds of easy purchases
Standard substance (including amounting to 11 kinds of chemical compositions in 5 kinds of composition Chinese crude drugs that " Chinese Pharmacopoeia " 2015 editions regulation needs measure) are set up
SANHUANG square preparation chemical fingerprint method of quality control, analyze method with the HPLC-UV of existing report compared with, the most significantly
Shorten analysis time, improve separation efficiency and detection sensitivity；And give the information of molecular weight, by building can to each peak
Vertical chromatography of ions figure, can accurately calculate the peak area at each peak, and if when laboratory monitoring reappears retention time deviate, can lead to
The molecular weight information crossing each peak is accurately positioned, thus ensure that the reproducibility of fingerprint atlas detection method, can be used for three
Yellow party is about the quality control of preparation and analysis.
(2) 18 can be measured in three yellow party simultaneously and enter blood component, for studying the serum medicine of SANHUANG square preparation further
Neo-Confucianism, pharmacokinetics, developing new drug have important effect.
(3) this programme can also be used for the chemical substance fundamental analysis of 5 kinds of composition Chinese crude drugs.
(4) not containing other interference component under testing conditions of the present invention in sample in addition to purpose composition, result shows this
The detection method precision of bright offer is high, more stable, reproducible, and good tolerance.
Accompanying drawing explanation
Fig. 1 is that the UPLC-Q-TOF-MS cation of SANHUANG square preparation and 5 kinds of composition Chinese medicines extracts chromatography of ions figure (HQ=
The Radix Astragali, HL=Rhizoma Coptidis, ZX=Rhizoma Alismatis, PH=Pollen Typhae, YC=Herba Artemisiae Scopariae, SHD=tri-yellow party extracting solution).
Fig. 2 is that the UPLC-Q-TOF-MS anion of SANHUANG square preparation and 5 kinds of composition Chinese medicines extracts chromatography of ions figure (HQ=
The Radix Astragali, HL=Rhizoma Coptidis, ZX=Rhizoma Alismatis, PH=Pollen Typhae, YC=Herba Artemisiae Scopariae, SHD=tri-yellow party extracting solution).
Fig. 3 is the UPLC-Q-TOF-MS finger printing (cation mode) of SANHUANG square preparation.
Fig. 4 is that the UPLC-Q-TOF-MS cation of SANHUANG square preparation rat Contained Serum extracts chromatography of ions figure.
Fig. 5 is that the UPLC-Q-TOF-MS anion of SANHUANG square preparation rat Contained Serum extracts chromatography of ions figure.
Detailed description of the invention
Below by specific embodiment, the present invention is carried out detailed and concrete introduction, so that being better understood from the present invention,
But following embodiment is not limiting as the scope of the invention.
Embodiment 1
The present embodiment is the assay method of SANHUANG square preparation UPLC-Q-TOF-MS finger printing, and method detailed is as follows:
1. instrument and reagent chemicals
1.1. instrument
Waters company AcquityTM Ultra Performance LC system (Waters, Milford, USA),
System is MassLynx (V 4.1) equipped with quaternary pump, vacuum degassing machine, automatic sampler, control software.
1.2. reagent chemicals
Acetonitrile and formic acid are chromatographically pure, and water is ultra-pure water, and other reagent is analytical pure；
Astragaloside IV, formononetin, calycosin, calycosin-7-O-β-D-glycoside, berberine, bar
Ma Ting, jateorhizine, Alisol B monoacetate, chlorogenic acid, scopoletin, 6,7-dimethoxy coumarin, typhaneoside, different Mus
Li Su-3-O-neohesperidin, isorhamnetin, naringenin, Quercetin, kaempferide is purchased from Chinese food Drug Administration；Table
Berberine, coptisine, Alisol A, alisol F, 24-alisol acetyl A, 6-hydroxyl-7-methoxyl group-coumarin is purchased from Shanghai China
One Bioisystech Co., Ltd；5 kinds of Chinese crude drugs are purchased from prepared slices of Chinese crude drugs company limited of Hongqiao in Shanghai.
