CN107991399A - Method that is a kind of while measuring 31 kinds of components in compound Salviae Miltiorrhizae extract or related medicinal material - Google Patents
Method that is a kind of while measuring 31 kinds of components in compound Salviae Miltiorrhizae extract or related medicinal material Download PDFInfo
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Abstract
The present invention relates to method that is a kind of while measuring 31 kinds of components in compound Salviae Miltiorrhizae extract or related medicinal material, the method is using reverse phase hydrophilic action chromatography mass spectrometry combination method of directly connecting, and described method includes following steps：The preparation of step 1 sample solution；The preparation of step 2 standard serial solution；The preparation of step 3 inner mark solution；The preparation of step 4 test solution；Step 5 takes step 4 resulting solution injection directly series connection reverse phase hydrophilic effect liquid chromatography mass combined system, obtains the extraction ion flow graph of 31 kinds of analytes, the content of 31 kinds of components in compound Salviae Miltiorrhizae extract or related medicinal material is calculated according to extraction ion flow graph.
Description
Technical field：
It is more particularly to a kind of to measure compound Danshen Root extraction at the same time the present invention relates to a kind of detection method of effective component of chinese medicine
The method of 31 kinds of components in thing or related medicinal material.
Background technology：
The research of mass balance is of great significance for illustrating the composition of Chemistry for Chinese Traditional Medicine material group.Chemistry for Chinese Traditional Medicine material group
Into extremely complex, contain numerous nascent metabolite and secondary metabolite.
Early-stage study shows that compound danshen dripping pills (is made, specific preparation method is shown in of Radix Salviae Miltiorrhizae, pseudo-ginseng, borneol《Middle traditional Chinese medicines
Allusion quotation》Version in 2015) mainly contain oligosaccharides, amino acid, nucleosides, organic acid, phenolic acid, saponin(e and tanshinone compound.Due to this
The structure of a little compounds, physical chemical differences are big, and researchers usually more uses various analysis to different types of
Compound carries out quantitative analysis, such as detects amino acid using amino-acid analyzer, uses HPLC ELSD
Detector detects oligosaccharides and saponin(e, using HPLC- UV detection device or liquid chromatography mass combined instrument to phenolic acid and
Tanshinone.The application of various analysis brings very big inconvenience for the research of mass balance.
Reverse-phase chromatography：Mobile phase polarity is more than the situation of stationary phase polarity, is known as reverse-phase chromatography.Non-polar linkage phase chromatography
It can make reverse-phase chromatography.It is most widely used in modern liquid chromatography, modern liquid chromatography analysis work 70% above is non-
Carried out in polar binding stationary phase.
Hydrophilic Interaction Chromatography：It can be counted as the continuity in normal-phase chromatography hydrotropism's mobile phase field.Mobile phase is that water mutually delays
Fliud flushing (＜ 40%) and organic solvent.Stationary phase is the polar adsorbent of strongly hydrophilic, and such as silica gel bonded phase, polar polymer are filled out
Material or ion exchange absorbent.The common feature of these stationary phases is that they are very strong with the active force of water, is consequently belonging to " hydrophilic
Property ".The gradient and rp mode used is opposite.Primary condition is including organic phase, typical concentration are 95% organic phases at high proportion
Such as acetonitrile, water phase is stepped down to.Therefore, be otherwise known as anti-reverse-phase chromatography.
Direct series connection-anti-phase-hydrophilic Interaction Chromatography mass spectrometry system：The chromatographic column of two kinds of clastotypes is directly gone here and there
Connection so that can preferably be retained from highly polar to the material of low pole, so as to reach separated purpose.
Compound Salviae Miltiorrhizae extract or related medicinal material are existing medicine, are prepared by pseudo-ginseng, Radix Salviae Miltiorrhizae and borneol by processing.
With promoting blood circulation and removing blood stasis, the effect of regulating qi-flowing for relieving pain.For the obstruction of qi in the chest of caused by energy stagnation and blood stasis, symptoms include uncomfortable in chest, pareordia shouting pain；Coronary heart disease
Angina pectoris is shown in above-mentioned patient.
Compound Salviae Miltiorrhizae extract of the present invention or related medicinal material include commercially available compound red sage root preparation, such as composite salvia dropping
Ball or Fufang Danshen Pian, common extract also including pseudo-ginseng and Radix Salviae Miltiorrhizae or extract the extract remixed, the extraction respectively
Thing is commercially available, such as compound Danshen Root medicinal extract, Radix Salviae Miltiorrhizae extractum, pseudo-ginseng medicinal extract, also refers to preparation method system of the prior art
It is standby to obtain, also including pseudo-ginseng and red rooted salvia, such as Danshen Root and Notoginseng Root.
Compound Salviae Miltiorrhizae extract of the present invention or related medicinal material, 31 kinds of active ingredient therein are respectively：Adenine, amber
Amber acid, pyroglutamic acid, adenosine, uridine, danshensu, valine, lysine, proline, protocatechualdehyde, glucose, sucrose, smart ammonia
Acid, salvianolic acid D, notoginsenoside R, ginsenoside Rg1, ginsenoside Re, Rosmarinic acid, trisaccharide, alkannic acid, tanshin polyphenolic acid B, pellet
Phenolic acid A, ginsenoside Rb1, salvianolic acid C, ginsenoside Rg2, ginsenoside Rh 1, ginsenoside Rd, tetrose, Cryptotanshinone, pellet
Join ketone I, tanshinone IIA.
For the deficiency of existing mass balance research method, this research establishes direct series connection-anti-phase-hydrophilic Interaction Chromatography
7 major classes in compound Salviae Miltiorrhizae extract or related medicinal material, 31 kinds of components can measure at the same time by mass spectrometry system.
The present invention establishes direct series connection-anti-phase-hydrophilic Interaction Chromatography mass spectrometry system, to compound Salviae Miltiorrhizae extract or
7 major classes of related medicinal material neutrality matter very different, 31 kinds of components are measured, and can be applied to compound Salviae Miltiorrhizae extract or Related Drug
The quality evaluation of material, has the characteristics that comprehensively, accurately and rapidly.
The content of the invention：
The present invention provides method that is a kind of while measuring 31 kinds of components in compound Salviae Miltiorrhizae extract or related medicinal material, the party
Method is using direct series connection-anti-phase-hydrophilic Interaction Chromatography mass spectrometry.
Method provided by the invention comprises the following steps：
The preparation of step 1 sample solution：Compound Salviae Miltiorrhizae extract or related medicinal material are taken, methanol solution is configured to methanol；
The preparation of step 2 standard serial solution：The standard items of 31 kinds of components are taken respectively, and methanol solution is configured to methanol；
The preparation of step 3 inner mark solution：Take internal standard compound L-lysine -4,4,5,5-d4, diazepam and chloramphenicol, use respectively
Methanol is configured to methanol solution；
The preparation of step 4 test solution：The inner mark solution of step 1 or step 2 resulting solution respectively with step 3 is mixed
Close；
Step 5 takes step 4 gained test solution to inject anti-phase-hydrophilic interaction liquid chromatography mass combination system of directly connecting
System, obtains the extraction ion flow graph of 31 kinds of analytes, and compound Salviae Miltiorrhizae extract or related medicinal material are calculated according to extraction ion flow graph
In 31 kinds of components content.
