CN106018577B - Three yellow party formulation ingredients detection methods and fingerprint map construction method - Google Patents
Three yellow party formulation ingredients detection methods and fingerprint map construction method Download PDFInfo
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Abstract
The invention belongs to Pharmaceutical Analysis fields, it is related to a kind of measuring method of traditional Chinese medicine fingerprint, the invention discloses a kind of three yellow party formulation ingredients detection methods, test solution is injected into UPLC-Q-TOF-MS combined instrument, it is measured with UPLC-Q-TOF-MS method using gradient elution, according to UPLC-Q-TOF-MS combined instrument testing result, the ingredient of three yellow party preparations is obtained.The invention also discloses utilize above-mentioned three yellow party formulation ingredients detection method, three yellow party preparation test solutions of different batches are taken to be detected by above-mentioned method, according to testing result, shared peak is demarcated, three yellow party preparation ultra performance liquid chromatography-high-resolution-flight time mass spectrums combination finger-print is established.This method and finger-print can be used as one of the control of the three yellow party qualities of the pharmaceutical preparations and the effective ways of analysis.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, it is related to the measuring method that Chinese medicine refers to tangerine green trichoderma bacterium spectrum, more particularly to a kind of
Three yellow party formulation ingredients detection methods and ultra performance liquid chromatography-high-resolution-flight time mass spectrum are combined fingerprint map construction side
Method.
Background technique
Traditional Chinese medicine fingerprint is certain class or a few classes that can reflect Chinese medicine or Chinese patent drug established using modern analytical technique
The chromatography of ingredient or the map of spectral information can more fully reflect the total chemical characteristics of Chinese medicine, embody in it entirety
Quality can make up only with a small number of ingredients come the drawbacks of controlling traditional Chinese medicine quality, it has also become internationally recognized control Chinese medicine or crude drug
The effective means of amount of substance.However, the diversity and complexity of chemical composition of Chinese materia medica and the uncertainty of effective substance,
Increase traditional Chinese medicine fingerprint foundation difficulty and finger-print it is effective and repeated.
Currently, it is Chemistry for Chinese Traditional Medicine finger-print that traditional Chinese medicine fingerprint, which is studied more, it is using chromatography, wave spectrum and other points
The finger-print for being used to characterize chemical composition of Chinese materia medica feature that analysis method is established.High performance liquid chromatography (HPLC) has separation efficiency
Height analyzes the characteristics of fast speed, favorable reproducibility, and can cooperate different detectors to analyze the advantage of various types sample,
As the generally acknowledged predominant methods for establishing traditional Chinese medicine fingerprint.The ultra performance liquid chromatography technology (UPLC) of rising in recent years has
The features such as super-pressure, hypersensitivity, superelevation separating degree, it by HPLC theory and principle, using the chromatography of 1.7 μm of partial sizes
Column packing can make the analysis time of a finger-print by shortening within original at least 1 hour as 10min or so, and chromatography
Peak capacity and stability also significantly improve, and UPLC has obvious excellent in the separation analysis of the complex systems such as Chinese medicine and natural products
Gesture provides new separation platform for traditional Chinese medicine fingerprint research.
Ultraviolet (UV) detector is to be combined to carry out the most widely used detection side of finger-print research at present with liquid chromatogram
Method.However the ultraviolet absorptivity of chemical component has additive property, so that measurement result be made to generate deviation, cannot really reflect 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 no UV absorption or
Absorb weaker ingredient, it is difficult to effectively be measured.Significant important chemical component in monarch drug in a prescription Radix Astragali in this special compound preparation
Significant important chemical component original note alkane triterpenes components are (such as in triterpene saponin (such as Astragaloside IV IV) and adjutant rhizoma alismatis
Alisol A etc.) ultra-violet (UV) band absorb it is very weak and interfere it is more, discomfort share UV detector measurement.
Mass spectrum (MS) detector can be used for the detection of various compounds in Chinese medicine, can produce preferable response, be a kind of
Highly sensitive all-purpose detector, it is often more important that the molecular weight and molecular structure information (tandem mass spectrum) of compound can be provided.
