CN109100431A - A kind of effective substance research method of palace of the Qing Dynasty Shoutao pills - Google Patents
A kind of effective substance research method of palace of the Qing Dynasty Shoutao pills Download PDFInfo
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Abstract
The present invention provides a kind of effective substance research methods of palace of the Qing Dynasty Shoutao pills, belong to technical field of traditional Chinese medicines, a kind of effective substance research method of palace of the Qing Dynasty Shoutao pills, characterized by the following steps: (1) carry out LC-MS for palace of the Qing Dynasty Shoutao pillsnGlobal analysis carries out Structural Identification to wherein main chemical component, determines series components type therein；(2) the LC-MS analysis method of series components is established respectively, and combines the quick Structural Identifications of carry out such as chromatographic retention, ultra-violet absorption spectrum feature, molecular weight and multistage cracking fragment ion information.One aspect of the present invention can farthest retain original ingredient in sample to avoid sample-pretreating method；On the other hand, for holistic method, the analysis method of each series components is established respectively, and the ingredient during reduction Mass Spectrometer Method flows out the inhibition to ingredient mass signal such as effect and ion depression effect altogether to a certain extent, to be conducive to illustrating for Chinese medicine complex material system.
Description
Technical field
The present invention relates to belong to technical field of traditional Chinese medicines, the effective substance research side of specifically a kind of palace of the Qing Dynasty Shoutao pills
Method.
Background technique
As the rarity of the Chinese nation for thousands of years, traditional Chinese medicine plays not the health of the people and national multiplying and living
Alternative effect.The chemical component and its bioactivity for how illustrating these precious legacy, preferably serve the mankind, to me
For be important task in shoulder, shoulder heavy responsibilities.With the development of modern technologies, Mechanism of TCM and chemical substance basis are ground
Study carefully and also deepens continuously.However, chemical composition of Chinese materia medica is extremely complex, containing largely needing qualitative and quantitative chemical component, and
Dynamic change easily occurs during processing, compatibility, product may be closely related with clinical efficacy.Therefore, Chemistry for Chinese Traditional Medicine at
The complexity divided becomes the bottleneck of Development of Chinese Medicine and modernization.
Currently, merely by traditional extracting and developing, the Natural products research method of Structural Identification trilogy and with work
Property tracking be core Natural products research method, although can comparison system, in depth study natural drug in chemical component
Or active constituent, but time-consuming, at high cost, blindness is strong, and can not obtain the structural information of all the components in natural drug,
With many limitations.Therefore, the research of complex material system anlysis method has become the advanced subject in Pharmaceutical Analysis field
One of.
The liquid chromatography-mass spectrometry (LC-MS) of the nineties later period in last century maturation is by outstanding separating capacity
It is organically combined with the detection method of highly sensitive and high specificity, has become the most powerful material foundation of tcm research hand
One of section.In modern analytical technique, with the characteristics of quick, highly sensitive, specific and multi information and can most effectively with
The technologies such as chromatography are combined and the attention by scientist.Especially in recent years, with electrospray ionisation and ground substance assistant laser solution
The appearance of the soft ionizations mass-spectrometric techniques such as ionization is inhaled, application of the mass spectrum in the numerous areas such as drug and life science has obtained pole
The earth is expanded.Soft ionization mass spectrum, tandem mass spectrum and the combination with chromatography are analyzed in conjunction with high resolution mass spectrum, in effective component of chinese medicine
Structural confirmation in terms of there is the incomparable advantage of other analytical technologies, have become Study of Traditional Chinese Medicine chemical substance basis, explain
State one of the best approach of Chinese medicine preparation principle and compatibility of medicines in a prescription principle scientific meaning.
But during the detection and identification of Chinese medicine constant and micro constitutent, ingredient flows out altogether and ion inhibition is two
It is a to be difficult to overcome the problems, such as.This is that is, the Chinese medicine complex material system anlysis based on LC-MS technology is still limited by constant
It is difficult to micro constitutent while key issues of detection and identification.In recent years, although the fast development of chromatographic isolation filler and
The commonly used separation selectivity that can improve to a certain extent between substance of multidimensional liquid separation technology, makes ion identification degree
With belongingness enhance, but excessive chromatographic isolation may result in the micro especially trace constituent concentration in part it is too low and by mass spectrum
Detector missing inspection can not still overcome the ion depression effect of total effluent in Soft ionization techniques.Meanwhile as mass spectrograph detects speed
The raising of degree and precision, the data that mass spectrum generates also sharply increase, and how correctly efficiently parsing these data also becomes Chinese medicine
A significant challenge in material base research field.Therefore, how around " Chinese medicine constant and traces component measure and identification "
" fast resolving of magnanimity mass spectrometric data " two critical issues, building be suitable in the big compound of Chinese medicine especially Chinese medicine chemistry at
Strategy and the method for dtex point are current research hotspots.
The research object palace of the Qing Dynasty of the present invention Shoutao pills be by the fructus alpiniae oxyphyllae of kidney-replenishing, the radix rehmanniae preparata of tonifying kidney-yin, tonifying both YIN and YANG Chinese holly
The selected formula composition of more than ten kidney-nourishing tcm drugs such as Diaphragma juglandis of matrimony vine, reinforcing the kidney and controlling noctural emission has kidney-tonifying sperm-generating, the strong function of benefit member
Effect, it is tired to can be used for dizziness caused by kidney deficiency aging, memory loss, soreness and weakness of waist and knees, Hiccough and deaf, and dim eyesight is shed tears, frequent urination at night, urinates
There is the heeltap.Since its flavour of a drug composition is extremely complex, contained chemical component is also quite abundant, illustrates its material base with important
Meaning.In the course of the research, the chemistry that chemical composition of Chinese materia medica Structural Identification strategy comes in the Rapid identification compound is constructed first
Ingredient: the Structural Identification strategy based on Chinese medicine series components, it is intended to by improving chromatographic isolation effect in situ, make micro constitutent
Concentration is enriched with and is detected, it is desirable to for the analysis of Chinese medicine complex material system and the research provider of material foundation of tcm
The science of law is used for reference.