The order of above-mentioned steps 1-4 is not the limitation in time sequencing, can phase double replacement.
In the above method, the compound Salviae Miltiorrhizae extract or related medicinal material are selected from：Compound Danshen Root medicinal extract, Radix Salviae Miltiorrhizae extractum, three
Seven medicinal extract, Radix Salviae Miltiorrhizae or pseudo-ginseng.
Preferably, the step 1：Sample solution is prepared as：Compound Danshen Root medicinal extract, Radix Salviae Miltiorrhizae extractum, pseudo-ginseng leaching are taken respectively
Cream, Danshen Root or Notoginseng Root 0.10g, accurately weighed, addition 60-80% methanol 8-12mL, are ultrasonically treated 20-40min, press
8-12min is centrifuged according to 16,000rpm, crosses 0.22 μm of filter membrane, all solution are maintained in 4 DEG C of refrigerators before analysis；
Further preferably, step 1：Sample solution is prepared as：Compound Danshen Root medicinal extract, Radix Salviae Miltiorrhizae extractum, pseudo-ginseng leaching are taken respectively
Cream, Danshen Root and Notoginseng Root 0.10g, it is accurately weighed, 70% methanol 10mL is added, is ultrasonically treated 30min, according to 16,
000rpm centrifuges 10min, crosses 0.22 μm of filter membrane, and all solution are maintained in 4 DEG C of refrigerators before analysis；
Preferably, step 2：Standard serial solution is prepared as：It is appropriate to weigh determinand standard items, it is accurately weighed, respectively
Add 60-80% methanol-waters and the solution that concentration is 450-550 μ g/mL is made, it is accurate respectively to measure as each determinand storing solution
Each determinand storing solution is appropriate, and the tetrose, concentration 50000ng/mL that concentration is 70000ng/mL is made with 60-80% methanol-waters
Sucrose, concentration be 12000ng/mL tanshin polyphenolic acid B, concentration be 8000ng/mL ginsenoside Rg1, concentration 6000ng/mL
Glucose, concentration be 4000ng/mL trisaccharide, concentration be 4000ng/mL ginsenoside Rb1, concentration be 4000ng/mL
Rosmarinic acid that pyroglutamic acid that ginsenoside Rd, concentration are 2000ng/mL, concentration are 2000ng/mL, concentration 2000ng/
The Panax Notoginseng saponin R of mL1Hybrid standard product storing solution with concentration for remaining determinand of 1000ng/mL；Hybrid standard product are taken to store up
Standby liquid is appropriate, is made serial dense according to extension rate 2,5,10,20,50,100,200,500 and 1000 using 60-80% methanol
Mixed standard solution is spent, 4 DEG C of refrigerators save backup；
Further preferably, step 2：Standard serial solution is prepared as：It is appropriate to weigh determinand standard items, is separately added into
The solution that concentration is 500 μ g/mL is made in 70% methanol-water, accurate respectively to measure each determinand storage as each determinand storing solution
Standby liquid is appropriate, and sucrose, concentration that tetrose, concentration that concentration is 70000ng/mL are 50000ng/mL, which is made, with 70% methanol-water is
The tanshin polyphenolic acid B of 12000ng/mL, the ginsenoside Rg that concentration is 8000ng/mL1, concentration be the glucose of 6000ng/mL, concentration
The ginsenoside Rb that trisaccharide, concentration for 4000ng/mL are 4000ng/mL1, ginsenoside Rd, dense that concentration is 4000ng/mL
Spend the Panax Notoginseng saponin R that the pyroglutamic acid for 2000ng/mL, the Rosmarinic acid that concentration is 2000ng/mL, concentration are 2000ng/mL1
Hybrid standard product storing solution with concentration for remaining determinand of 1000ng/mL；Take hybrid standard product storing solution appropriate, use
Series concentration mixed standard solution is made according to extension rate 2,5,10,20,50,100,200,500 and 1000 in 70% methanol, and 4
DEG C refrigerator saves backup；
Preferably, step 3：Inner mark solution is prepared as：Weigh L-lysine -4,4,5,5-d4(IS1), diazepam
(IS2) and chloramphenicol (IS3) in right amount, accurately weighed, it is 450-550 μ g/mL to be separately added into 60-80% methanol-waters and concentration is made
Solution, as each internal standard storing solution, accurate each internal standard storing solution of measurement is appropriate respectively, and each internal standard concentration is made with 70% methanol-water
For the IS reference substance solutions of 100ng/mL, 4 DEG C of refrigerators save backup；
Further preferably, step 3 is：The preparation of inner mark solution：Weigh L-lysine -4,4,5,5-d4(IS1), diazepam
(IS2) and chloramphenicol (IS3) in right amount, it is accurately weighed, be separately added into 70% methanol-water be made concentration be 500 μ g/mL solution, make
For each internal standard storing solution, accurate each internal standard storing solution of measurement is appropriate respectively, and each internal standard concentration, which is made, with 70% methanol-water is
The IS reference substance solutions of 100ng/mL, 4 DEG C of refrigerators save backup；
Preferably, step 4：Test solution is prepared as：Each concentration mixing reference substance of accurate measurement equivalent series is molten respectively
Liquid or sample solution are mixed with IS reference substance solutions, and the serial mixed reference substance solution or sample solution of containing the internal standard, 4 DEG C of ice are made
Case saves backup, and serial mixed reference substance solution is inner mark solution extension rate 2,4,10,20,40,100,200,400,1000
With 2000 solution, the volume ratio that sample solution is mixed with IS reference substance solutions is 1-10: 1-10；
Further preferably, step 4：Test solution is prepared as：It is accurate respectively to measure each concentration mixing control of equivalent series
Product solution or sample solution are mixed with IS reference substance solutions, the serial mixed reference substance solution or sample solution of obtained containing the internal standard, and 4
DEG C refrigerator saves backup, serial mixed reference substance solution is inner mark solution extension rate 2,4,10,20,40,100,200,400,
1000 and 2000 solution, sample solution are mixed with IS reference substance solutions for equal proportion；
Preferably, step 5：Above-mentioned test solution injecting chromatograph is taken, obtains chromatogram, compound is calculated according to chromatogram
The content of 31 kinds of components, wherein content are calculated by internal standard method in Salvia root P.E or related medicinal material；Wherein chromatographic condition is such as
Under：
Reverse-phase chromatographic column uses the chromatographic column of sub- 2 μm of C18 fillers, and guard column uses Waters pot strainers (2 μm),
Mobile phase is made of 0.08-0.15% formic acid or acetic acid water (A1) and acetonitrile (B1), and Gradient program is：0min, 25%B；
0.5min, 25%B；1.0min, 35%B；5.0min, 60%B；10.0min 80%B；15.0min, 80%B, flow velocity 0.1-
0.12mL/min, 25-45 DEG C of column temperature, sample size 1-3 μ L；
Chromatographic column with hydrophilic function uses Waters pot strainers (2 μm) using sub- 2 μm of hydrophilic chromatographic columns, guard column, flows
Dynamic to be mutually made of 0.08-0.15% formic acid or acetic acid water (A2) and acetonitrile (B2), Gradient program is：0min, 97%B；
10.0min 50%B；10.5min 50%B；11.0min 97%B；15.0min 97%B；Flow velocity 0.35-0.45mL/min,
25-45 DEG C of column temperature；Reverse-phase chromatographic column is connected with chromatographic column with hydrophilic function by a threeway；
The Mass Spectrometry Conditions are as follows：
It is connected using electro-spray ionization (ESI) interface with chromatographic column with hydrophilic function, cation capillary voltage is arranged to
5500V, anion capillary voltage are arranged to -4500V；Ion source temperature is arranged to 550 DEG C；Atomization gas, heating gas and gas curtain
Gas uses high-purity nitrogen, and it is respectively 55,55 and 25psi to set pressure, and combine time-histories using negative ions switching reacts prison more
Survey (MRM) method quantitative analysis determinand.