Using mass detector, by determining the mass scan range of ion, it can make that it includes the information of all target compounds.It answers
With total ion current detection pattern, all information of compound can be integrated；In turn, believed by examining or check the mass spectrum of shared chromatographic peak
Breath can determine the composition situation of chromatographic peak, guarantee that chromatographic peak for single compound composition, has preferable scientific.Due to
The complexity of chemical composition of Chinese materia medica, guarantee traditional Chinese medicine fingerprint in chromatographic peak be single compound peak be difficult it is several in other words
Can not, and mass detector is used, the ion of specific m/z can be extracted from total ion chromatogram (TIC), foundation mentions
Chromatography of ions figure (EIC) is taken, to ensure that chromatographic peak for single compound composition.In addition, by carrying out mass spectrum to shared peak
It is qualitative, the extraction chromatography of ions figure at shared peak is established, and then pass through the one-to-one relationship of chromatographic peak and compound m/z, realized
Accurate positionin and measurement to each shared peak, the shortcomings that so as to make up the poor reproducibility between other methods, laboratory.
Flight time tandem mass spectrometer (Q-TOF-MS) is high-resolution tandem mass spectrum, and distinguishing feature is highly sensitive, high
Selectivity, can obtain high quality mass spectrogram and compound accurate molecular weight (Mass accuracy < 3 × 106), compound can be greatly improved
The accuracy of Structural Identification.UPLC-Q-TOF-MS combines very good chromatogram separating capacity and very sensitive, very intelligent
Detectability, great development prospect and space in Chemistry for Chinese Traditional Medicine finger-print, advantage is prominent, and feature is obvious, will become
The development trend of traditional Chinese medicine fingerprint and direction.
Three yellow party be Huashan Hospital Affiliated To Fudan Univ cipher prescription, by Radix Astragali, the coptis, rhizoma alismatis, cattail pollen, oriental wormwood compatibility and
At clinical application for many years is dissipated in treatment metabolic syndrome and nonalcoholic fatty liver for department of traditional Chinese medicine early stage cipher prescription QI invigorating
(patent No.: the side of simplifying 200810034507.3) (number of patent application: 201511018056.0), there is QI invigorating to dissipate poly- function for poly- side
Effect can improve patient sign and internal insulin-resistant states, can promote the precise and tiny normal transporting of qi and blood, it is different to correct organism metabolism
Often, that is, have the function of that turbid, Tiaozhi Huayu drops in QI invigorating dissipating bind, clearing heat and eliminating phlegm, clearing damp.Since effective component important in Radix Astragali is such as yellow
The purple of the protostanes triterpenes such as important effective component such as alisol and its esters 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 also poor, limitation
The finger-print research of compound preparation containing both Chinese medicines.We report so far is contained using 2 ingredients, 4 ingredients
Quantity measuring method controls the content of three yellow party preparations, but still without its multicomponent assay and utilizes UPLC-Q-TOF-
The report of MS progress quality control.5 kinds are formed the present invention is based on " Chinese Pharmacopoeia " 2015 editions and is surveyed needed for the Quality Identification of Chinese medicine
(Radix Astragali is Astragaloside IV IV, calycosin glucoside to fixed 11 kinds of chemical components of kind；The coptis be epiberberine, coptisine,
Palmatine, jamaicin；Rhizoma alismatis is Alisol B monoacetate；Pollen Typhae is Isorhamnetin-3-O-neohespeidoside and typhaneoside；Mattress
Old is chlorogenic acid and escoparone) and State Food and Drug Administration's standard items catalogue, and refer to three yellow party preparation blood
Clear chemical research (18 kinds of ingredients can enter blood), the 23 kinds of determining fingerprint pattern indexs selected are in addition to covering the above chemical component
And 12 kinds have been increased newly for disadvantage less to detection target component needed for the special Pollen Typhae of 5 kinds of Chinese medicines, rhizoma alismatis in pharmacopeia respectively
Chemical component, and the measurement through 10 batches of three yellow party solution finger-prints, primarily determine total chemical ingredient, as three yellow party preparations
The reference method of quality control.
This technology is based on high-end liquid phase measurement technology, and required equipment is not yet a large amount of universal at home, but for generally comprehensive
Property university and large-scale Chinese medicine production medicine enterprise be all equipped with to be indispensable.Three yellow party preparations are basically completed preclinical study at present, will be first
Step is not yet mass produced as preparation, preparation is developed in Huashan Hospital Affiliated To Fudan Univ institute, current finger-print master
It to be used as and refer to map, but this measuring method can be used as the method for quality control of three yellow party preparations.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology and insufficient, provides a kind of simple using UPLC-Q-TOF-MS
Single method for quickly carrying out the control of the three yellow party qualities of the pharmaceutical preparations, and thus three yellow party preparation finger constructed by method.
