CN108680654A - A kind of novel separation method of Chinese medicine - Google Patents
A kind of novel separation method of Chinese medicine Download PDFInfo
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- CN108680654A CN108680654A CN201810125967.0A CN201810125967A CN108680654A CN 108680654 A CN108680654 A CN 108680654A CN 201810125967 A CN201810125967 A CN 201810125967A CN 108680654 A CN108680654 A CN 108680654A
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- acid
- microemulsion
- sodium danshensu
- protocatechualdehyde
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
Abstract
The invention discloses a kind of novel separation methods of Chinese medicine.The method is：The accurate n-butanol for weighing the oil phase of 0.1 0.4%w/v, the SDS of 0.5 2.0%w/v and 2.0 5.0%w/v is mixed with pure water, and ultrasonic 30min forms the microemulsion of transparent and stable.Then the pH of microemulsion is adjusted with the ammonium hydroxide of phosphoric acid or 10%.Finally, microemulsion is filtered by 0.2 μm of nylon leaching film, obtained filtrate can be used as after ultrasound degassing 20min the hybrid standard product (Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A) or Radix Salviae Miltiorrhizae sample extraction solution or danshen injections progress separation detection of 8 kinds of phenolic acid of mobile phase pair of high performance liquid chromatography.Method used in the present invention is easy to operate, and consumption of organic solvent is few, reduces the murder by poisoning to operating personnel and the pollution to environment.Separating effect is notable, can liposoluble ingredient of the efficient and sensible ground in separation detection Radix Salviae Miltiorrhizae simultaneously.
Description
Technical field
The invention belongs to the separation field of Chinese medicine, it is related to a kind of side of active ingredient in separation determination Radix Salviae Miltiorrhizae and its preparation
Method, and in particular to a kind of Microemulsion liquid chromatography using water packet ionic liquid micro emulsion as mobile phase has in separation detection Radix Salviae Miltiorrhizae simultaneously
The method for imitating ingredient.
Background technology
The ionic liquid (IL) for being referred to as fuse salt is by large volume organic cation such as alkyl imidazole, alkylammonium, alkane
Ji Phosphonium, alkyl pyridine, alkyl pyrrolidine and various inorganic (such as chloride, bromide, hexafluorophosphate, tetrafluoroborates
With trifluoromethanesulfonic acid root anion) or organic anion (such as [(CF3SO2)2N]-[(CF3CO2)]-) constitute.Pass through choosing
Physicochemical properties can be adjusted by selecting suitable anion and cation.Since it is with various structures, IL is to polarity
The solubility of wider range is all had with non-polar compound.In recent years, due to its unique physicochemical properties, for example, it is variable
Range of viscosities, low volatility, low melting point, without effective vapour pressure, high chemically and thermally stability and recycling property, IL has caused
More and more extensive concern.IL can be mainly since they can be neglected as the environmentally friendly non-volatile solvents of Conventional solvents are substituted
The volatility and incombustibility slightly disregarded.Also, IL can be interacted by hydrogen bond, π-π, n- π interactions, and electrostatic is mutual
Effect and dipolar interaction are had an effect with analyte.Further, since its unique property, IL are urged in such as sample preparation
It is had a wide range of applications in the various analysis fields of change and separation.In chromatography, IL can be used as gas-chromatography (GC), liquid phase
Stationary phase in chromatography (LC) and supercritical fluid chromatography (SFC), electrolyte solution in mobile phase and Capillary Electrophoresis in LC
Additive.Moreover, peak shape can be improved as the additive of mobile phase in LC using IL, and influence column effect and theoretical cam curve.