Summary of the invention
In view of this, the present invention is intended to provide a kind of effective substance research method of palace of the Qing Dynasty Shoutao pills.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows: a kind of effective substance of palace of the Qing Dynasty Shoutao pills
Basic research method, includes the following steps:
(1) LC-MS is carried out for palace of the Qing Dynasty Shoutao pillsnGlobal analysis carries out Structural Identification to wherein main chemical component,
Determine series components type therein；
(2) the LC-MS analysis method of series components is established respectively, and combines chromatographic retention, ultra-violet absorption spectrum special
The quick Structural Identifications of carry out such as sign, molecular weight and multistage cracking fragment ion information.
Further, the preparation of sample solution includes the following steps: in the LC-MS analysis method
(1) preparation of preparation test solution: palace of the Qing Dynasty Shoutao pills coarse powder (being free of the intermediate of auxiliary material) 0.3g is taken to be placed in
In 50mL conical flask, adding methanol 25mL, weighs, room temperature ultrasound (250W, 70kHz) handles 30min, and standing is cooled to room temperature, then
Secondary weighing adds methanol to supply the weight of less loss, crosses 0.22 μm of miillpore filter, take subsequent filtrate to get；
(2) preparation of each flavour of a drug test solution: taking each flavour of a drug of palace of the Qing Dynasty Shoutao pills, crushes, and crosses No. four sieves, weighs 0.8g and set
In 50mL conical flask, adding methanol 10mL, weighs, room temperature ultrasound (250W, 70kHz) handles 30min, and standing is cooled to room temperature,
Weigh again, methanol added to supply the weight of less loss, cross 0.22 μm of miillpore filter, take subsequent filtrate to get；
(3) preparation of standard solution: taking above-mentioned reference substance appropriate respectively, adds methanol that every 1mL is made and contains each reference substance about
The solution of 50 μ g, under the conditions of 4 DEG C sealing be kept in dark place to get.
Further, UPLC condition is as follows in the LC-MS analysis method:
With T3,2.1 × 100mm, 1.8 μm of Waters ACQUITY UPLC HSS for chromatographic column；It is stream with 0.1% formic acid
Dynamic phase A, using acetonitrile as Mobile phase B, gradient elution, gradient are as follows: 0-2min, 8%B；2-20min, 8-26%B, 20-
22min, 26%B；22-30min, 26-42%B；30-50min, 42-60%B；50-55min, 60-95%B；55-58min,
95%B, 58-60min, 95-8%B；60-65min, 8%B；Room temperature；Sample volume is 3 μ L, flow velocity 0.3mL/min.
Further, MS condition is as follows in the LC-MS analysis method:
Negative ion mode, 350 DEG C of capillary temperature, sheath gas (nitrogen) 30arb, secondary air speed (nitrogen) 10arb,
Spray voltage: 3kV, capillary voltage: -35V, pipe lens voltage: -110V；
High resolution mass spectrum quality axis accuracy using caffeine, lauryl sodium sulfate, natrium taurocholicum,
Tetrapeptide MRFA and Ultramark hybrid standard product solution is corrected, and Mass accuracy error is within 5ppm；
Quality testing range: m/z 100-1200；Sample is scanned by the way of full scan, is swept entirely with high-resolution FT
It retouches, resolution ratio R is set as 30000, detects fragment ion with ion trap dynode；
Setting dynamic excludes, number of repetition: 3, the repetition time: and 10s, exclusion inventory size: 100, exclude the time: 20s.
Compared with the existing technology, present invention has the advantage that
Research method of the invention: on the one hand ingredient can be caused to hand over to avoid sample-pretreating method, such as stage extraction method
The loss of especially micro (trace) ingredient of chemical component caused by serious, the open column chromatography of fork, farthest retains sample
Original ingredient in product；On the other hand, for holistic method, the analysis method of each series components is established respectively, it can
To obtain be more good chromatographic isolation effect, to be obtained in micro (trace) ingredient mass spectrometric data collection process more
Detection time, the ingredient during reduction Mass Spectrometer Method flows out effect and ion depression effect etc. to ingredient altogether to a certain extent
The inhibition of mass signal, to be conducive to illustrating for Chinese medicine complex material system.
Detailed description of the invention
Fig. 1 is palace of the Qing Dynasty Shoutao pills material base research strategy figure.
Fig. 2 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of palace of the Qing Dynasty Shoutao pills.
Fig. 3 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of silkworm excrement medicinal material.
Fig. 4 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of Radix Angelicae Sinensis medicinal material.
Fig. 5 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of Diaphragma juglandis medicinal material.
Fig. 6 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of fructus lycii medicinal material.
Fig. 7 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of Radix Ophiopogonis medicinal material.
Fig. 8 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of ginseng crude drug.
Fig. 9 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of radix rehmanniae preparata medicinal material.
Figure 10 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of semen ziziphi spinosae medicinal material.
Figure 11 is the UPLC-LTQ-Orbitrap high resolution mass spectrum total ion current figure of lucid asparagus medicinal material.
Figure 12 is palace of the Qing Dynasty Shoutao pills and each flavour of a drug UPLC-LTQ-Orbitrap total ion current figure (negative ion mode).
Figure 13 is the possible lytic pathway of di-coffee mesitoyl quinine acid ingredient.
Figure 14 is the possible ESI-MS/MS lytic pathway of Ginsenoside Rc.
Figure 15 be using acetonitrile precipitation method treated administration rat plasma in effective constituents analyze (A: palace of the Qing Dynasty Shoutao pills sample
Product；B: blank rat plasma sample；C: administration rat plasma sample).
Figure 16 be using methanol extraction method treated administration rat plasma in effective constituents analyze (A: palace of the Qing Dynasty Shoutao pills sample
Product；B: blank rat plasma sample；C: administration rat plasma sample).
Figure 17 be using solid phase extraction treated administration rat plasma in effective constituents analyze (A: palace of the Qing Dynasty Shoutao pills sample
Product；B: blank rat plasma sample；C: administration rat plasma sample).
Figure 18 be using acetonitrile precipitation method treated administration rat urine in effective constituents analyze (A: palace of the Qing Dynasty Shoutao pills sample
Product；B: blank rat urine sample；C: administration rat urine sample).
Figure 19 be using methanol extraction method treated administration rat urine in effective constituents analyze (A: palace of the Qing Dynasty Shoutao pills sample
Product；B: blank rat urine sample；C: administration rat urine sample.
Figure 20 be using solid phase extraction treated administration rat urine in effective constituents analyze (A: palace of the Qing Dynasty Shoutao pills sample
Product；B: blank rat urine sample；C: administration rat urine sample.