The reverse-phase chromatographic column is preferably Shim-Pack XR-ODS III (1.6 μm, 2.0mm × 50mm) or Waters
Acquity BEH C18 (1.7 μm, 2.1 × 50mm).
The hydrophilic chromatographic column preferred amide chromatographic column, more preferably Waters Acquity BEH
Amide (1.7 μm, 2.1mm × 100mm).
Explain：IS reference substance solutions are corresponding L-lysine -4,4,5,5-d4(IS1), diazepam (IS2) and chloramphenicol
(IS3) reference substance solution.
Method provided by the invention is obtained by a large amount of screening experiments：
1st, 31 kinds of components in detection compound Salviae Miltiorrhizae extract and medicine, proportionately sub-category point：Amino acid, nucleotide, have
Machine acid, polysaccharide belong to nascent metabolite, and danshinolic acid, saponin(e, tanshinone belong to secondary metabolite.
Polarity size is sequentially from small to large：Tanshinone, danshinolic acid, saponin(e, organic acid, nucleosides, amino acid, sugar are (no
It is absolute size order, is simply probably arranged according to property sequentially may have with this according to the particularity of respective material
Enter).
It is equal per the most general method of one kind material in view of the otherness of the ready availability property between material of instrument and equipment
Differ, but the purpose of this experiment is to disclose the mass balance that Chinese medicine forms, and the result between each measuring each respectively is not
Possess comparativity, therefore, need to consider to detect at the same time.
2nd, the selection of the method for the present invention：Reverse phase hydrophilic chromatography in series technology is applicable to the chemical combination that separating polar differs greatly
Thing, does not apply it to the report in terms of compound Salviae Miltiorrhizae extract or pseudo-ginseng, Radix Salviae Miltiorrhizae at present.
Inventor's first Application is in the field of compound Salviae Miltiorrhizae extract and related medicinal material.Seven constituents that this experiment is chosen
The main component (mass ratio accounts for more than 50% beyond moisture removal) belonged in compound Salviae Miltiorrhizae extract, and common quality control
Index processed, wherein oligosaccharides, amino acid, nucleoside compound belong to the larger component of polarity, if individually preferably selecting parent if measure
Water action chromatography, but pair do not retain in hydrophilic Interaction Chromatography with middle polarity or nonpolar material, it is unsuitable for analyzing.Phase
Over the ground, selected organic acid, phenolic acid, saponin(e and tanshinone compound, preferably using reverse-phase chromatography.In view of time economy into
This, if wanting the so complicated component of polarity difference reaching good separating effect, need to use reverse phase hydrophilic chromatography in series technology.
Not interfereing with each other mutually between substance-measuring plus tandem mass spectrum MRM patterns.
3rd, the difficult point of anti-phase-hydrophilic detection method：
1) for detection object：When being detected for Chinese medicine, multi-purpose greatly is Vavle switching method, according to appearance time,
Hydrophilic post separation is switched to anti-phase post separation, unsuitable part in the part of suitable reverse phase separation, can only be combined at last
Rather than full two dimension series connection, and problem caused by full two dimension series connection is that reverse phase hydrophilic column shares same flow visualizing.For
Detection object it is different, testing result also slightly influences, and such as is detecting organic acid, saponin(e, oligosaccharides, amino acid, nucleosides material
When, test result indicates that this few class material is in newly-established train, with each individually preferably compared with chromatographic column, peak shape
Do not influence, be only to have certain difference on appearance time, belong to normal phenomenon；For liposoluble ingredient, compare
In exclusive use reversed-phase column, situation about slightly trail, but this case is had by the acid concentration of additive in mobile phase and changed
It is kind；For tanshinone component, since it retains most by force in reversed-phase column, so last is eluted in reversed-phase column,
But it needs just be eluted out in the case of organic phase at high proportion in hydrophilic column, and the actual elution side according to hydrophilic column
Formula should be from the ratio of high organic phase, to organic phase：Water phase/50: 50 ratio carries out gradient elution.Inventor has found to wait until pellet
After ginseng ketone is eluted out from the first dimension reversed-phase column, Radix Salviae Miltiorrhizae has not been met to mobile phase ratio when the second dimension hydrophilic column
The appearance condition of ketone, it is thus possible to disperse or non-appearance occur.For this case, the solution that inventor uses is root
According to appearance time, ensureing that other materials all after normal appearance, improve the ratio of the second hydrophilic column mobile phase organic phase of dimension
Example, makes tanshinone normally retain, result of the test meets expection.Summarize：For apolar substance, due to full two-dimentional system, there are
It is one-dimensional after elution and with second dimension after run mobile phase ratio uncoordinated situation, the above method can be taken to be groped.
2) equilibration time：Due to being the system of full two dimension series connection and accurate quantitative analysis.Therefore equilibration time will more independent chromatography
Column is longer, the situation of appearance time drift otherwise occurs, depending on specific time length is because of situation.
During the 3rd, anti-phase-hydrophilic chromatographic combination, it is desirable to which meeting the sample of Mass Spectrometer Method condition (cannot contain PEG, surface-active
Agent etc. cannot be into mass spectrographic material presence) it can be detected in theory.And pass through discussion above, danshinolic acid and pellet
Ginseng letones have certain difficulty when using this method, investigate and prove by many experiments, are finally determined that one is fitted
For compound Salviae Miltiorrhizae extract, anti-phase-hydrophilic method for combined use of three kinds of samples of red rooted salvia and pseudo-ginseng, in this experiment condition
Under, by methodology validation, accurate quantitative analysis work can be carried out.Other similar samples are needed after being verified again to method,
Also can be measured with the method.
Specific screening process is as follows：
First, the selection of chromatographic condition, directly series connection reverse-phase chromatography and the hydrophilic Interaction Chromatography system established have to examine
Consider solvent compatibility and the order of connection.