Microemulsion is a kind of organic phase (oil) and water phase containing by surfactant and cosurfactant stabilization
Thermodynamically stable, optically transparent and isotropic liquid dispersion system of (water).Hydrophilic and lyophobic dust can
It is dissolved in microemulsion.Microemulsion has been used as the flowing in the false stationary phase in Capillary Electrophoresis (CE) and liquid chromatogram (LC)
Phase.In addition, micro emulsion is divided into oil-in-water (O/W) and Water-In-Oil (W/O), wherein O/W types micro emulsion is due to its High water cut
Amount and low viscosity, the mobile phase being widely used as in reversed-phase high performance liquid chromatography.In recent years, due to its unique selectivity and
High separating efficiency without gradient elution and its can overcome the ability of the environmental problem that Traditional liquid phase brings (consumption largely has
Solvent), Microemulsion liquid chromatography method (MELC) receives more and more attention and is successfully applied to separation science.
Common oil phase is to generate dysgenic hydrophobic alkane, such as ethyl acetate, octane and hexamethylene to environment in MELC, according to us
Known, the ionic liquid using environmental protection not yet appears in the newspapers as the oil phase in MELC.
Radix Salviae Miltiorrhizae (Salvia Miltiorrhiza Bunge) has special bioactivity, is during one kind is widely used
Herbal medicine.Radix Salviae Miltiorrhizae and its preparation such as Radix Salviae Miltiorrhizae tablet, Danshen root injection, capsule of red sage root and Danshen Root dropping ball are widely used in cerebrovascular disease
Disease, coronary artery disease, atherosclerosis, hyperlipidemia, hypertension, hepatitis, chronic renal failure, hepatic sclerosis and bone are lost
The clinical treatment of the various diseases such as mistake.Primary bioactive components in Radix Salviae Miltiorrhizae can be divided into hydrophilic phenolic acid compound (such as
Danshensu, caffeic acid, protocatechualdehyde, Rosmarinic acid and tanshin polyphenolic acid B) and lipophilic diterpene-kind compound (including dihydro Radix Salviae Miltiorrhizae
Ketone I, Cryptotanshinone, tanshinone IIA and Tanshinone I).Therefore, these active ingredients are extracted and measured from Radix Salviae Miltiorrhizae and its preparation
Object is very important.Currently, various analysis methods such as high performance liquid chromatography connects UV detector (HPLC-UV), HPLC connects two
Pole pipe array detector (DAD) and ultra performance liquid chromatography and Quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF/MS) are
It is used to measure the active constituent in Radix Salviae Miltiorrhizae and its preparation.However, using water and organic solvent as stream in these methods
Dynamic phase, consumes a large amount of toxic organic solvents, results in the serious pollution of environment.Therefore, it is few to establish a kind of consumption of organic solvent
And the analysis that the MELC systems with high separation capacity carry out chemical composition to actual sample is significantly.
The present invention establish it is a kind of it is simple, efficiently, the MELC methods of environmental protection, using water packet ionic liquid (IL/W) micro emulsion as
Mobile phase, for measuring the liposoluble ingredient in Radix Salviae Miltiorrhizae and its preparation.The present invention is to influencing the separating properties of target analytes
Several important parameters, include the type of oil phase, oily concentration, the concentration of lauryl sodium sulfate (SDS), n-butanol concentration and micro-
The pH of lotion is evaluated and optimized.The verification of this method be according to linear, precision, reproducibility, accuracy, detection limit and
These aspects of quantitative limit carry out.Finally, the IL/W MELC methods of exploitation are successfully applied to target in Radix Salviae Miltiorrhizae sample and its preparation
The separation of compound and measurement.
Invention content
Radix Salviae Miltiorrhizae is detected as liquid chromatogram mobile phase quick separating using water packet ionic liquid micro emulsion the present invention provides a kind of
In liposoluble ingredient method, solve in current separation detection red rooted salvia and its preparation and deposited in the method for liposoluble ingredient
Take, separation selectivity is low, consumption of organic solvent is big the problems such as.The liposoluble ingredient be Sodium Danshensu, protocatechuic acid,
One or more of protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A.