Specific embodiment
The effective substance research method of 1 palace of the Qing Dynasty Shoutao pills
1.1 materials and reagent
Palace of the Qing Dynasty Shoutao pills and each single medicinal material are provided by commission producer.Chlorogenic acid, neochlorogenic acid, Cryptochlorogenic acid, coffee
Acid, echinacoside, rutin, quercitin, isoquercitrin, acteoside, 3,4- cynarin, bis- caffeoyl Kui of 3,5-
Buddhist nun's acid, notoginsenoside R, ginsenoside Rg1, ginsenoside Re, ginsenoside Rh 1, ginsenoside Rb1, Ginsenoside Rc, people
Join the reference substances such as saponin(e Rd, Spine Date Seed jujubosideB, ginseng sapoglycoside Rg 3 to examine purchased from National Institute for Food and Drugs Control's biological products
Fixed institute and Chengdu Purification Technology Development Co., Ltd., structure are determined through means such as 1H-NMR, 13C-NMR, purity warp
The detection of HPLC normalization method, is all larger than 95%, can be used for qualitative research.
Chromatographic grade acetonitrile and methanol are purchased from Fisher Scientific (Fair Lawn, NJ, USA), analysis level methanol, first
Acid is purchased from Sigma Aldrich (St.Louis, MO, USA), and ultrapure water is by Millipore Synergy UV ultrapure water mechanism
It is standby；0.22 μm of miillpore filter is purchased from Beijing Hua Zhi chromatography Science and Technology Ltd..
1.2 instrument
Thermo Scientific Accela 600pump high performance liquid chromatograph, be equipped with on-line degassing machine, automatically into
Sample device, high pressure binary gradient pump；HPLC by electrospray interface and LTQ-Orbitrap mass spectrograph (Thermo Scientific,
Bremen, Germany) connection；2.1 work station of Thermo Xcaliber；Mass Frontier 7.0(Thermo
Scientific).KQ-250DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；Millipore
Synergy UV type ultrapure water machine (Millipore company of the U.S.)；(German Sartorius is public for R200D type electronic analytical balance
Department).
The preparation of 1.3 sample solutions
(1) preparation of preparation test solution: palace of the Qing Dynasty Shoutao pills coarse powder (being free of the intermediate of auxiliary material) 0.3g is taken to be placed in
In 50mL conical flask, adding methanol 25mL, weighs, room temperature ultrasound (250W, 70kHz) handles 30min, and standing is cooled to room temperature, then
Secondary weighing adds methanol to supply the weight of less loss, crosses 0.22 μm of miillpore filter, take subsequent filtrate to get；
(2) preparation of each flavour of a drug test solution: taking each flavour of a drug of palace of the Qing Dynasty Shoutao pills, crushes, and crosses No. four sieves, weighs 0.8g and set
In 50mL conical flask, adding methanol 10mL, weighs, room temperature ultrasound (250W, 70kHz) handles 30min, and standing is cooled to room temperature,
Weigh again, methanol added to supply the weight of less loss, cross 0.22 μm of miillpore filter, take subsequent filtrate to get；
(3) preparation of standard solution: taking above-mentioned reference substance appropriate respectively, adds methanol that every 1mL is made and contains each reference substance about
The solution of 50 μ g, under the conditions of 4 DEG C sealing be kept in dark place to get.
1.4 LC-MS analysis methods
UPLC condition is as follows:
With T3,2.1 × 100mm, 1.8 μm of Waters ACQUITY UPLC HSS for chromatographic column；It is stream with 0.1% formic acid
Dynamic phase A, using acetonitrile as Mobile phase B, gradient elution, gradient are as follows: 0-2min, 8%B；2-20min, 8-26%B, 20-
22min, 26%B；22-30min, 26-42%B；30-50min, 42-60%B；50-55min, 60-95%B；55-58min,
95%B, 58-60min, 95-8%B；60-65min, 8%B；Room temperature；Sample volume is 3 μ L, flow velocity 0.3mL/min.
MS condition is as follows:
Negative ion mode, 350 DEG C of capillary temperature, sheath gas (nitrogen) 30arb, secondary air speed (nitrogen) 10arb,
Spray voltage: 3kV, capillary voltage: -35V, pipe lens voltage: -110V；
High resolution mass spectrum quality axis accuracy using caffeine, lauryl sodium sulfate, natrium taurocholicum,
Tetrapeptide MRFA and Ultramark hybrid standard product solution is corrected, and Mass accuracy error is within 5ppm；
Quality testing range: m/z 100-1200；Sample is scanned by the way of full scan, is swept entirely with high-resolution FT
It retouches, resolution ratio R is set as 30000, detects fragment ion with ion trap dynode；
Setting dynamic excludes, number of repetition: 3, the repetition time: and 10s, exclusion inventory size: 100, exclude the time: 20s.
1.5 LC-MS analyzes result
According to 1.4 lower LC-MS analysis methods, the palace of the Qing Dynasty Shoutao pills of acquisition and the total ion current figure of each flavour of a drug are shown in figure
2-12.According to the literature, in conjunction with chromatography retention behavior, molecular weight information and cleavage of mass spectrum rule, tentatively to thereinization
It studies and point is identified, determine chemical component classification therein, determine series components type contained in the peach offered as a birthday present pill of the palace of the Qing Dynasty
Mainly have: the big constituents such as flavones, triterpenoid saponin, steroid saponin, organic acid, iridoid glycoside and benzyl carbinol glycosides.
Meanwhile in conjunction with document report and related control product, each chromatographic peak in MS figure is identified, the results are shown in Table 1.
Structural Identification of the table 1 based on chemical component in the palace of the Qing Dynasty Shoutao pills of UPLC-LTQ-Orbitrap HRMS
Δ: using reference substance identification；It * is [M+HCOO]-Ion
(1) identification of organic acid ingredient
Organic acid ingredient type in the Shoutao pills of the palace of the Qing Dynasty is more, and basic component units have chinic acid (Q), caffeic acid
(C), cinnamic acid (pCo), ferulic acid (F) etc..By comparing the chemical component in preparation and flavour of a drug, in the peach offered as a birthday present pill of the palace of the Qing Dynasty
Organic acid ingredient structure carry out Rapid identification.