The first purpose of the invention is to provide a kind of three yellow party formulation ingredients detection methods, comprising the following steps:
Step 1: the preparation of test solution
Radix Astragali, the coptis, rhizoma alismatis, Pollen Typhae and oriental wormwood are taken, is extracted with water and ethyl alcohol, constant volume obtains test solution；
Step 2: the measurement of test solution
Draw test solution inject UPLC-Q-TOF-MS combined instrument, with UPLC-Q-TOF-MS method using gradient elution into
Row measurement obtains the ingredient of three yellow party preparations according to UPLC-Q-TOF-MS combined instrument testing result.
Preferably, above-mentioned step 1 specifically includes the following steps:
Step a: after Radix Astragali, the coptis, rhizoma alismatis, Pollen Typhae and oriental wormwood are impregnated with purified water, purified water is added with boiling, so
Simultaneously concentration obtains concentrated medicament to leaching medical fluid afterwards；
Step b: being added ethyl alcohol after mixing evenly into the concentrated medicament that step a is obtained, and room temperature precipitates 24 hours, then takes
Supernatant recycling ethyl alcohol simultaneously carries out concentration, takes supernatant recycling ethyl alcohol again and carries out concentration, obtains concentrate；
Step c: into the concentrate that step b is obtained be added water after mixing evenly, precipitate 24 hours, then by supernatant into
Row concentration, constant volume obtain test solution.
Preferably, by weight calculating in above-mentioned step 1, each component content is respectively as follows: 1-4 parts of Radix Astragali；1 part of the coptis；
1-3 parts of rhizoma alismatis；1-3 parts of Pollen Typhae；1-3 parts of oriental wormwood.
Preferably, UPLC chromatographic condition in above-mentioned steps 2 are as follows: the formic acid second for the formic acid water -0.1% that mobile phase is 0.1%
Nitrile；Flow velocity 0.3-0.5mL/min；35-50 DEG C of column temperature；Sample volume 1-5 μ L.
Preferably, UPLC chromatographic determination mode is negative ions full scan, electron spray (ESI) ion source in above-mentioned steps 2
Detection pattern；Scanning quality range is 100-1000Da.
Preferably, the chromatographic column of UPLC chromatography is ACQUITYUPLC BEH C18 chromatographic column in above-mentioned steps 2.
Preferably, the chromatographic condition of above-mentioned ACQUITY UPLC BEH C18 chromatographic column are as follows: flow velocity 0.4mL/min, column
Warm 45-50 DEG C, sample volume 1-5 μ L.
Second object of the present invention provides a kind of three yellow party preparation finger construction methods, takes the three of different batches yellow
Square preparation test solution is measured by three above-mentioned yellow party formulation ingredients detection methods, and according to testing result, calibration is shared
Three yellow party preparation fingers are established at peak.
It is compared by 10 batches of three yellow party preparation test solutions, it is determined that 23 kinds of shared ingredients, and mark shared peak.This hair
Other interference components are not contained under bright testing conditions in sample in addition to purpose ingredient, as the result is shown detection method provided by the invention
Precision is high, more stable, reproducible, good tolerance, map of the invention can be used for three yellow party preparations quality control with
Analysis.
The present invention characterizes the chemical component of three yellow party preparations using finger-print on the whole, and passes through serum drug chemistry
It is the ingredient for being absorbed into blood that method, which proves that this method can be used for measuring 18 shared peaks, it may be possible to which three yellow party preparations play medicine
The potential effective component of effect.
Compared with prior art, the invention has the following advantages that
(1) present invention is easy purchase using 23 kinds at home and abroad for the first time using UPLC-Q-TOF-MS as analysis means
Standard items (amounting to 11 kinds of chemical components in 5 kinds of composition Chinese medicines for needing to measure including " Chinese Pharmacopoeia " 2015 editions regulations) are established
Three yellow party formulation chemist fingerprint pattern quality control methods, compared with the HPLC-UV analysis method of existing report, not only significantly
Analysis time is shortened, separative efficiency and detection sensitivity are improved；And the information of molecular weight can be assigned to each peak, by building
Vertical chromatography of ions figure, can accurately calculate the peak area at each peak, and if when laboratory monitoring reappears, retention time deviates, can lead to
The molecular weight information for crossing each peak is accurately positioned, to ensure that the reproducibility of fingerprint atlas detection method, can be used for three
Quality control and analysis of the yellow party in relation to preparation.