Weak eluting solvent in reverse-phase chromatography is strong eluting solvent in hydrophilic Interaction Chromatography, and vice versa.Solvent is compatible
Sex chromosome mosaicism can be solved by the method for on-line dilution, i.e. the first root chromatogram column carries out small flow velocity elution, and the second root chromatogram column carries out
Big flow velocity elution (3-8 times) so that the mobile phase into the second root chromatogram column is compatible with chromatographic.Since two root chromatogram columns are straight
Capable series connection is tapped into, system pressure can have a distinct increment.Compared with reverse-phase chromatographic column, chromatographic column with hydrophilic function is due to the use of at high proportion
Organic phase is eluted, lower in high flow rate lower prop pressure.Meanwhile the Ionization Efficiency higher of organic phase at high proportion, this is conducive to
Lift the sensitivity of analysis method.In summary consider, tested using the combination of anti-phase-hydrophilic Interaction Chromatography.
Secondly, chromatographic column is investigated
The optimization selection of chromatographic column specification and filler, variable, need to will flow equal factor and fix in order to control.Chromatographic column has
The selection of machine phase is common for acetonitrile and methanol, and rule of thumb using acetonitrile, column pressure is relatively low, refers to hereinafter.And for
0.1% formic acid is universal method for Mobile Phase Additives, is suitable for method and gropes initial conditions.
Test experiments use following mobile phase：A phases, 0.1% formic acid water；B phases, acetonitrile.Reverse-phase chromatography investigates different size
C18 chromatographic columns (column length 50mm and 100mm) are for the separating effect of analyte.Due to the first root chromatogram column need under small flow velocity into
Row separation, setting flow velocity is 0.1mL/min.The chromatographic column (50mm and 100mm) of two kinds of specifications can be to phenolic acid, saponin(e and pellet
Ginseng ketone compounds have preferable separation, but amino acid, oligosaccharides and nucleosides isopolarity compound are retained poor.In low flow velocity
Under, 50mm chromatographic columns can complete separation in 8min, and peak stretching phenomenon does not occur for chromatographic peak；100mm chromatographic columns need
The separation of 12min is carried out, while chromatography peak stretching is serious.Hydrophilic Interaction Chromatography investigates silica gel (HILIC) and acid amides (Amide) two
Kind stationary phase, carries out under 0.4mL/min flow velocitys.Silica gel solid phase hardly retains other analytes in addition to oligosaccharides,
And acid amides stationary phase retains oligosaccharides and saponin(e preferably, there is certain reservation to the type compound such as phenolic acid, nucleosides and amino acid, it is right
Tanshinone material does not retain.Acid amides stationary phase embodies good separation orthogonality with C18 stationary phases, therefore uses C18 chromatographies
Column and amide chromatogram column are combined.
3rd, flow visualizing
Compared with methanol aqueous systems, the system pressure of acetonitrile aqueous systems is lower, while hydrophilic Interaction Chromatography is in acetonitrile water body
Separating property in system is more preferable, therefore selects acetonitrile water as mobile phase.The selection of Mobile Phase Additives includes acid adding or adds salt
Or hydrochlorate combination, therefore, inventor investigates the combination of three kinds of Mobile Phase Additives, including A.0.1% formic acid, B.10mM ammonium formate and
C.0.1% formic acid -10mM ammonium formates.
All additives are added in water phase.Under anti-phase and two kinds of clastotypes of hydrophilic interaction, 0.1% formic acid can
The peak type of phenolic acid compound is enough significantly improved, and ammonium formate makes phenolic acid compound seriously trail.Three kinds of additives to oligosaccharides,
The reservation and response of saponin(e and tanshinone compound have little to no effect.The addition of formic acid can reduce anion response, but right
Have the function that in improvement chromatographic peak peak type irreplaceable.In direct train further investigate 0.00%, 0.02%,
0.05% and 0.10% 4 formic acid pitch-based sphere responds the separating effect of analyte with peak area evaluation compound.Do not add
When adding formic acid, phenolic acid and tanshinone response are relatively low, and the former trails seriously；When adding 0.02% formic acid, oligosaccharides, amino acid, core
The response of glycosides, saponin(e and tanshinone increases, but the improvement of phenolic acid peak type is limited, it is difficult to meets quantitative requirement；Addition
During 0.05% formic acid, phenolic acid peak type makes moderate progress, and tanshin polyphenolic acid B, salvianolic acid D and alkannic acid response lifting are obvious, but still cannot examine
Measure danshensu；When adding 0.10% formic acid, phenolic acid peak type improves substantially, and all phenolic acid responses reach maximum, while saponin(e,
Pyroglutamic acid and adenine response reduce (minimum to be down to the 28.6% of the response of 0.02% pitch-based sphere).Analyte in comprehensive sample
Quantity and content information, method disclosure satisfy that analysis demand in 0.10% formic acid aqueous systems.
4th, mobile phase hybrid mode is investigated
Threeway connection mode and mixed pipe line length can influence the mixing of reverse-phase chromatography and hydrophilic Interaction Chromatography mobile phase
Effect.As shown in figure 3, three are connected with tri- kinds of connection modes of ABC.
Test result indicates that C mode chromatographic peak has obvious broadening, this is probably that the mobile phase that convection current connects goes out in threeway
Existing vortex phenomenon so that the result of the separation loss of reverse-phase chromatography.A and B-mode can obtain good chromatography peak type, and A
The response of pattern is higher than B-mode, this is probably preferably to be mixed because the perpendicular direction access threeway of big flow rate can obtain
Close effect.Mixed effect is of great significance for the separating property and stability of hydrophilic Interaction Chromatography.Mixed process should try one's best
Reduce diffusion and separating degree loss of the analyte in system, therefore mixing effect is improved by the way of pipeline after extending mixing
Rate.Investigation internal diameter is 0.01mm, and length is respectively the metal tubes of 5,10 and 15cm on the separated influence of hydrophilic Interaction Chromatography.When
When pipeline between threeway and chromatographic column with hydrophilic function extends to 15cm, each analyte peak type is good, and method can stablize repetition,
Illustrate that two kinds of mobile phases obtain enough mixing.According to result above, subsequently ground using mode A and 15cm mixed pipe lines
Study carefully.
5th, flow velocity and gradient elution program optimize
Mobile phase compatibility issue is solved using the method for on-line dilution.Flow velocity and ratio to the chromatographic behavior of analyte and
Response has considerable influence.It is 0.50mL/min to set hydrophilic Interaction Chromatography flow velocity first, investigates reverse-phase chromatography flow velocity respectively and is
0.06th, 0.08,0.10,0.12 and 0.14mL/min (velocity ratio is respectively 1: 8.33,1: 6.25,1: 5.00,1: 4.16 and 1:
3.57) chromatographic behavior and response condition of each analyte when.With the raising of reverse-phase chromatography flow velocity, each compound, which retains, to be weakened,
Response gradually rises at the same time.When flow velocity is promoted to 0.12mL/min, chromatography peak response is basicly stable, therefore is carried out under the flow velocity
Reversed phase chromatography separation.Then hydrophilic Interaction Chromatography flow velocity is investigated, 0.30,0.40 and 0.50mL/min, tri- levels are set.