Compound 2,3 and 4 retention times are very short, illustrate that polarity is very big.ESI-MS spectrum under anionic textiles mode
In, [M-H] can be observed-Ion (m/z 515.1395), thus it is speculated that its molecular formula is C22H27O14, quality error is respectively less than 5ppm,
Simultaneously it can be observed that [M-H-glc] in their ESI-MS/MS spectrum-(m/z 353)、[M-H-glc-caffeoyl]-(m/z
191)、[M-H-glc-caffeoyl-H2O]-Fragment ions such as (m/z 173).It analyzes its possible molecular formula and cracking ion is broken
Piece is accredited as caffeoyl guinic acid glucoside.In conjunction with m/z 191, m/z 173 and m/z 179 3 it is important son from
These three compounds can be inferred as respectively 3-caffeoylquinic acid glucoside, 4-caffeoyl-quinic acid by the relative abundance of son
Glucoside and 4-caffeoyl-quinic acid glucoside.Since glucose residue the position of substitution is difficult to determine, only by them
Caffeoyl guinic acid glucoside compounds simply are accredited as, do not indicate the position of substitution of glucosyl group.
ESI-MS spectrum of the compound 5,11 and 16 under anionic textiles mode generates the quasi-molecule of m/z179.0339 respectively
Quasi-molecular ions, deduction molecular formula are C9H7O4, error is less than 5ppm, and produces respectively and coffee in their ESI-MS/MS spectrum
Thus they are identified respectively as caffeic acid isomerism such as m/z 135 and m/z 161 by the identical daughter ion of sour reference substance
Body, caffeic acid isomer and caffeic acid (precise Identification).
ESI-MS spectrum of the compound 18 under anionic textiles mode generates the quasi-molecular ion peak of m/z 193.0495, and
Daughter ion identical with ferulic acid reference substance is produced in its ESI-MS/MS spectrum, such as [M-H-CO2]-、[M-H-CH3]-Equal spies
Thus its precise Identification is ferulic acid by sign property ion.Meanwhile the ESI-MS/MS fragment ion of compound 9 and ferulic acid phase
Seemingly, in conjunction with its molecular weight information, it is inferred as ferulic acid glucoside.
ESI-MS spectrum of the compound 6,12,14 under anionic textiles mode generates identical [M-H]-Ion (m/z
353.0867), molecular formula C16H17O9, error is respectively less than 5ppm.Neochlorogenic acid (3- is accredited as and comparing with reference substance
Caffeoylquinic acid), chlorogenic acid (5-Caffeoylquinic acid), Cryptochlorogenic acid (4-Caffeoylquinic
acid).And reference literature is reported, by comparing ESI-MS2The difference of fragment ion in spectrum, it can determine acylated on chinic acid
Position.It is, in general, that can all generate [Quinic acid-H] in 3- or 5- when acylated–Ion (m/z 191)
Base peak ion, important difference between the two are [Caffeic acid-H] when acylation location is at 3-–Ion (m/
Z179) abundance is high compared with 5-.And when acylation location is in 4-, [Quinic acid-H-H2O]-(m/z 173) is ESI-MS2Spectrum
Base peak ion.Whereupon it may be inferred that compound 6,12,14 be respectively neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, this and by with
The conclusion that reference substance obtains more afterwards is consistent.
Under anionic textiles mode, the ESI-MS spectrum of compound 20 and 21 generates identical [M-H]-Ion, matter
Accurate molecular weight (337.0928) error of amount number and such theoretical compound is respectively less than 5ppm.In their ESI-MS2Spectrum
It is middle to produce base peak ion m/z 163 and m/z 191 respectively.Bibliography, the MS of 3-pCoQA2Base peak is m/z 163 in spectrum,
The MS of 4-pCoQA2Base peak is m/z 173, the MS of 5-pCoQA in spectrum2Spectrum base peak is m/z 191, thus by compound 20 and 21 point
It is not accredited as 3-pCoQA (3- p-coumaroylquinic acids) and 5-pCoQA (5- p-coumaroylquinic acids).
ESI-MS spectrum of the compound 23,24 under anionic textiles mode generates identical [M-H]-Ion m/z
367.1024 deduction molecular formula is C17H19O9, quality error is below 5ppm.Meanwhile in their ESI-MS2It is produced respectively in spectrum
Base peak ion m/z 193 and m/z 191 is given birth to.Bibliography, the MS of 3-FQA2Base peak ion is m/z 193 in spectrum, 4-FQA's
MS2Base peak ion is m/z 173, the MS of 5-FQA in spectrum2Spectrum base peak ion is m/z 191, thus by compound 23 and 24 two
Compound is identified respectively as 3- feruloyl quinic acid and 5- feruloyl quinic acid.
Cynarin: ESI-MS spectrum of the compound 41,44,45 and 46 under anionic textiles mode generates m/
[M-H] of z 515.1184-Ion, thus it is speculated that their molecular formula is C25H23O12, quality error is below 5ppm.Meanwhile at it
ESI-MS2M/z 353 [CQA-H] is generated in spectrum-Ion, illustrating them is dicaffeoylquinic acid compound.Such
The ESI-MS/MS lytic pathway of compound is shown in Figure 13.From the point of view of the type and quantity of fragment ion, they are essentially identical, but
From the point of view of generated abundance of ions, then there is bigger difference between them.It, can be by chemical combination by being compared with reference substance
44,45,46 precise Identification of object is 3,4-DiCQA, 3,5-DiCQA and 1,5-DiCQA.Up to the present the two caffeoyl Kuis found
In Buddhist nun's acid compounds, with the polarity highest of 1,3-DiCQA, therefore it can be eluted first in reverse-phase chromatographic column, therefore
Compound 41 is accredited as 1,3-DiCQA.
(2) identification of iridoids chemical component
[M-H] can be observed in the ESI-MS spectrum under anionic textiles mode in compound 7-Ion m/z375.1295,
The molecular formula for speculating it is C16H23O10, quality error 2.40ppm.In its multi-stage ms, [M-H]-Ion produces [M-
H-glc]-(m/z 213)、[M-H-glc-CO2]-(m/z 169)、[M-H-H2O]-Fragment ions such as (m/z 357).According to its point
Son amount and cleaved fragment are accredited as 8- table vomiting nut acid in conjunction with compound reported in the literature.