2. the preparation of need testing solution
The 30g Radix Astragali of Chinese medicine processing specification, 9g Rhizoma Coptidis, 15g Rhizoma Alismatis, 15g Pollen Tyjphae, the weighing of 15g Herba Artemisiae Scopariae, ingredients will be met
Standby, wherein the Radix Astragali, Rhizoma Coptidis, Rhizoma Alismatis, Pollen Tyjphae, the weight ratio of Herba Artemisiae Scopariae are 1-4:1:1-3:1-3:1-3.Above medicine is first soaked
Bubble 0.5h, adds purified water and boils (wherein Pollen Tyjphae is decocted a drug wrapped) together, extract 2 times, add water 8-12 times every time, boil 2 times, each 1h, filter
Taking liquid, concentrates crude drug, adds 95% ethanol of 1 times amount, stir, and room temperature precipitates 24 hours, takes supernatant and reclaims ethanol
And concentrate；Add 5 times amount 95% ethanol, stand 24 hours, take supernatant and reclaim clean ethanol；Add 2 times of water (70-80
DEG C), stirring, 10-15 DEG C of environment precipitates 24 hours, Aspirate supernatant, and makes after mixing homogeneously with medicinal correctives, preservative
Oral administration solution, filters precipitate, concentrates as 20mL solution (i.e. 4.2g Chinese medicine/mL solution).
Drawing the 50 above-mentioned solution of μ L, add 950 μ L methanol-acetonitrile (1:1) solution, 4h placed by 4 DEG C of refrigerators, be centrifuged (4 DEG C,
20,267g, 15min), take supernatant, sample size is 2 μ L.
3. finger printing testing conditions
Chromatographic condition: analytical column is ACQUITY UPLCTM BEH C18column (100mm × 2.1mm i.d., 1.7 μ
M, waters), column temperature 45 DEG C, [A is the formic acid solution of 0.1% (v/v) to gradient elution, and B is the acetonitrile containing 0.1% (v/v) formic acid
Solution, graded situation is as follows: 0 0.5min, 5%B；0.5 4min, 5 20%B；4 6min, 20 25%B；6–8min,
25 50%B；8 12.5min, 50 85%B；12.5 13.5min, 85 100%B；13.5 15min, 100%B；15–
15.5min, 100 5%B；15.5 17min, 5%B].
Mass Spectrometry Conditions: electron spray (ESI) ion source (Waters Corporation, Milford, MA), full scan data
Being set to 100-1000Da, interval time is 0.02s, and scanning time second is 0.3s, and capillary voltage is 3000V (cation mould
Formula) and 2800v (ion mode), desolventizing temperature is 350 DEG C, and sample cone voltage is that 35v (cation mode) and 50v is (cloudy
Ion mode), collision energy is 4eV, and extracting cone voltage is 4.0V, and source temperature is 115 DEG C, and cone gas flow rate is 50L/h, precipitation
Agent gas velocity is 600L/h.0.2ng/mL leucine enkephalin solution (v=100 μ L/min) on-line correction.Experimental result such as Fig. 1
Shown in Fig. 2.Embodiment 2
The present embodiment is foundation and the evaluation of SANHUANG square preparation finger printing
1.1. the determination at total peak
Collect 10 batches of SANHUANG square preparations, operate by the method in embodiment 1, be analyzed by the testing conditions of embodiment 1.
From the point of view of testing result, having 23 compound peaks is that each batch of test sample has, therefore using these 23 compound peaks as three yellow party
The total compound peaks of preparation, obtains the extraction chromatography of ions figure of these 23 compound peaks, such as Fig. 3 from total ions chromatogram
Shown in.Wherein the peak area of palmatine is maximum, elects as with reference to peak.