What this method was specifically completed according to the following steps：
(1) the accurate n-butanol for weighing the oil phase of 0.1-0.4%w/v, the SDS of 0.5-2.0%w/v and 2.0-5.0%w/v
It is mixed with pure water, ultrasonic 30min forms the microemulsion of transparent and stable.Then phosphoric acid or 10% ammonium hydroxide is used to adjust microemulsion
pH.Finally, microemulsion is filtered by 0.2 μm of nylon leaching film, after obtained filtrate carries out ultrasound degassing 20min
Hybrid standard product (Sodium Danshensu, protocatechuic acid, protocatechualdehyde, coffee of 8 kinds of phenolic acid of mobile phase pair as high performance liquid chromatography
Acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A) or Radix Salviae Miltiorrhizae sample extraction solution or danshen injections carry out separation inspection
It surveys.
(2) a series of reference substance solution of various concentrations is configured to Sodium Danshensu reference substance, with optimal MELC conditions
It is detected, the chromatogram of Sodium Danshensu reference substance is obtained, with a concentration of abscissa of Sodium Danshensu reference substance solution, with Radix Salviae Miltiorrhizae
The peak area of chromatographic peak is that ordinate makes Sodium Danshensu standard curve in plain sodium MELC chromatograms, makes former youngster in the same way
The standard curve of boheic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A.According to red sage root extract
Or in danshen injections chromatogram the peak area of each chromatographic peak and each ingredient standard curve, be calculated in extracting solution respectively at
The content divided.And then content of the active ingredient extracted relative to red rooted salvia powder that can accordingly convert.
Wherein oil phase described in (1) includes：1- butyl -3- methylimidazoles hexafluorophosphates ([BMIM] PF6), 1- hexyls-
3- methylimidazoles hexafluorophosphate ([HMIM] PF6), 1- octyl -3- methylimidazoles hexafluorophosphates ([OMIM] PF6), 1- hexyls-
3- methyl imidazolium tetrafluoroborates ([HMIM] BF4), 1- butyl -3- methylimidazole bis-trifluoromethylsulfoandimide salt ([BMIM]
TF2N), 1- octyls -3- methylimidazole bis-trifluoromethylsulfoandimides salt ([OMIM] TF2N), preferably [HMIM] PF6。
The ratio of the oil phase is：0.1%w/v, 0.2%w/v, 0.3%w/v, 0.4%w/v, preferably 0.2%w/v.
The ratio of the SDS is：0.5%w/v, 1.0%w/v, 1.5%w/v, 2.0%w/v, preferably 1.0%w/v.
The ratio of the n-butanol includes：2.0%w/v, 3.0%w/v, 4.0%w/v, 5.0%w/v, preferably 3.0%
w/v。
The pH is：1.5,2.0,2.5,5.0,5.5, preferably 2.5.
Advantage of the invention is that：The present invention is examined ionic liquid as the separation of oil of microemulsion mobile phase in MELC for the first time
The liposoluble ingredient in Radix Salviae Miltiorrhizae and its preparation is surveyed, there is originality.This method it is easy to operate, consumption of organic solvent is few, subtracts
The murder by poisoning to operating personnel and the pollution to environment are lacked.Its water packet ionic liquid micro emulsion mobile phase selected can rapidly and efficiently simultaneously
Liposoluble ingredient in ground separation detection Radix Salviae Miltiorrhizae and its preparation.
Description of the drawings
Fig. 1 is Microemulsion liquid chromatography flow chart of the water packet ionic liquid micro emulsion of the present invention as mobile phase.
Fig. 2 is the chromatogram investigated the type of oil phase and influenced on 8 kinds of phenolic acid separating effects.In figure, (a) [BMIM] PF6；
(b)[HMIM]PF6；(c)[OMIM]PF6；(d)[BMIM]TF2N；(e)[HMIM]BF4；(f)[OMIM]TF2N, 1,2,3,4,5,6,
7,8 Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A are respectively represented.