(3) identification of benzyl carbinol glycoside chemical component
Through analyzing, compound 8,25,26,27,38 and 43 can be accredited as benzyl carbinol glycoside compound.With compound
25, for 26 and 27, illustrate the Structural Identification process of such compound.ESI-MS spectrum of the three under anionic textiles mode can
It generates [M-H]-Ion m/z 785.2509, m/z 785.2512, m/z785.2510, thus it is speculated that their molecular formula is
C35H45O20, quality error is respectively less than 5ppm.Three's isomer each other can pass through continuous neutral loss 162Da and generate m/
The fragment ion of z 623 and m/z 461.In conjunction with above-mentioned cracking law-analysing, and reference substance is combined to compare, it can be by these three chemical combination
The Structural Identification of object is echinacoside and its isomer.Similarly, compound 8, compound 38 and compound 43 also can by by
One identification.
(4) identification of saponin component
The identification of triterpene saponin chemical component: triterpenoid saponin is main effective component in ginseng crude drug.In the palace of the Qing Dynasty longevity
It can detecte the more constituents in peach pill.Method by being compared with standard items, can by compound 47,49,50,
57,58,61,66 and 79 it is identified respectively as Panax Notoginseng saponin R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rh1, ginseng soap
Glycosides Rb1, Ginsenoside Rc, ginsenoside Rd and ginsenoside Rg3.These compounds are most important in MS/MS cracking process
Characteristic fragmentation approach is [M-H]-Ion or [M+HCOO]-Ion successively loses the sugar unit connected on aglycon, generates a series of
Fragment ion, and obtain corresponding aglycon ion.By taking Ginsenoside Rc as an example, [M-H]-quasi-molecular ion (m/z
1077.5860) base peak ion is generated first by losing an arabinose residues on aglycon in its ESI-MS/MS spectrum
945 ion of m/z ([M-H-arab]-), and supervene 783 ion of m/z ([M-H-arab-glc]-)；783 ion of m/z into
One step sloughs glucose residue and generates m/z 621 ([M-H-arab-2glc]-) and ion m/z 459 ([M-H-arab-3glc]-)
Deng.Its possible lytic pathway is shown in Figure 14.In conjunction with cleavage of mass spectrum data reported in the literature, other such compounds can be carried out
Structure Deduction.
For example, the quasi-molecular ion [M-H] of compound 62-(m/z 955.4908), its molecular formula of deducibility are
C45H73O17, quality error 1.99ppm.In its ESI-MSnTwo molecule glucose residues are continuously lost in cracking process, are gone forward side by side
One step takes off H2O、CO2Deng generation m/z 793 [M-H-glc]-、m/z 613[M-H-2glc-H2O]-、m/z 569[M-H-2glc-
CO2-H2O]-With 523 [M-H-2glc-CO of m/z2-CO-2H2O]-Plasma.In conjunction with its retention time and bibliography, thus it is speculated that change
The structure for closing object 62 may be ginsenoside Ro.
For another example, the fragmentation pathways of compound 77 and compound 78 are almost the same, illustrate their isomers each other.They
Quasi-molecular ion m/z 783 in MS2In continuously lose glucose, generate m/z 621 [M-H-glc]-With 459 [M-H- of m/z
2glc]-Ion.In summary analysis and bibliography, deduction compound 77 are 20 (S)-ginsenoside Rgs3, compound 78 is 20
(R)-ginsenoside Rg3.Other triterpenoid saponins in ginseng and semen ziziphi spinosae medicinal material can also successively be reflected as a result,
It is fixed.
The identification of steroid saponin chemical component: steroid saponin is the characteristic chemical constituent in Radix Ophiopogonis medicinal material.Through analyzing, chemical combination
Object 56 and 70 belongs to such compound.By taking compound 56 as an example, [M-H] is generated in first mass spectrometric-Quasi-molecular ion (m/z
901.4807), infer that its molecular formula is C45H73O18, quality error 1.77ppm.In multi-stage ms fragmentation, first
The glucose residue on sugar chain is lost, is generated [M-H-Glc]-Ion (m/z 739), the rhamnose for hereafter losing connection again generate
[M-H-glc-rha]-Ion (m/z 593).This sequence consensus lost with steroid saponin compound glycosyl: glucose (rock
Algae sugar) rhamnose that the xyloses of 3 or 4 connections are always connected compared with 2 preferentially loses.Thus it is accredited as
Protobioside or its isomer.Similarly, compound 70 can also be accredited as partially sapogenin 3-O- [α-L- pyrans
Rhamnopyranosyl-(1 → 2)] [β-D- xylopyranosyl-(1 → 4)]-β-D- glucopyranose.
(5) identification of flavones ingredient
In the Shoutao pills of the palace of the Qing Dynasty contain a large amount of flavones ingredient, since flavones ingredient is relatively conventional, and document report compared with
More, this research mainly uses following method: (1) Literature Consult in qualification process, in each flavour of a drug of system summary palace of the Qing Dynasty Shoutao pills
Reported flavones ingredient；(2) high-resolution EIC data processing method is used, in the peach offered as a birthday present pill test solution of the palace of the Qing Dynasty
Chemical component carry out rapidly extracting；(3) the multi-stage ms fragmentation rule of each flavonoids candidate compound of careful analysis, is into one
It walks and determines that structure provides foundation；(4) reference substance Comparison Method is used, above-mentioned inferred results are further verified.For example, to change
For closing object 33, [M-H] is generated in first mass spectrometric-Quasi-molecular ion (m/z 609.1450) infers that its molecular formula is
C27H29O16, quality error is -0.82ppm.At its [M-H]-In the MS/MS cracking process of ion, quasi-molecular ion peak passes through
Neutral loss rue saccharide residue also produces aglycon ion m/z 301, and further generate respectively in cracking process m/z179 [1, 2A]-、m/z151[1,2A-CO]-、m/z193[M-H-B]-、m/z 107[0,4A]-Plasma: wherein m/z193 [M-H-B]-Show
There are two hydroxyls to replace on B ring for it, and m/z107 [0,4A]-Then illustrating aglycon, there are two hydroxyls to replace on A ring.Thus may be used
To infer compound 33 for rutin, this is consistent with the result compared using reference substance.And so on, to other flavonoids
It closes object and has carried out Structural Identification.
(6) identification of other constituents
In the peach offered as a birthday present pill of the palace of the Qing Dynasty, it was found that some other constituents, such as compound 1,10,31,37,48.This
A little compounds are mostly from fructus lycii medicinal material.In conjunction with document report, first mass spectrometric accurate mass number, the multistage of each compound are analyzed
Mass spectrometric fragment ion etc. infers their structure.