Flight time mass spectrum is high resolution mass spectrum, it is possible to measures the accurate mass of ion, and then is less than according to measured deviation
The principle of 5PPM and isotope degree of fitting minimum calculates the molecular formula at each shared peak.
As shown in table 1, arranging by appearance time order, the karyoplasmic ratio at 23 total peaks is respectively as follows: compound 2 (peak 1), m/
z 354；Compound 11 (peak 2), m/z 192；Compound 13 (peak 3), m/z 192；Compound 14 (peak 4), m/z 770；Chemical combination
Thing 15 (peak 5), m/z 446；Compound 18 (peak 6), m/z 624；Compound 25 (peak 7), m/z 206；Compound 27 (peak 8),
m/z 320；Compound 30 (peak 9), m/z 336；Compound 31 (peak 10), m/z 338；Compound 33 (peak 11), m/z 302；
Compound 34 (peak 12), m/z 284；Compound 35 (peak 13), m/z 336；Compound 36 (peak 14), m/z 352；Compound 37
(peak 15), m/z 272；Compound 40 (peak 16), m/z 316；Compound 41 (peak 17), m/z 268；Compound 42 (peak 18),
m/z 784；Compound 44 (peak 19), m/z 300；Compound 51 (peak 20), m/z 488；Compound 52 (peak 21), m/z 490；
Compound 54 (peak 22), m/z 532；Compound 56 (peak 23), m/z 514.
The karyoplasmic ratio additionally having 33 chemical compositions is respectively as follows: compound 1, m/z 354；Compound 3, m/z 368；
Compound 4, m/z 594；Compound 5, m/z 516；Compound 6, m/z 684；Compound 7, m/z 342；
Compound 8, m/z 164；Compound 9, m/z 368；Compound 10, m/z 464；Compound 12, m/z 464；
Compound 16, m/z 740；Compound 17, m/z 594；Compound 19, m/z 624；Compound 20, m/z 286；
Compound 21, m/z 322；Compound 22, m/z 478；Compound 23, m/z 284；Compound 24, m/z 188；
Compound 26, m/z 316；Compound 28, m/z 354；Compound 29, m/z 338；Compound 32, m/z 430；
Compound 38, m/z 334；Compound 39, m/z 350；Compound 43, m/z 504；Compound 45, m/z 528；
Compound 46, m/z 826；Compound 47, m/z 486；Compound 48, m/z 826；Compound 49, m/z 488；
Compound 50, m/z 528；Compound 53, m/z 532；Compound 55, m/z 472.
It addition, the relative peak area at each total peak of SANHUANG square preparation finger printing is as shown in table 2, the most not
The relative peak area similarity having peak with the test sample of batch is the highest, and the coefficient of variation is the least, illustrates that this method is for mensuration three
Yellow party formulation content repeatability is high, and result is accurate, can be used for the mensuration of SANHUANG square preparation component content.
The chemical composition of the SANHUANG square preparation that table 1 UPLC-ESI-Q-TOF-MS is measured
a)Astragalus membranaceus(Fisch.)Bunge,b)Coptis chinensis Franch.,c)
Alismao rientale(Sam.)Juz.d)Typha angustifolia L.,e)
Artemisia capillaries Thunb.；
Glc:β-D-glucose；Rha:rhamnopyrano；Xyl:xylose
The relative peak area at each total peak of table 2 SANHUANG square preparation finger printing
Embodiment 3
The present embodiment is the determination that SANHUANG square preparation enters blood component, specifically comprises the following steps that
Take Wistar rat (200 ± 20g), fasting 12h, oral SANHUANG square preparation liquid, 1h sodium phenobarbital after administration
Physiological salt liquid intraperitoneal injection of anesthesia (concentration 100mg/mL, 2.0mL/kg body weight), takes a blood sample through ventral aorta, centrifugal
(3000rpm, 10min, 4 DEG C), takes serum 200 μ L, adds 800 μ L methanol-acetonitrile (1:1) solution, and 4h placed by 4 DEG C of refrigerators, from
The heart (4 DEG C, 20,267g, 15min), takes supernatant, and sample size is 6 μ L, by the chromatographic condition detection drafted, it is thus achieved that 18 enter blood
The extraction chromatography of ions figure of the mass-to-charge ratio divided, experimental result is as shown in figs. 4 and 5.