Fig. 3 is the chromatogram that the concentration of oil phase influences 8 kinds of phenolic acid separating effects.In figure, (a) 0.1%w/v；(b)
0.2%w/v；(c) 0.3%w/v；(d) 0.4%w/v, 1,2,3,4,5,6,7,8 respectively represent Sodium Danshensu, protocatechuic acid, original
Catechu aldehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A.
Fig. 4 is the chromatogram investigated different SDS concentration and influenced on 8 kinds of phenolic acid separating effects.In figure, (a) 0.5%w/v；
(b) 1.0%w/v；(c) 1.5%w/v；(d) 2.0%w/v, 1,2,3,4,5,6,7,8 respectively represent Sodium Danshensu, protocatechuic acid,
Protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A.
Fig. 5 is the chromatogram investigated different n-butanol concentration and influenced on 8 kinds of phenolic acid separating effects.In figure, (a) 5%w/v；
(b) 4%w/v；(c) 3%w/v；(d) 2%w/v, 1,2,3,4,5,6,7,8 respectively represent Sodium Danshensu, protocatechuic acid, former catechu
Aldehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A.
Fig. 6 is the chromatogram investigated different pH and influenced on 8 kinds of phenolic acid separating effects.In figure, (a) 1.5；(b)2.0；(c)
2.5；(d)5.0；(e) 5.5,1,2,3,4,5,6,7,8 respectively represent Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, purple
Oxalic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A.
Fig. 7 A are that danshen injections dilute the chromatogram after 5 times, and B is the chromatogram of red rooted salvia extracting solution.In figure, 1,3,
4,5,6,7 Sodium Danshensu, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B are respectively represented.
Fig. 8 is that the 8 kinds of phenolic acid hybrid standard product of oil phase pair for using traditional ethyl acetate as microemulsion in MELC detach
Chromatogram.In figure, 1,3,4,5,6,7 respectively represent Sodium Danshensu, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, red phenol
Sour B.
Specific implementation mode
Method provided by the present invention is described further by specific embodiment below.
Since it has a wide range of application, therefore specific embodiment is also more, makees to present disclosure with reference to several examples
It is further elucidated above.
The preparation method of Radix Salviae Miltiorrhizae reference substance solution is：Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, purple are taken respectively
Oxalic acid, Rosmarinic acid, tanshin polyphenolic acid B, the reference substance of salviandic acid A are appropriate, accurately weighed, are respectively placed in brown measuring bottle, add respectively
The reference substance solution that every lmL contains 1000 μ g is made in methanol.
Embodiment 1
Accurate 6 kinds of different types of ionic liquid ([BMIM] PF for weighing 0.2%w/v6, [HMIM] PF6, [OMIM] PF6,
[HMIM]BF4, [BMIM] TF2N, [OMIM] TF2N) and the n-butanol of SDS and 6 part of 3%w/v of 6 parts of 1%w/v is in 6 vials
In, it is separately added into pure water and is mixed with, ultrasonic 30min forms the microemulsion of transparent and stable.Then microemulsion is adjusted with phosphoric acid
PH to 2.5.Finally, microemulsion is filtered by 0.2 μm of nylon leaching film, obtained filtrate carries out ultrasound degassing 20min
It can be used as hybrid standard product (Sodium Danshensu, protocatechuic acid, former catechu of 8 kinds of phenolic acid of mobile phase pair of high performance liquid chromatography afterwards
Aldehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A) carry out separation detection.
The instrument that this experiment uses is Agilent ultra performance liquid chromatography (Agilent 1290Infinity LC, Santa
Clara, CA, USA) it is equipped with vacuum pump, autosampler, thermostatted column compartment, UV detector.Chromatographic column Agilent
Reverse-phase SB-C18 columns (4.6mm × 150mm, 5 μm), 30 DEG C, flow velocity 0.6mL/min of column temperature, 2 μ L of sampling volume.