2 palace of the Qing Dynasty Shoutao pills blood plasma and urine effective constituents identification research
2.1 materials and instrument
Palace of the Qing Dynasty Shoutao pills coarse powder is provided by pharmaceutical factory；Mass spectrum level acetonitrile, mass spectrum grade methanol, mass spectrum grade formic acid are purchased from Fisher
Scientific (Fair Lawn, NJ, USA), ultrapure water are prepared by Millipore Synergy UV ultrapure water machine.
Thermo Scientific Accela 600pump UPLC system: including on-line degassing machine, autosampler,
High pressure binary gradient pump；HPLC passes through electrospray interface and LTQ-Orbitrap high-resolution mass spectrometer (Thermo
Scientific, Bremen, Germany) connection；2.1 work station of Thermo Xcalibur；KQ-250DE type numerical control supersonic
Washer (Chinese Kunshan Ultrasonic Instruments Co., Ltd.)；The Millipore Synergy UV type ultrapure water machine (U.S.
Millipore company)；R200D type electronic analytical balance (ten a ten thousandths, German Sartorius company)；Grace PureTM
SPE C18-Low solid phase extraction column (500mg/3mL)；21 microcentrifuge of FRESCO (U.S. Thermo Fisher
Scientific company)；TARGIN VX-II type multitube turbula shaker (Beijing Ta Jin Science and Technology Ltd.)；TTL-DCI type
Nitrogen evaporator (Beijing Tongtailian Technology Development Co., Ltd.)；50-200 μ L micropipettor, 200-1000 μ L micropipettor.
2.2 experimental method
2.2.1 prepared by solution
The preparation of palace of the Qing Dynasty Shoutao pills administration sample: palace of the Qing Dynasty Shoutao pills coarse powder 36g is taken, appropriate 0.5%CMC-Na solution is added
120ml is made into the sample solution that concentration is 300mg/mL, 4 DEG C of storages.
2.2.2 experimental animal and dosage regimen
Sprague Dawley (SD) rat, weight (220 ± 10) g, being purchased from Beijing dimension tonneau China experimental animal technology has
Limit company, credit number SCXK (capital) 2011-0004, raise in Beijing University of Chinese Medicine's animal house (SPF grades): 12h is followed round the clock
Ring, room temperature is constant at 22-24 DEG C, and relative humidity is maintained at 55-65%.
The collection of blood and urine specimen: 8 normal male rats adapt to raising one week under experimental situation.Before experiment,
Mouse is placed in metabolic cage, fasting 12 hours, free water was collected simultaneously blank diaper.Rat adapts to environment 3 days, freely
Diet drinking-water.Fasting 12 hours before being administered, when experiment, are administered with the dosage oral administration gavage of 1.25g/kg, twice daily, are continuously given
Medicine three days.Last time be administered after, each rat upon administration 0,0.5,1,2,4h respectively orbital vein take blood 0.5ml (0h acquisition
Blood sample is blank sample).Blood sample collected is placed in the centrifuge tube for filling heparin sodium, and 3500rpm is centrifuged 10min.Merge institute
There is supernatant, and is frozen in -80 DEG C of refrigerators.
Meanwhile the administration collected after gastric infusion for 24 hours is urinated.Period subsists and water.Merge all urine samples, with
After 3500rpm is centrifuged 10min, -80 DEG C of refrigerators of supernatant is taken to freeze.
2.2.3 the processing of Biological Samples of Rats
In order to identify the Metabolite in rat plasma and urine as much as possible, it is heavy that all biological samples are all made of methanol
Shallow lake method, acetonitrile precipitation method and SPE solid phase extraction column handle three kinds of methods.
(1) the acetonitrile precipitation processing method of Biological Samples of Rats
The processing method of blood sample: the blood sample that each group is frozen is put into thaw at room temperature, takes different group Contained Serums
And each 200 μ L of blank serum, it is separately added into 600 μ L acetonitriles, vortex oscillation 3min is centrifuged (14300r/min) 30min, after standing
Supernatant is taken, is dried up at room temperature with N2, the residue initial liquid phase of 100 μ L is redissolved, it is vortexed after concussion 3min, 14000rpm
It is centrifuged 15min, supernatant is taken to analyze into UPLC-LTQ-Orbitrap.
The processing method of urine sample: the urine specimen that each group is frozen is put into thaws at room temperature, and drug containing urine sample and blank is taken to urinate
Each 200 μ L of sample, is separately added into 600 μ L acetonitriles, and vortex oscillation 3min is centrifuged (14300r/min) 30min, takes supernatant after standing
Liquid is dried up with N2 at room temperature, and the residue initial liquid phase of 100 μ L is redissolved, and is vortexed after concussion 3min, 14000rpm centrifugation
15min takes supernatant to analyze into UPLC-LTQ-Orbitrap.
(2) the methanol extraction processing method of Biological Samples of Rats
The processing method of blood sample: the blood sample that each group is frozen is put into thaw at room temperature, takes different group Contained Serums
And each 200 μ L of blank serum, it is separately added into 600 μ L acetonitriles, vortex oscillation 3min is centrifuged (14300r/min) 30min, after standing
Supernatant is taken, uses N at room temperature2Drying, the residue initial liquid phase of 100 μ L are redissolved, are vortexed after concussion 3min, 14000rpm
It is centrifuged 15min, supernatant is taken to analyze into UPLC-LTQ-Orbitrap.
The processing method of urine sample: the urine specimen that each group is frozen is put into thaws at room temperature, and drug containing urine sample and blank is taken to urinate
Each 200 μ L of sample, is separately added into 600 μ L acetonitriles, and vortex oscillation 3min is centrifuged (14300r/min) 30min, takes supernatant after standing
Liquid uses N at room temperature2Drying, the residue initial liquid phase of 100 μ L are redissolved, are vortexed after concussion 3min, 14000rpm centrifugation
15min takes supernatant to analyze into UPLC-LTQ-Orbitrap.
(3) the SPE processing method of Biological Samples of Rats
The processing method of blood sample: taking GracePureTM SPE solid phase extraction column, first activates pillar with the methanol of 5mL, after
The deionized water balance solid phase extraction column of 5mL is added.The plasma sample of 1mL is placed in the solid phase extraction column activated, so
Impurity is rinsed with 2mL water afterwards, is then eluted with 3mL methanol, meoh eluate is collected.By above-mentioned all meoh eluates, in room
It is dried up under temperature with N2, the residue initial liquid phase of 100 μ L is redissolved, and is vortexed after concussion 3min, and 14000rpm is centrifuged 15min, takes
Supernatant is analyzed into UPLC-LTQ-Orbitrap.