The result shows that flow velocity has little to no effect each compound chromatographic behavior, but chromatography peak response increases with flow velocity
Add and slightly increase.When flow velocity is promoted to 0.40mL/min, chromatography peak response is basicly stable.Therefore, two dimensional separation is set
Flow velocity is 0.40mL/min.Gradient elution program can significantly affect each compound chromatographic behavior.Since reverse-phase chromatography is in low stream
Separated under speed, it is necessary to which higher proportion organic phase in a short time could separate analyte.When reverse-phase chromatography is initial
When the organic Phase Proportion of mobile phase is less than 20%, there is obvious peak stretching phenomenon in each analyte, and phenolic acid compound chromatographic peak drags
Tail, and elution time is grown；When the organic Phase Proportion of liquid phase is improved to 25%, chromatographic peak peak type improves obvious and all
Analyte can be eluted out in 15min.Hydrophilic Interaction Chromatography can further separate reverse-phase chromatography efflux, but
It is that tanshinone compound can not be eluted out by unidirectional gradient elution program.In hydrophilic Interaction Chromatography, tanshinone chemical combination
Thing can be eluted out in organic phase at high proportion, but diffusing phenomenon occur in low ratio organic phase.In reverse-phase chromatography,
Tanshinone compound is eluted out after 10min, to ensure the peak type of tanshinone, in 10.5min by hydrophilic interaction color
Organic Phase Proportion of spectrum is promoted to rapidly 97%.
6th, the optimization of Mass Spectrometry Conditions
1) ion pair optimizes
Amino acid, nucleosides, tanshinone are preferable in response in the positive-ion mode, are able to detect that obvious quasi-molecular ion
Peak.Oligosaccharides, organic acid, phenolic acid and saponin(e are responded compared with anion height in the negative ion mode, are able to detect that obvious quasi-molecule
Quasi-molecular ions.Pyroglutamic acid and adenine are respectively amino acid and nucleoside compound, but both respond height in the negative ion mode
In cation, therefore using negative ion mode detection both the above material.Cluster voltage and incident voltage are removed respectively so that each chemical combination
Thing quasi-molecular ion peak response reaches maximum.Daughter ion scanning, optimization further are carried out to the quasi-molecular ion peak of each compound
Impact energy and collision cell escape voltage so that daughter ion response reaches maximum.The highest ion pair of Response to selection as it is quantitative from
Son is right, and the high ion pair of response time is as qualitative ion pair.
2) internal standard screening compound
The selection of internal standard compound is of great significance for the stability and reliability of ensuring method.Due to measuring substance classes
More, each compound structure and physical chemical differences are larger, and analysis method is established using more internal standard substances.Positive ion mode
Lower use L-lysine -4,4,5,5-d4As the internal standard substance of amino acid and nucleosides, internal standard compound of the diazepam as tanshinone
Matter, internal standard substance of the chloramphenicol as whole compounds is used under negative ion mode.L-lysine -4,4,5,5-d4Structure with
The amino acid of measure is similar, and retention behavior is close with nucleosides；Due to tanshinone appearance time rearward, thus using polarity it is similar
Diazepam as internal standard substance；The testing compound of negative ion mode detection is in 2-7min appearances, chloramphenicol (5.55min)
The ionization situation of compound similar in retention time can be reacted.The response of three kinds of internal standard substances is high and stablizes, peak type it is sharp and
Symmetry is good, disclosure satisfy that the requirement of each compound analysis measure.
Method provided by the invention has the following advantages：
1st, at present, there has been no a kind of method can at the same time to the oligosaccharides contained by compound danshen dripping pills, amino acid, nucleosides,
A variety of chemical physical properties such as organic acid, phenolic acid, saponin(e and tanshinone and the very big compound of architectural difference are quantitatively divided
Analysis.The present invention develop accurately and reliably, simple and rapid assay method, compound danshen dripping pills Multiple components quantitative analysis can be used as
Means, 7 major classes in compound Salviae Miltiorrhizae extract, 31 kinds of components can be measured at the same time.Time cost, labour cost are saved, method is fast
It is fast accurate, it can more fully assess the quality of the pharmaceutical preparations.
2nd, Chemistry for Chinese Traditional Medicine material composition and its complexity, contain numerous nascent metabolite and secondary metabolite.Explain
The composition of bright Chemistry for Chinese Traditional Medicine material group, is unreasonable only by measure single component or the other component of unitary class.And with not Tongfang
Method respectively gets up Multiple components measure recombinant the process analyzed, not only time-consuming and laborious, it is often more important that a variety of methods
Between (common method of each Autonomous test is shown in background technology second segment) measure result there is no comparativity.
3rd, the method established of the present invention, can cover the measurement range of bigger by once measuring, make up it is described above not
Foot, lays the first stone for Study of Traditional Chinese Medicine mass balance.
Brief description of the drawings
Fig. 1：The structure of 31 kinds of analytes and 3 kinds of internal standard compounds；
Fig. 2：Directly connect anti-phase-hydrophilic interaction liquid chromatography mass combined system schematic diagram；
Fig. 3：Three kinds of threeway connection mode schematic diagrames, wherein：Mobile phase 1 is reverse-phase chromatography flow visualizing；Mobile phase 2 is
Hydrophilic Interaction Chromatography flow visualizing；
Fig. 4：The extraction ion flow graph of 31 kinds of analytes in compound Salviae Miltiorrhizae extract, the numbering in figure and the chemical combination in table 4
Thing numbering is consistent.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.
Laboratory apparatus and material
1. instrument
Shimadzu Ultra Performance Liquid Chromatography instrument (Shimadzu, Japan), be equipped with platform four high-pressure pumps, on-line degassing machine, from
Dynamic injector, chromatographic column stability controller；The triple level Four bar mass spectrographs of Qtrap 5500 (American AB company)；KQ-500DE ultrasounds
Instrument (Kunshan Ultrasonic Instruments Co., Ltd.)；XS105DU assay balances (METTLER companies of Switzerland).
2. reagent and medicine
HPLC grades are purchased from Merck companies of Germany with MS grades of acetonitriles.HPLC grades of formic acid (purity ＞ 95%) are purchased from U.S. Sigma
Company.
High-purity deionized water is made (Milli-Q companies of the U.S.) by Milli-Q systems.
Adenine, adenosine, uridine, butanedioic acid, glucose, sucrose, pyroglutamic acid, proline, valine, arginine, rely
Propylhomoserin, protocatechualdehyde, protocatechuic acid, danshensu, Rosmarinic acid, tanshin polyphenolic acid B, Panax Notoginseng saponin R1, ginsenoside Rb1, ginsenoside
Rd, ginsenoside Re, ginsenoside Rg1, ginsenoside Rg2, ginsenoside Rh1, Tanshinone I, tanshinone IIA, Cryptotanshinone,
Diazepam (IS2) and chloramphenicol (IS3) are purchased from national drug biological product determination research institute；
Salviandic acid A, salvianolic acid C, salvianolic acid D and alkannic acid are purchased from one side Technology Co., Ltd. of Tianjin；
L-lysine -4,4,5,5-d4(IS1) it is purchased from Sigma Co., USA；
α-D- glucopyranoses-(1 → 2)-beta-D-fructofuranose-(1 → 1)-β-D- galactopyranoses (trisaccharide) and O- α-
D- galactopyranoses-(1 → 6)-O- α-D- galactopyranoses-(1 → 6)-O- α-D- glucopyranoses-(1 → 2)-O- α-D- furans
Fructose (tetrose) of muttering is made by Tian Shili modern Chinese herbal medicines Resources Co., Ltd.