The present embodiment utilizes finger printing to characterize the chemical composition of SANHUANG square preparation on the whole, and by serum drug
Method proves that the method can be used for measuring 18 total peaks and is the composition being absorbed into blood, it may be possible to SANHUANG square preparation plays
The potential effective ingredient of drug effect.
Embodiment 4
The present embodiment mainly chooses 7 important chemical marker compositions and to its Simultaneous Quantitative Analysis for the present invention
Assay method further verified.
(1) range of linearity is investigated with detection limit, quantitative limit: linearity and range is studied by joining 7 selected chemical compositions
The working solution of 6 variable concentrations of system is investigated, tested 2 times of each sample.Detection limit is respectively signal to noise ratio (S/ with quantitative limit
N) be 3 and 10 standard sample concentration.The results are shown in Table 2.
(2) repeatability is investigated: same sample is by replicate analysis 6 times, for investigating the repeatability of method.The results are shown in Table 2.
(3) precision is investigated: withinday precision by 5 samples being measured 1 time at interval of certain time in one day, totally 5
Secondary mensuration；Day to day precision measured same concentration sample by continuous 5 days at synchronization and obtains.The results are shown in Table 2.
(4) accuracy is investigated: investigated by average recovery.I.e. in the sample of 3 parts of known content, it is separately added into difference
The standard substance of the same race of content (the 80% of known content, 100% and 120%), measure the content of target criteria product in gained sample,
And calculate the difference of actually detected amount and theoretical addition.Computing formula is: the response rate (%)=100 × (detection limit-theory adds
Enter amount)/theoretical addition amount；And represent the coefficient of variation with relative standard deviation (RSD).The results are shown in Table 3.
By 7 important chemical marker compositions as comparison contrast, demonstrate the assay method of the present invention further
Accuracy is high, and repeatability is good.
The quantitative analysis of 37 kinds of significant compounds of table
Being described in detail the specific embodiment of the present invention above, but it is intended only as example, the present invention does not limit
It is formed on particular embodiments described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and
Substitute the most all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Amendment, all should contain within the scope of the invention.
Claims (10)
1. a SANHUANG square preparation component detection method, it is characterised in that comprise the following steps:
Step 1: the preparation of need testing solution
Take the Radix Astragali, Rhizoma Coptidis, Rhizoma Alismatis, Pollen Tyjphae and Herba Artemisiae Scopariae, carry out concentration with water and ethanol, obtain need testing solution；
Step 2: the mensuration of need testing solution
Draw need testing solution and inject UPLC-Q-TOF-MS combined instrument, use gradient elution to survey with UPLC-Q-TOF-MS method
Fixed, according to UPLC-Q-TOF-MS combined instrument testing result, it is thus achieved that the composition of SANHUANG square preparation.
SANHUANG square preparation component detection method the most according to claim 1, it is characterised in that described step 1 is specifically wrapped
Include following steps:
Step a: after the Radix Astragali, Rhizoma Coptidis, Rhizoma Alismatis, Pollen Tyjphae and Herba Artemisiae Scopariae being soaked by purified water, adds purified water and boils together, then filter
Taking liquid concentration obtain concentrated medicament；
Step b: after addition ethanol stirs in the concentrated medicament that step a obtains, room temperature precipitates 24 hours, then takes supernatant
Liquid reclaims ethanol and carries out concentration, again takes supernatant and reclaims ethanol and carry out concentration, obtains concentrated solution；
Step c: after addition water stirs in the concentrated solution that step b obtains, precipitate 24 hours, then supernatant is carried out dense
Contracting processes, and constant volume obtains need testing solution.