Mobile phase is microemulsion.Equilibration time 5min.
The separating effect chromatogram of 8 kinds of phenolic acid of mobile phase pair of the ionic liquid containing variety classes is as shown in Figure 2, the results showed that,
With [HMIM] PF68 substances can be made to reach to be kept completely separate.And when the anion of ionic liquid is identical, increase the alkane of cation
The retention time of base chain length, phenolic acid integrally extends.Reason may be that phenolic acid is polar compound, and the increase of alkyl chain length makes oil phase
Drop hydrophobicity enhances so that distribution of the target compound in micro emulsion drop is reduced, and then extends retention time.It selects
TF2N-Type ionic liquid replaces PF6 -Type ionic liquid shows poor separation selectivity.And use [HMIM] BF4As oil phase, 5,6 Hes
7, No. 8 peaks occur being overlapped and time lengthening.So preferably [HMIM] PF6As oil phase.
Embodiment 2
Accurate [HMIM] PF for weighing various concentration6(0.1%w/v, 0.2%w/v, 0.3%w/v, 0.4%w/v) and 4 parts
The n-butanol of SDS and 4 part of 3%w/v of 1%w/v is separately added into pure water and is mixed in 4 vials, ultrasonic 30min, shape
At the microemulsion of transparent and stable.Then the pH to 2.5 of microemulsion is adjusted with phosphoric acid.Finally, microemulsion is passed through into 0.2 μm of nylon
Filter membrane is filtered, and can be used as 8 kinds of phenol of mobile phase pair of high performance liquid chromatography after obtained filtrate progress ultrasound degassing 20min
Hybrid standard product (Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, the red phenol of acid
Sour A) carry out separation detection.
Difference [HMIM] PF6The separating effect chromatogram of 8 kinds of phenolic acid of mobile phase pair of concentration is as shown in Figure 3.When oil phase
6 and No. 7 overlap of peaks are at a peak when a concentration of 0.1%w/v.With the increase of oil phase concentration, the retention time of phenolic acid extends, this
May be since the increase of oil phase concentration makes the hydrophobicity of microemulsion enhance, to reduce phenolic acid solubility wherein and subtract
Allocation proportion is lacked.As [HMIM] PF6Best separating effect can be obtained when a concentration of 0.2%w/v.Concentration is further increased,
Retention time also further increases, and the eluting order at 5,6,7,8 peaks also changes, and as a concentration of 0.3%w/v, and 5 and 6
And 7 and 8 overlap.Therefore, 0.2%w/v is best oil phase concentration.
Embodiment 3
Accurate [HMIM] PF for weighing 4 parts of 0.2%w/v6, 4 parts of various concentrations SDS (0.5%w/v, 1.0%w/v,
1.5%w/v, 2.0%w/v) and 4 parts of 3%w/v n-butanol in 4 vials, be separately added into pure water and be mixed with, ultrasound
30min forms the microemulsion of transparent and stable.Then the pH to 2.5 of microemulsion is adjusted with phosphoric acid.Finally, microemulsion is passed through 0.2
μm nylon leaching film filtered, obtained filtrate can be used as after ultrasound degassing 20min the flowing of high performance liquid chromatography
Hybrid standard product (Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, the pellet of opposite 8 kinds of phenolic acid
Phenolic acid B, salviandic acid A) carry out separation detection.
The separating effect chromatogram of 8 kinds of phenolic acid of mobile phase pair of different SDS concentration is as shown in Figure 4.By can see in figure,
With the increase of SDS concentration, the retention time and separation selectivity of 8 phenolic acid all decrease.Reason may be SDS concentration
Increase the surface for promoting target compound distribution into the increased micro emulsion drop of volume or being left in drop, it is molten to reduce
The reservation of matter.When a concentration of 1.0%w/v of SDS obtains best separating effect.As a concentration of 0.5%w/v of SDS, when reservation
Between long be difficult to detect.When concentration is higher than 1.0%w/v, separating degree and separation selectivity all have dropped.Therefore, 1.0%w/v is
Best surfactant concentration.