Determinand structural formula is shown in Fig. 1.Calculated using HPLC and ELDS by areas of peak normalization method, all reference substance purity are big
In 97%.
Embodiment 1：Detection method
1st, the preparation of standard solution and inner mark solution
The preparation of 1.1 standard serial solutions
It is appropriate to weigh determinand standard items, accurately weighed, it is 500 μ g/mL's to be separately added into 70% methanol-water and concentration is made
Solution, as each determinand storing solution.Accurate each determinand storing solution of measurement is appropriate respectively, and tetrose is made with 70% methanol-water
(concentration is about 70000ng/mL), sucrose (concentration is about 50000ng/mL), tanshin polyphenolic acid B (concentration is about 12000ng/mL), people
Join saponin(e Rg1(concentration is about 8000ng/mL), glucose (concentration is about 6000ng/mL), trisaccharide, ginsenoside Rb1, ginseng soap
Glycosides Rd (concentration is about 4000ng/mL), pyroglutamic acid, Rosmarinic acid, Panax Notoginseng saponin R1(concentration is about 2000ng/mL) and remaining
The hybrid standard product storing solution of determinand (concentration is about 1000ng/mL).Take hybrid standard product storing solution appropriate, use 70% first
Series concentration mixed standard solution, 4 DEG C of refrigerators are made according to extension rate 2,5,10,20,50,100,200,500 and 1000 in alcohol
Save backup.
The preparation of 1.2 inner mark solutions
IS1, IS2 and appropriate IS3 are weighed, accurately weighed, it is 500 μ g/mL's to be separately added into 70% methanol-water and concentration is made
Solution, as each internal standard storing solution.Accurate each internal standard storing solution of measurement is appropriate respectively, and each internal standard concentration is made with 70% methanol-water
For the IS reference substance solutions of 100ng/mL, 4 DEG C of refrigerators save backup.
Accurate respectively to measure each concentration mixed reference substance solution of equivalent series and IS reference substance solutions, mixing, is made containing interior
Target series mixed reference substance solution (extension rate 2,4,10,20,40,100,200,400,1000 and 2000), 4 DEG C of refrigerators
Save backup.
2nd, chromatographic condition and Mass Spectrometry Conditions
2.1 directly connect reverse-phase chromatography-hydrophilic Interaction Chromatography
Reverse-phase chromatographic column uses Shim-Pack XR-ODS III (1.6 μm, 2.0mm × 50mm), and guard column uses
Waters pot strainers (2 μm).Mobile phase is made of 0.1% formic acid water (A1) and acetonitrile (B1), and Gradient program is：0min,
25%B；0.5min, 25%B；1.0min, 35%B；5.0min, 60%B；10.0min 80%B；14.0min 80%B.Flow velocity
0.12mL/min, 30 DEG C of column temperature, 2 μ L of sample size.
Chromatographic column with hydrophilic function uses Waters AcquityBEH Amide (1.7 μm, 2.1mm ×
100mm), guard column uses Waters pot strainers (2 μm).Mobile phase is made of 0.1% formic acid water (A2) and acetonitrile (B2),
Gradient program is：0min, 97%B；10.0min 50%B；10.5min 50%B；11.0min 97%B；14.0min 97%
B.Flow velocity 0.4mL/min, 30 DEG C of column temperature.Reverse-phase chromatographic column is connected with chromatographic column with hydrophilic function by a threeway, sees Fig. 2.
2.2 Mass Spectrometry Conditions
It is connected using electro-spray ionization (ESI) interface with chromatographic column with hydrophilic function.Cation capillary voltage is arranged to
5500V, anion capillary voltage are arranged to -4500V；Ion source temperature is arranged to 550 DEG C；Atomization gas, heating gas and gas curtain
Gas uses high-purity nitrogen, and it is respectively 55,55 and 25psi to set pressure.Time-histories, which is combined, using negative ions switching reacts prison more
(MRM) method quantitative analysis determinand is surveyed, and cluster voltage (DP), incident voltage (EP), impact energy (CE) is separately optimized and touches
Hit room escape voltage (CXP) parameter.Qualitatively and quantitatively parameter is shown in Table 1.
The retention time and associated mass spectrometry parameter of 1 analyte of table and internal standard compound
*Internal standard compound；aQuota ion pair；bQualitative ion pair.
DP, removes cluster voltage；EP, incident voltage；CE, impact energy；CXP, collision cell escape voltage.
The assay of 2 31 kinds of components of embodiment
1st, the preparation of sample test solution
(lot number is respectively 20131205,20141103,20120201,20150310 and to compound Salviae Miltiorrhizae extract S01-S05
20150310) provided by Tasly Pharmaceutical Group Co., Ltd.；Red rooted salvia S06-S27 collected from the multiple provinces of China,
Pseudo-ginseng S28-S52 is crushed collected from Yunnan Province, and in Tasly Institute.
Compound Danshen Root medicinal extract, Radix Salviae Miltiorrhizae extractum, pseudo-ginseng medicinal extract, Danshen Root and Notoginseng Root 0.10g are taken respectively, and precision claims
It is fixed, 70% methanol 10mL is added, is ultrasonically treated 30min, centrifugation 10min (16,000rpm), crosses 0.22 μm of filter membrane, it is accurate respectively
Measure equivalent sample solution and IS reference substance solutions, mixing, the sample test solution of obtained containing the internal standard, for RP-HILIC-MS/
MS is analyzed.All solution are maintained in 4 DEG C of refrigerators before analysis.
2nd, analysis method is verified
2.1 method specificities
31 kinds of analytes are ionized under Mass Spectrometry Conditions in 1.3 and Collision-induced dissociation.By taking compound Salviae Miltiorrhizae extract as an example,
Observe daughter ion scanning mass spectrogram of each analyte in different samples.As shown in figure 4, sample substrate is in more reflection monitoring analyses
The measure of 31 kinds of analytes is not disturbed in window, illustration method specificity is good.
2.2 standard curves and lower limit of quantitation
Standard curve is obtained using series concentration hybrid standard product solution, and each concentration carries out two-sample analysis.With analysis
Thing concentration (ng/mL) is abscissa x, and the peak area ratio of analyte and internal standard compound is ordinate y, with formaldehyde (w=1/x2) most
Small square law carries out recurrence calculating, tries to achieve the linear regression equation of standard curve.Test limit and quantitative limit are respectively 3 times and 10 times
Analyte concentration during signal-to-noise ratio.Internal standard compound, regression equation, quantitative limit and detection limit information are shown in Table 2.