SANHUANG square preparation component detection method the most according to claim 1, it is characterised in that the Radix Astragali in described step 1,
Rhizoma Coptidis, Rhizoma Alismatis, Pollen Tyjphae and Herba Artemisiae Scopariae, calculate by weight, and each constituent content is respectively as follows: Radix Astragali 1-4 part；Rhizoma Coptidis 1 part；Rhizoma Alismatis 1-
3 parts；Pollen Tyjphae 1-3 part；Herba Artemisiae Scopariae 1-3 part.
SANHUANG square preparation component detection method the most according to claim 1, it is characterised in that UPLC color in described step 2
Spectral condition is: flowing is the formic acid acetonitrile of the formic acid water-0.1% of 0.1% mutually；Flow velocity 0.3-0.5mL/min；Column temperature 35-50 DEG C；
Sample size 1-5 μ L.
SANHUANG square preparation component detection method the most according to claim 1, it is characterised in that UPLC color in described step 2
Spectrum mensuration mode is negative ions full scan, electron spray (ESI) ion source detection pattern；Quality of scanning scope is 100-
1000Da。
SANHUANG square preparation component detection method the most according to claim 1, it is characterised in that UPLC color in described step 2
The chromatographic column of spectrum is ACQUITYUPLC BEH C18 chromatographic column.
SANHUANG square preparation component detection method the most according to claim 6, it is characterised in that described ACQUITYUPLC
The chromatographic condition of BEH C18 chromatographic column is: flow velocity 0.4mL/min, column temperature 45-50 DEG C, sample size 1-5 μ L.
8. a SANHUANG square preparation fingerprint map construction method, it is characterised in that take the SANHUANG square preparation test sample of different batches
Solution method as described in claim 1-7 any one detects, and according to testing result, demarcates total peak, sets up SANHUANG
Square preparation finger printing.
SANHUANG square preparation finger printing the most according to claim 8 is in SANHUANG square preparation quality control and analysis method
Application.
SANHUANG square preparation finger printing the most according to claim 8 enters blood component setting analysis side really at SANHUANG square preparation
Application in method.
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| CN111624267A (en) * | 2020-04-30 | 2020-09-04 | 广东经典名方科技有限公司 | High performance liquid detection method for 16-oxo-alisol A in alisma orientale |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102028840A (en) * | 2009-09-28 | 2011-04-27 | 中国药品生物制品检定所 | Ultrahigh pressure liquid phase detection method for Chinese patent medicine |
| CN103575819A (en) * | 2012-08-02 | 2014-02-12 | 河北以岭医药研究院有限公司 | Method for measuring fingerprint spectrum of cardiac traditional Chinese medicine preparation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102028840A (en) * | 2009-09-28 | 2011-04-27 | 中国药品生物制品检定所 | Ultrahigh pressure liquid phase detection method for Chinese patent medicine |
| CN103575819A (en) * | 2012-08-02 | 2014-02-12 | 河北以岭医药研究院有限公司 | Method for measuring fingerprint spectrum of cardiac traditional Chinese medicine preparation |
Non-Patent Citations (4)
| Title |
|---|
| YAO QI等: "Chemical profiling ofWu-toudecoctionbyUPLC–Q-TOF-MS", 《TALANTA》 * |
| 刘庆丰等: "HPLC-ELSD 测定三黄合剂中黄芪甲苷的含量", 《中国现代应用药学》 * |
| 徐璐扬等: "HPLC 测定三黄汤中4 种成分含量", 《中国现代应用药学》 * |
| 芮雯等: "不同产地黄芪药材的UPLC/Q-TOF-MS 指纹图谱研究", 《药物分析杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111624267A (en) * | 2020-04-30 | 2020-09-04 | 广东经典名方科技有限公司 | High performance liquid detection method for 16-oxo-alisol A in alisma orientale |
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