(2) 18 can be measured in three yellow party simultaneously and enters blood component, for further studying the serum medicine of three yellow party preparations
Neo-Confucianism, pharmacokinetics, developing new drug have the function of important.
(3) this programme can also be used for the chemical substance fundamental analysis of 5 kinds of composition Chinese medicines.
(4) other interference components are not contained in addition to purpose ingredient in sample under testing conditions of the present invention, as the result is shown this hair
The detection method precision of bright offer is high, more stable, reproducible, and good tolerance.
Detailed description of the invention
Fig. 1 is the UPLC-Q-TOF-MS cation extraction chromatography of ions figure (HQ=of three yellow party preparations and 5 kinds of composition Chinese medicines
Radix Astragali, the HL=coptis, ZX=rhizoma alismatis, PH=cattail pollen, YC=oriental wormwood, tri- yellow party extracting solution of SHD=).
Fig. 2 is the UPLC-Q-TOF-MS anion extraction chromatography of ions figure (HQ=of three yellow party preparations and 5 kinds of composition Chinese medicines
Radix Astragali, the HL=coptis, ZX=rhizoma alismatis, PH=cattail pollen, YC=oriental wormwood, tri- yellow party extracting solution of SHD=).
Fig. 3 is the UPLC-Q-TOF-MS finger-print (cation mode) of three yellow party preparations.
Fig. 4 is that the UPLC-Q-TOF-MS cation of three yellow party preparation rat Contained Serums extracts chromatography of ions figure.
Fig. 5 is that the UPLC-Q-TOF-MS anion of three yellow party preparation rat Contained Serums extracts chromatography of ions figure.
Specific embodiment
The present invention is described in more detail below by specific embodiment, for a better understanding of the present invention,
But following embodiments are not intended to limit the scope of the invention.
Embodiment 1
The present embodiment is the measuring method of three yellow party preparation UPLC-Q-TOF-MS finger-prints, 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, autosampler, control software.
1.2. reagent chemicals
Acetonitrile and formic acid are chromatographically pure, and water is ultrapure water, and other reagents are that analysis is pure；
Astragaloside IV IV, onocerin, calycosin, calycosin-7-O-β-D-glycoside, jamaicin, bar
Ma Ting, jateorrhizine, Alisol B monoacetate, chlorogenic acid, scopolactone, 6,7- dimethoxycoumarins, typhaneoside, different mouse
Li Su -3-O- neohesperidin, Isorhamnetin, naringenin, Quercetin, Kaempferol are purchased from Chinese food Drug Administration；Table
Jamaicin, coptisine, alisol A, alisol F, 24- acetylalisol A, 6- hydroxyl -7- methoxyl group-cumarin is purchased from Shanghai China
One Bioisystech Co., Ltd；5 kinds of Chinese medicines are purchased from Hongqiao in Shanghai's prepared slices of Chinese crude drugs Co., Ltd.
2. the preparation of test solution
The 30g Radix Astragali for meeting Chinese medicine preparation specification, the 9g coptis, 15g rhizoma alismatis, 15g Pollen Typhae, 15g oriental wormwood are weighed, ingredients
It is spare, wherein Radix Astragali, the coptis, rhizoma alismatis, Pollen Typhae, oriental wormwood weight ratio be 1-4:1:1-3:1-3:1-3.The above drug is first soaked
0.5h is steeped, adds purified water with boiling (wherein Pollen Typhae is decocted a drug wrapped), extracts 2 times, add 8-12 times of water every time, boil 2 times, each 1h, filter
Crude drug is concentrated in taking liquid, and 95% alcohol of 1 times of amount is added, stirs evenly, and room temperature precipitates 24 hours, and supernatant is taken to recycle ethyl alcohol
And it is concentrated；5 times of 95% alcohol of amount are added, stand 24 hours, supernatant is taken to recycle clean ethyl alcohol；2 times of water (70-80 are added
DEG C), stirring, 10-15 DEG C of environment precipitates 24 hours, Aspirate supernatant, and is made after mixing with medicinal corrigent, preservative
Oral administration solution filters out sediment, is concentrated as 20mL solution (i.e. 4.2g Chinese medicine/mL solution).