The internal standard compound, regression equation, quantitative limit (LOD) and test limit (LOQ) of 2 31 analytes of table
2.3 precision are investigated with sample stability
Methodological study is carried out using compound Salviae Miltiorrhizae extract, Radix Salviae Miltiorrhizae and pseudo-ginseng.Precision is used and in a few days and in the daytime made a variation
Property is evaluated.6 pin of withinday precision METHOD FOR CONTINUOUS DETERMINATION same sample, continuous three days measure same samples of day to day precision, daily 3
Pin.Repeatability is analyzed by 6 parts of samples of parallel preparation.4 DEG C of 0,2,4,8,10 and 12h of placement of study on the stability sample solution
When analyte content.All experiments calculate according to the standard curve on the same day and measure concentration.Precision, repeatability and stability
As a result represented using RSD, the results are shown in Table 3.
Precision, repeatability and the stability result of 31 analytes in 3 compound Salviae Miltiorrhizae extract of table, Radix Salviae Miltiorrhizae and pseudo-ginseng
3 the results show of table：Test sample is in a few days respectively smaller than 4.87% and 5.41% with day to day precision RSD, repeats
Property RSD be less than 5.58%, sample stability RSD be less than 4.90%.
2.4 accuracy are investigated
Accuracy is assessed using sample recovery rate.Respectively known quantity is added into compound Salviae Miltiorrhizae extract, Radix Salviae Miltiorrhizae and pseudo-ginseng
Standard items so that analyte concentration is about 50%, 100% and the 200% of original concentration in sample after addition, each concentration system
Standby 3 parts of samples are analyzed.The rate of recovery is calculated according to formula：The rate of recovery (%)=((measured amount-original vol)/additive amount) ×
100%.Investigation the results are shown in Table 4.
The rate of recovery (%) (RSD, %, n=3) of 31 analytes in 4 Radix Salviae Miltiorrhizae of table, pseudo-ginseng and compound Danshen Root medicinal extract
The rate of recovery of sample is respectively 92.3-106.8% (RSD ＜ in 50%, 100% and 200% 3 pitch-based sphere
5.48%), 95.1-105.0% (RSD ＜ 4.39%) and 95.6-106.4% (RSD ＜ 5.34%).
3rd, sample measurement result
Using the directly series connection of foundation it is anti-phase-hydrophilic Interaction Chromatography mass spectrometry combination method measure 5 batches of extracts, 22 batches of Radix Salviae Miltiorrhizaes
31 kinds of analyte contents with 25 batches of pseudo-ginseng samples, the results are shown in Table 5.
5 the results show of table：31 kinds of compounds are detected in compound Salviae Miltiorrhizae extract, measure content of material accounts for extract weight
55.5-71.5%.S03 and S04 is Salvia root P.E, is not detected by saponins compound；S05 is Notogineng Extract, is not examined
Measure arginine, trisaccharide, tetrose, phenolic acid and tanshinone compound.Oligosaccharides, soap in compound Salviae Miltiorrhizae extract (S01 and S02)
Glycosides, phenolic acid and amino acid content are higher, account for 40.3-46.6%, 11.3-11.9%, 6.0- of extract gross weight respectively
6.8%.Detect 20 kinds of compounds in Radix Salviae Miltiorrhizae (S06-S27), including 5 kinds of amino acid, 4 kinds of oligosaccharides, a kind of nucleosides, a kind it is organic
Acid, 6 kinds of phenolic acid and 3 kinds of tanshinones, measure content of material account for the 16.4-38.2% of Radix Salviae Miltiorrhizae weight.Detection in pseudo-ginseng (S28-S52)
To 18 kinds of compounds, including 5 kinds of amino acid, 2 kinds of oligosaccharides, 3 kinds of nucleosides, a kind of organic acid and 7 kinds of saponin(es, measure content of material accounts for
The 8.6-35.9% of three seven weights.
Claims (10)
1. method that is a kind of while measuring 31 kinds of components in compound Salviae Miltiorrhizae extract or related medicinal material, this method is using direct
Series connection-anti-phase-hydrophilic Interaction Chromatography Mass Spectrometry.
2. according to the method described in claim 1, it is characterized in that, the detection method comprises the following steps：
The preparation of step 1 sample solution：Compound Salviae Miltiorrhizae extract or related medicinal material are taken, methanol solution is configured to methanol；
The preparation of step 2 standard serial solution：The standard items of 31 kinds of components are taken respectively, and methanol solution is configured to methanol；
The preparation of step 3 inner mark solution：Take internal standard compound L-lysine -4,4,5,5-d4, diazepam and chloramphenicol, use methanol respectively
It is configured to methanol solution；
The preparation of step 4 test solution：Step 1 or step 2 resulting solution are mixed with the inner mark solution of step 3 respectively；
Step 5 takes step 4 resulting solution to inject anti-phase-hydrophilic interaction liquid chromatography mass combined system of directly connecting, and obtains 31
The extraction ion flow graph of kind analyte, 31 kinds of components in compound Salviae Miltiorrhizae extract or related medicinal material are calculated according to extraction ion flow graph
Content.
3. according to the method described in claim 2, it is characterized in that, the preparation method of wherein step 1 sample solution is：Take respectively
Compound Danshen Root medicinal extract, Radix Salviae Miltiorrhizae extractum, pseudo-ginseng medicinal extract, Danshen Root or Notoginseng Root 0.10g, accurately weighed, addition 60-80% first
Alcohol 8-12mL, is ultrasonically treated 20-40min, and 8-12min is centrifuged according to 16,000rpm, crosses 0.22 μm of filter membrane, and all solution are dividing
It is maintained in before analysis in 4 DEG C of refrigerators.
4. according to the method described in claim 3, it is characterized in that, the preparation method of wherein step 1 sample solution is：Take respectively
Compound Danshen Root medicinal extract, Radix Salviae Miltiorrhizae extractum, pseudo-ginseng medicinal extract, Danshen Root or Notoginseng Root 0.10g, accurately weighed, 70% methanol of addition
10mL, is ultrasonically treated 30min, centrifuges 10min according to 16,000rpm, crosses 0.22 μm of filter membrane, all solution preserve before analysis
In 4 DEG C of refrigerators.