The processing method of urine sample: the urine specimen that each group is frozen is put into be dissolved at room temperature, is taken the urine of 2mL to be placed in and has been lived
Then the solid phase extraction column of change rinses impurity with 3mL water, is then eluted with 3mL methanol, collects meoh eluate.It will be above-mentioned
All meoh eluates, are dried up with N2 at room temperature, and the residue initial liquid phase of 100 μ L is redissolved, and are vortexed after concussion 3min,
14000rpm is centrifuged 15min, and supernatant is taken to analyze into UPLC-LTQ-Orbitrap.
2.2.4 testing conditions
UPLC condition: with Waters ACQUITY UPLC HSS T3 (2.1 × 100mm, 1.8 μm) for chromatographic column；With
0.1% formic acid is mobile phase A, using acetonitrile as Mobile phase B, gradient elution；Room temperature；Sample volume is 3 μ L, flow velocity 0.3mL/min.
Gradient are as follows: 0-2min, 8%B；2-20min, 8-26%B, 20-22min, 26%B；22-30min, 26-42%B；30-
50min, 42-60%B；50-55min, 60-95%B；55-58min, 95%B, 58-60min, 95-8%B；60-65min, 8%
B。
MS condition: negative ion mode, 350 DEG C of capillary temperature, sheath gas (nitrogen) 30arb, secondary air speed (nitrogen
Gas) 10arb, spray voltage: 3kV, capillary voltage: -35V, pipe lens voltage: -110V.High resolution mass spectrum quality axis accuracy
It is molten using caffeine, lauryl sodium sulfate, natrium taurocholicum, tetrapeptide MRFA and Ultramark hybrid standard product
Liquid is corrected, and Mass accuracy error is within 5ppm.Quality testing range: m/z 100-1200；Sample using full scan-from
The mode that sublist-dynamic excludes (Full scan-Parent ions list-Dynamic exclusion, FPD) scans,
With high-resolution FT carry out full scan (Full scan, FS), resolution ratio R is set as 30000, with ion trap dynode detection fragment from
Son.Setting dynamic excludes (Dynamic exclusion, DE), number of repetition (Repeat count): 3, the repetition time
(Repeat duration): 10s is excluded inventory size (Exclusion list size): 100, exclude the time
(Exclusion duration): 20s.Data collection system is Xcalibur 2.1.
2.3 experimental result
2.3.1 after being administered in rat plasma effective constituents identification
In order to preferably identify internal effective constituents, we mainly use three kinds not in the treatment process of plasma sample
With processing method: acetonitrile precipitation method, methanol extraction method and solid phase extraction, thus guarantee detect effective constituents can
By property and comprehensive.
The identification of blood plasma effective constituents after rat oral gavage is mainly acquiring high-resolution using UPLC-LTQ-Orbitrap
After mass spectrometric data, Figure 15-17 is seen.Pass through comparison administration rat plasma and palace of the Qing Dynasty Shoutao pills original preparation, blank plasma data, screening
Identify relevant internal effective constituents.
Finally, from identifying effective constituents totally 20 after administration in rat plasma, 2 are shown in Table.
Effective constituents qualification result in rat plasma is administered in 2 palace of the Qing Dynasty Shoutao pills of table
2.3.2 after being administered in rat urine effective constituents identification
In order to preferably identify internal effective constituents, we are also main in the treatment process of urine sample to use three kinds
Different processing methods: acetonitrile precipitation method, methanol extraction method and solid phase extraction, to guarantee the effective constituents detected
Reliability and comprehensive.
The identification of urine effective constituents after rat oral gavage is mainly acquiring high-resolution using UPLC-LTQ-Orbitrap
After mass spectrometric data, Figure 18-20 is seen.Pass through comparison administration rat urine and palace of the Qing Dynasty Shoutao pills original preparation, blank diaper data, screening
Identify relevant internal effective constituents.
Finally, from identifying effective constituents totally 38 after administration in rat urine, 3 are shown in Table.
Effective constituents qualification result in rat urine is administered in 3 palace of the Qing Dynasty Shoutao pills of table
Chromatography is carried out to each constituents in the Shoutao pills of the palace of the Qing Dynasty using UPLC-LTQ-Orbitrap high-resolution LC-MS instrument
The Structural Identification of separation, mass spectrometric data acquisition and proper constituent, finally identifies triterpenoid saponin, Huang from the peach offered as a birthday present pill of the palace of the Qing Dynasty
Each constituents such as ketone, steroid saponin, iridoid, benzyl carbinol glycosides totally 84.By the coarse powder of palace of the Qing Dynasty Shoutao pills with 1.25g/kg's
Dosage is to Sprague Dawley Oral Administration in Rats gastric infusion, twice daily, successive administration three days, takes the urine of serum and accumulation
Metabolite detection is carried out, effective constituents 20 are as a result identified from the rat plasma after successive administration, after successive administration
Identify that effective constituents 38 tentatively illustrate the material base of palace of the Qing Dynasty Shoutao pills in rat urine, further to study its drug effect object
Matter basis provides foundation and premise.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of effective substance research method of palace of the Qing Dynasty Shoutao pills, characterized by the following steps:
(1) LC-MS is carried out for palace of the Qing Dynasty Shoutao pillsnGlobal analysis carries out Structural Identification to wherein main chemical component, determines
Series components type therein；
(2) the LC-MS analysis method for establishing series components respectively, carries out quick Structural Identification.
2. the effective substance research method of palace of the Qing Dynasty Shoutao pills according to claim 1, it is characterised in that: the LC-
The preparation of sample solution includes the following steps: in MS analysis method
(1) preparation of preparation test solution: taking palace of the Qing Dynasty Shoutao pills coarse powder 0.3g to be placed in 50mL conical flask, add methanol 25mL,
Weighing, room temperature are ultrasonically treated 30min, and standing is cooled to room temperature, weighs again, and methanol is added to supply the weight of less loss, cross 0.22 μm
Miillpore filter, take subsequent filtrate to get；
(2) preparation of each flavour of a drug test solution: taking each flavour of a drug of palace of the Qing Dynasty Shoutao pills, crushes, and crosses No. four sieves, weighs 0.8g and be placed in
In 50mL conical flask, add methanol 10mL, weigh, room temperature is ultrasonically treated 30min, and standing is cooled to room temperature, weighs again, adds methanol
Supply the weight of less loss, cross 0.22 μm of miillpore filter, take subsequent filtrate to get；
(3) preparation of standard solution: taking above-mentioned reference substance appropriate respectively, adds methanol that every 1mL about 50 μ g containing each reference substance is made
Solution, under the conditions of 4 DEG C sealing be kept in dark place to get.