The 50 above-mentioned solution of μ L to be drawn, 950 μ L methanol-acetonitrile (1:1) solution are added, 4 DEG C of refrigerators place 4h, be centrifuged (4 DEG C,
20,267g, 15min), supernatant is taken, sample volume is 2 μ L.
3. finger-print testing conditions
Chromatographic condition: analytical column is ACQUITY UPLCTM BEH C18column (100mm × 2.1mm i.d., 1.7 μ
M, waters), 45 DEG C of column temperature, [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, change of gradient situation are 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
It is set as 100-1000Da, interval time 0.02s, scanning time second is 0.3s, and capillary voltage is 3000V (cationic mould
Formula) and 2800v (ion mode), desolventizing temperature is 350 DEG C, and sample cone voltage is 35v (cation mode) and 50v is (negative
Ion mode), collision energy 4eV, 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
With shown in Fig. 2.Embodiment 2
The present embodiment is the foundation and evaluation of three yellow party preparation fingers
1.1. the determination at peak is shared
10 batches of three yellow party preparations are collected, operates by the method in embodiment 1, is analyzed by the testing conditions of embodiment 1.
From the point of view of testing result, 23 compound peaks are that each batch of test sample is shared, therefore using this 23 compound peaks as three yellow party
The shared compound peaks of preparation obtain the extraction chromatography of ions figure of this 23 compound peaks, such as Fig. 3 from total ion chromatogram
It is shown.Wherein the peak area of palmatine is maximum, is selected as referring to peak.
Flight time mass spectrum is high resolution mass spectrum, can measure the accurate mass of ion, and then be less than according to measured deviation
5PPM and the smallest principle of isotope degree of fitting calculate the molecular formula at each shared peak.
As shown in table 1, it is arranged by appearance time sequence, the karyoplasmic ratio at 23 shared 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
Object 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.
In addition the karyoplasmic ratio of 33 chemical components 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.
In addition, the relative peak area at each shared peak of three yellow party preparation fingers is as shown in table 2, as can be seen from Table 2 not
With batch test sample share peak relative peak area similarity it is very high, coefficient of variation very little, illustrate this method for measure three
Yellow party formulation content repeatability is high, as a result precisely, can be used for the measurement of three yellow party formulation ingredients contents.
The chemical component for the three yellow party preparations that 1 UPLC-ESI-Q-TOF-MS of table 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 shared peak of 2 three yellow party preparation finger of table
Embodiment 3
The present embodiment is the determination that three yellow party preparations enter blood component, the specific steps are as follows:
It takes Wistar rat (200 ± 20g), fasting 12h, takes orally three yellow party preparation liquid, 1h sodium phenobarbital after administration
Physiological salt liquid intraperitoneal injection of anesthesia (concentration 100mg/mL, 2.0mL/kg weight), takes a blood sample through abdominal aorta, centrifugation
(3000rpm, 10min, 4 DEG C) takes 200 μ L of serum, and 800 μ L methanol-acetonitrile (1:1) solution are added, and 4 DEG C of refrigerators place 4h, from
The heart (4 DEG C, 20,267g, 15min), takes supernatant, and sample volume is 6 μ L, is detected by the chromatographic condition drafted, obtain 18 enter blood at
The extraction chromatography of ions figure for the mass-to-charge ratio divided, experimental result is as shown in figs. 4 and 5.
The present embodiment characterizes the chemical component of three yellow party preparations using finger-print on the whole, and passes through serum drug
It is the ingredient for being absorbed into blood that method, which proves that this method can be used for measuring 18 shared peaks, it may be possible to which three yellow party preparations play
The potential effective component of drug effect.
Embodiment 4
The present embodiment mainly chooses 7 important chemical marker ingredients and is used for its Simultaneous Quantitative Analysis to the present invention
Measuring method further verified.
(1) range of linearity and detection limit, quantitative limit are investigated: linearity and range is studied by matching to 7 selected chemical components
The working solution for making 6 various concentrations is investigated, and each sample is 2 times tested.Detection limit and quantitative limit are respectively signal-to-noise ratio (S/
N) the standard sample concentration for being 3 and 10.It the results are shown in Table 2.