5. according to the method described in claim 2, it is characterized in that, the preparation method of wherein step 2 standard serial solution is：Claim
Take determinand standard items appropriate, accurately weighed, it is the molten of 450-550 μ g/mL to be separately added into 60-80% methanol-waters and concentration is made
Liquid, as each determinand storing solution, accurate each determinand storing solution of measurement is appropriate respectively, and concentration is made with 60-80% methanol-waters
Tanshin polyphenolic acid B that sucrose that tetrose, concentration for 70000ng/mL are 50000ng/mL, concentration are 12000ng/mL, concentration are
The ginsenoside Rg of 8000ng/mL1, concentration be 6000ng/mL glucose, concentration be 4000ng/mL trisaccharide, concentration be
The ginsenoside Rb of 4000ng/mL1, concentration be 4000ng/mL ginsenoside Rd, concentration be 2000ng/mL pyroglutamic acid,
The Panax Notoginseng saponin R that Rosmarinic acid that concentration is 2000ng/mL, concentration are 2000ng/mL1With concentration be 1000ng/mL remaining
The hybrid standard product storing solution of determinand；Take hybrid standard product storing solution appropriate, using 60-80% methanol according to extension rate 2,
5th, 10,20,50,100,200,500 and 1000 series concentration mixed standard solution is made, 4 DEG C of refrigerators save backup；
Preferably, the preparation method of step 2 standard serial solution is：It is appropriate to weigh determinand standard items, it is accurately weighed, point
70% methanol-water not being added the solution that concentration is 500 μ g/mL being made, as each determinand storing solution, accurate measurement respectively is respectively treated
Survey that thing storing solution is appropriate, with 70% methanol-water be made sucrose that tetrose, concentration that concentration is 70000ng/mL are 50000ng/mL,
The ginsenoside Rg that tanshin polyphenolic acid B that concentration is 12000ng/mL, concentration are 8000ng/mL1, concentration be 6000ng/mL grape
Sugar, concentration be 4000ng/mL trisaccharide, concentration be 4000ng/mL ginsenoside Rb1, concentration be 4000ng/mL ginseng soap
Rosmarinic acid that pyroglutamic acid that glycosides Rd, concentration are 2000ng/mL, concentration are 2000ng/mL, three that concentration is 2000ng/mL
Seven saponin(e R1Hybrid standard product storing solution with concentration for remaining determinand of 1000ng/mL；Hybrid standard product storing solution is taken to fit
Amount, series concentration hybrid standard is made using 70% methanol according to extension rate 2,5,10,20,50,100,200,500 and 1000
Solution, 4 DEG C of refrigerators save backup.
6. according to the method described in claim 2, it is characterized in that, the preparation method of wherein step 3 inner mark solution is：Weigh L-
Lysine -4,4,5,5-d4, diazepam and appropriate chloramphenicol, it is accurately weighed, be separately added into 60-80% methanol-waters concentration be made and be
The solution of 450-550 μ g/mL, as each internal standard storing solution, accurate each internal standard storing solution of measurement is appropriate respectively, with 70% methanol-water
The IS reference substance solutions that each internal standard concentration is 100ng/mL are made, 4 DEG C of refrigerators save backup.
7. according to the method described in claim 6, it is characterized in that, the preparation method of wherein step 3 inner mark solution is：Weigh L-
Lysine -4,4,5,5-d4, diazepam and appropriate chloramphenicol, it is accurately weighed, be separately added into 70% methanol-water and concentration be made as 500
The solution of μ g/mL, as each internal standard storing solution, accurate each internal standard storing solution of measurement is appropriate respectively, is made respectively with 70% methanol-water
Internal standard concentration is the IS reference substance solutions of 100ng/mL, and 4 DEG C of refrigerators save backup.
8. according to the method described in claim 2, it is characterized in that, the preparation method of wherein step 4 test solution is：Respectively
Precision measures each concentration mixed reference substance solution of equivalent series or sample solution is mixed with IS reference substance solutions, and containing the internal standard is made
Serial mixed reference substance solution or sample solution, 4 DEG C of refrigerators save backup, and serial mixed reference substance solution dilutes for inner mark solution
The solution of multiple 2,4,10,20,40,100,200,400,1000 and 2000, the body that sample solution is mixed with IS reference substance solutions
Product ratio is 1-10：1-10.
9. according to the method described in claim 8, it is characterized in that, the preparation method of wherein step 4 test solution is：Respectively
Precision measures each concentration mixed reference substance solution of equivalent series or sample solution is mixed with IS reference substance solutions, and containing the internal standard is made
Serial mixed reference substance solution or sample solution, 4 DEG C of refrigerators save backup, and serial mixed reference substance solution dilutes for inner mark solution
The solution of multiple 2,4,10,20,40,100,200,400,1000 and 2000, sample solution and IS reference substance solutions are equal proportion
Mixing.
10. according to the method described in claim 2, it is characterized in that, wherein step 5 chromatographic condition is as follows：
Reverse-phase chromatographic column uses the chromatographic column of sub- 2 μm of C18 fillers, and guard column uses Waters pot strainers (2 μm), flows
Mutually it is made of 0.08-0.15% formic acid or acetic acid water (A1) and acetonitrile (B1), Gradient program is：0min, 25%B；0.5min,
25%B；1.0min, 35%B；5.0min, 60%B；10.0min 80%B；15.0min, 80%B, flow velocity 0.1-0.12mL/
Min, 25-45 DEG C of column temperature, sample size 1-3 μ L；
Chromatographic column with hydrophilic function uses Waters pot strainers (2 μm), mobile phase using sub- 2 μm of hydrophilic chromatographic columns, guard column
It is made of 0.08-0.15% formic acid or acetic acid water (A2) and acetonitrile (B2), Gradient program is：0min, 97%B；10.0min,
50%B；10.5min 50%B；11.0min 97%B；15.0min 97%B；Flow velocity 0.35-0.45mL/min, column temperature 25-45
℃；Reverse-phase chromatographic column is connected with chromatographic column with hydrophilic function by a threeway；
The Mass Spectrometry Conditions are as follows：
It is connected using electro-spray ionization (ESI) interface with chromatographic column with hydrophilic function, cation capillary voltage is arranged to
5500V, anion capillary voltage are arranged to -4500V；Ion source temperature is arranged to 550 DEG C；Atomization gas, heating gas and gas curtain
Gas uses high-purity nitrogen, and it is respectively 55,55 and 25psi to set pressure, and combine time-histories using negative ions switching reacts prison more
Survey (MRM) method quantitative analysis determinand.
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| CN109374779A (en) * | 2018-12-17 | 2019-02-22 | 杭州奕安济世生物药业有限公司 | A kind of rapid detection method of cane sugar content |
| CN109856267A (en) * | 2019-01-23 | 2019-06-07 | 牡丹江友搏药业有限责任公司 | The method for measuring Multiple components content in Chinese materia medica preparation simultaneously based on LC-MS technology |
| CN109900826A (en) * | 2019-03-26 | 2019-06-18 | 广州莱泰制药有限公司 | A kind of Fufang Danshen Pian Radix Notoginseng Content Test method |
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| CN109856267A (en) * | 2019-01-23 | 2019-06-07 | 牡丹江友搏药业有限责任公司 | The method for measuring Multiple components content in Chinese materia medica preparation simultaneously based on LC-MS technology |
| CN109856267B (en) * | 2019-01-23 | 2022-02-18 | 牡丹江友搏药业有限责任公司 | Method for simultaneously determining contents of multiple components in Chinese medicinal preparation based on LC-MS technology |
| CN109900826A (en) * | 2019-03-26 | 2019-06-18 | 广州莱泰制药有限公司 | A kind of Fufang Danshen Pian Radix Notoginseng Content Test method |
| CN111812221A (en) * | 2020-05-29 | 2020-10-23 | 天津中医药大学 | Method for separating saponin component from Chinese medicinal materials of Panax |
| CN111812221B (en) * | 2020-05-29 | 2022-08-16 | 天津中医药大学 | Method for separating saponin component from Chinese medicinal materials of Panax |
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