3. the effective substance research method of palace of the Qing Dynasty Shoutao pills according to claim 1, it is characterised in that: the LC-
UPLC condition is as follows in MS analysis method:
With T3,2.1 × 100mm, 1.8 μm of Waters ACQUITY UPLC HSS for chromatographic column；Using 0.1% formic acid as mobile phase
A, using acetonitrile as Mobile phase B, gradient elution, gradient are as follows: 0-2min, 8%B；2-20min, 8-26%B, 20-22min,
26%B；22-30min, 26-42%B；30-50min, 42-60%B；50-55min, 60-95%B；55-58min, 95%B,
58-60min, 95-8%B；60-65min, 8%B；Room temperature；Sample volume is 3 μ L, flow velocity 0.3mL/min.
4. the effective substance research method of palace of the Qing Dynasty Shoutao pills according to claim 1, it is characterised in that: the LC-
MS condition is as follows in MS analysis method:
Negative ion mode, 350 DEG C of capillary temperature, sheath gas (nitrogen) 30arb, secondary air speed 10arb, spray voltage:
3kV, capillary voltage: -35V, pipe lens voltage: -110V；
High resolution mass spectrum quality axis accuracy uses caffeine, lauryl sodium sulfate, natrium taurocholicum, tetrapeptide
MRFA and Ultramark hybrid standard product solution is corrected, and Mass accuracy error is within 5ppm；
Quality testing range: m/z 100-1200；Sample is scanned by the way of full scan, carries out full scan with high-resolution FT,
Resolution ratio R is set as 30000, detects fragment ion with ion trap dynode；
Setting dynamic excludes, number of repetition: 3, the repetition time: and 10s, exclusion inventory size: 100, exclude the time: 20s.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112750509A (en) * | 2019-10-29 | 2021-05-04 | 昆药集团股份有限公司 | Establishment method of basic database of Xuesaitong injection material |
| CN115887593A (en) * | 2022-11-22 | 2023-04-04 | 津药达仁堂集团股份有限公司达仁堂制药厂 | Application of Qinggong Shoutao pills in preparation of autoimmune regulation medicine |
| CN117250300A (en) * | 2023-11-17 | 2023-12-19 | 江西中医药大学 | Method for detecting multiple components in Tibetan medicine twenty-five-flavor big soup pill |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101398417A (en) * | 2008-10-29 | 2009-04-01 | 中国药科大学 | Universal method for rapid detection and structural identification for Chinese traditional medicine complex component |
| CN102048990A (en) * | 2009-11-03 | 2011-05-11 | 天津中新药业集团股份有限公司达仁堂制药厂 | Quality control method for Chinese medicinal preparation Qinggong Shoutao pills |
| CN106324120A (en) * | 2016-08-09 | 2017-01-11 | 西藏藏医学院 | Volatile component measuring method for Tibetan medicine heracleum millefolium diels |
| CN107389813A (en) * | 2017-07-10 | 2017-11-24 | 北京中医药大学 | Rascal, dried orange peel, the dried immature fruit of citron orange and the method for Fructus Aurantii are differentiated based on chemical classification and UPLC Tof MS |
-
2017
- 2017-12-27 CN CN201711447051.9A patent/CN109100431B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101398417A (en) * | 2008-10-29 | 2009-04-01 | 中国药科大学 | Universal method for rapid detection and structural identification for Chinese traditional medicine complex component |
| CN102048990A (en) * | 2009-11-03 | 2011-05-11 | 天津中新药业集团股份有限公司达仁堂制药厂 | Quality control method for Chinese medicinal preparation Qinggong Shoutao pills |
| CN106324120A (en) * | 2016-08-09 | 2017-01-11 | 西藏藏医学院 | Volatile component measuring method for Tibetan medicine heracleum millefolium diels |
| CN107389813A (en) * | 2017-07-10 | 2017-11-24 | 北京中医药大学 | Rascal, dried orange peel, the dried immature fruit of citron orange and the method for Fructus Aurantii are differentiated based on chemical classification and UPLC Tof MS |
Non-Patent Citations (4)
| Title |
|---|
| JOÃO CARLOS LIMA RODRIGUES PITA 等: "Matrix effect and optimization of LC–MSn determination of trachylobane-360 in mice blood", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 刘思燚 等: "UHPLC-LTQ-Orbitrap鉴定苦碟子化学成分及其在大鼠血浆中的移行成分", 《中国中药杂志》 * |
| 张加余 等: "清火栀麦片中化学成分的SPE-HPLC-ESI-MSn快速识别与鉴定", 《中国中药杂志》 * |
| 韩彦琪 等: "HPLC-QTOF/MS方法分析元胡止痛方的化学成分", 《药学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112750509A (en) * | 2019-10-29 | 2021-05-04 | 昆药集团股份有限公司 | Establishment method of basic database of Xuesaitong injection material |
| CN115887593A (en) * | 2022-11-22 | 2023-04-04 | 津药达仁堂集团股份有限公司达仁堂制药厂 | Application of Qinggong Shoutao pills in preparation of autoimmune regulation medicine |
| CN115887593B (en) * | 2022-11-22 | 2024-03-26 | 津药达仁堂集团股份有限公司达仁堂制药厂 | Application of qinggongshou peach pill in preparing medicine for regulating autoimmune property |
| CN117250300A (en) * | 2023-11-17 | 2023-12-19 | 江西中医药大学 | Method for detecting multiple components in Tibetan medicine twenty-five-flavor big soup pill |
| CN117250300B (en) * | 2023-11-17 | 2024-02-20 | 江西中医药大学 | Method for detecting multiple components in Tibetan medicine twenty-five-flavor big soup pill |
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Address after: No.21, 10th Street, Binhai New Area Development Zone, Tianjin 300457 Patentee after: Jinyao Darentang Group Co.,Ltd. Darentang Pharmaceutical Factory Address before: No.21, 10th Street, Binhai New Area Development Zone, Tianjin 300457 Patentee before: DARENTANG PHARMACEUTICAL FACTORY, TIANJIN ZHONGXIN PHARMACEUTICAL GROUP Co.,Ltd. |