(2) repeatability is investigated: same sample is repeated analysis 6 times, for investigating the repeatability of method.It the results are shown in Table 2.
(3) precision is investigated: withinday precision is by measuring 1 time at interval of certain time to 5 samples in one day, and totally 5
Secondary measurement；Day to day precision measured same concentration sample in synchronization by continuous 5 days and obtains.It the results are shown in Table 2.
(4) accuracy is investigated: being investigated by sample recovery rate.Difference is separately added into the sample of 3 parts of known contents
The standard items of the same race of content (80%, 100% and the 120% of known content) measure the content of target criteria product in gained sample,
And calculate the difference of actually detected amount and theoretical additive amount.Calculation formula are as follows: the rate of recovery (%)=100 × (detection limit-theory adds
Enter amount)/theoretical addition amount；And the coefficient of variation is indicated with relative standard deviation (RSD).It the results are shown in Table 3.
It is compared by 7 important chemical marker ingredients as control, further demonstrates measuring method of the invention
Accuracy is high, and repeatability is good.
The quantitative analysis of 37 kinds of significant compounds of table
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and
Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and
Modification, all should be contained within the scope of the invention.
Claims (5)
1. a kind of three yellow party formulation ingredients detection methods, which comprises the following steps:
Step 1: the preparation of test solution
Radix Astragali, the coptis, rhizoma alismatis, Pollen Typhae and oriental wormwood are taken, concentration is carried out with water and ethyl alcohol, obtains test solution；
Step 2: the measurement of test solution
It draws test solution and injects UPLC-Q-TOF-MS combined instrument, surveyed with UPLC-Q-TOF-MS method using gradient elution
It is fixed, according to UPLC-Q-TOF-MS combined instrument testing result, obtain the ingredient of three yellow party preparations；
Wherein, the step 1 specifically includes the following steps:
Step a: after Radix Astragali, the coptis, rhizoma alismatis, Pollen Typhae and oriental wormwood are impregnated with purified water, purified water is added with boiling, is then filtered
Simultaneously concentration obtains concentrated medicament to taking liquid；
Step b: being added ethyl alcohol after mixing evenly into the concentrated medicament that step a is obtained, and room temperature precipitates 24 hours, then takes supernatant
Liquid recycling ethyl alcohol simultaneously carries out concentration, takes supernatant recycling ethyl alcohol again and carries out concentration, obtains concentrate；
Step c: being added water after mixing evenly into the concentrate that step b is obtained, and precipitates 24 hours, then carries out supernatant dense
Contracting processing, constant volume obtain test solution；
The finger-print testing conditions of UPLC-Q-TOF-MS combined instrument in the step 2:
Chromatographic condition: analytical column is ACQUITY UPLCTM BEH C18 column, sample volume 2 μ L, flow velocity 0.4mL/min, column temperature 45
DEG C, gradient elution: A is the formic acid solution of 0.1%v/v, and B is the acetonitrile solution of the formic acid containing 0.1%v/v, and change of gradient situation is such as
Under: 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: electric spray ion source, full scan data are set as 100-1000Da, interval time 0.02s, when scanning the second
Between be 0.3s, capillary voltage be cation mode 3000V and ion mode 2800V, desolventizing temperature be 350 DEG C, sample
Boring voltage is cation mode 35V and ion mode 50V, collision energy 4eV, and extracting cone voltage is 4.0V, and source temperature is
115 DEG C, cone gas flow rate is 50L/h, Desolvention gas velocity 600L/h.
2. three yellow party formulation ingredients detection method according to claim 1, which is characterized in that Radix Astragali in the step 1,
The coptis, rhizoma alismatis, Pollen Typhae and oriental wormwood, calculate by weight, and each component content is respectively as follows: 1-4 parts of Radix Astragali；1 part of the coptis；Rhizoma alismatis 1-
3 parts；1-3 parts of Pollen Typhae；1-3 parts of oriental wormwood.
3. a kind of three yellow party preparation finger construction methods, which is characterized in that take three yellow party preparation test samples of different batches
Solution is detected by method described in claim 1-2 any one, according to testing result, is demarcated shared peak, is established three Huangs
Square preparation finger-print.
4. three yellow party preparation finger according to claim 3 is in three yellow party formulation ingredients really setting analysis method
Using.
5. three yellow party preparation finger according to claim 3 enters blood component setting analysis method really in three yellow party preparations
In application.
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