Embodiment 4
Accurate [HMIM] PF for weighing 4 parts of 0.2%w/v6, the n-butanol of SDS and 4 part of various concentration of 4 parts of 1.0%w/v
(2.0%w/v, 3.0%w/v, 4.0%w/v, 5.0%w/v) is separately added into pure water and is mixed in 4 vials, ultrasound
30min forms the microemulsion of transparent and stable.Then the pH to 2.5 of microemulsion is adjusted with phosphoric acid.Finally, microemulsion is passed through 0.2
μm nylon leaching film filtered, obtained filtrate can be used as after ultrasound degassing 20min the flowing of high performance liquid chromatography
Hybrid standard product (Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, the pellet of opposite 8 kinds of phenolic acid
Phenolic acid B, salviandic acid A) carry out separation detection.
The separating effect chromatogram of 8 kinds of phenolic acid of mobile phase pair of different n-butanol concentration is as shown in Figure 5.The result shows that point
The retention time of analysis object extends with the increase of cosurfactant concentration, this may be since cosurfactant concentration increases
The organic solvent ratio for making mobile phase is added to increase, to slow down the elution of hydroaropic substance.And n-butanol can replace SDS
It is adsorbed on fixed phase surface, increases the polarity of stationary phase to enhance the reservation of analyte.Best separating effect is just
Butanol concentration be 3.0%w/v when obtain, when concentration be less than 3.0%, 6 and No. 7 peak complete-superposing, when concentration be higher than 3%, retain
Time lengthening is more and the separating degree at 7 and No. 8 peaks is deteriorated.Therefore, 3.0%w/v is best cosurfactant concentration.
Embodiment 5
Accurate [HMIM] PF for weighing 5 parts of 0.2%w/v6, the n-butanol of SDS and 5 part of 3.0%w/v of 5 parts of 1.0%w/v
It in 5 vials, is separately added into pure water and is mixed with, ultrasonic 30min forms the microemulsion of transparent and stable.Then phosphoric acid is used
Or 10% ammonium hydroxide adjust the pH of microemulsion to different numerical value (1.5,2.0,2.5,5.0,5.5).Finally, microemulsion is passed through
0.2 μm of nylon leaching film is filtered, and can be used as the stream of high performance liquid chromatography after obtained filtrate progress ultrasound degassing 20min
Dynamic opposite 8 kinds of phenolic acid hybrid standard product (Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid,
Tanshin polyphenolic acid B, salviandic acid A) carry out separation detection.
The separating effect chromatogram of 8 kinds of phenolic acid of mobile phase pair of different pH is as shown in Figure 6.It can be obtained from the figure that the guarantor of analyte
The time is stayed to shorten with the increase with pH value, when pH is 1.5 and 2.0,5 and 6 and 7 and 8 are overlapped into a peak, when pH increases
When adding to 5.0 and 5.5,8 analytes are difficult to realize efficiently separate.Reason may be since 8 phenols analytes are weak acid classes
The value of compound, pH determines that the degree of ionization of analyte, pH value more high ionization degree are bigger so that analyte is more difficult to
Retain in stationary phase.So it is best to provide optimal separation degree and the flowing phase pH value 2.5 of separation selectivity within a short period of time
Selection.
Withinday precision
Accurate [HMIM] PF for weighing 0.2%w/v6, the n-butanol of the SDS and 3.0%w/v of 1.0%w/v are in vial
In, it is separately added into pure water and is mixed with, ultrasonic 30min forms the microemulsion of transparent and stable.Then microemulsion is adjusted with phosphoric acid
PH to 2.5.Finally, microemulsion is filtered by 0.2 μm of nylon leaching film, obtained filtrate carries out ultrasound degassing 20min
It can be used as hybrid standard product (Sodium Danshensu, protocatechuic acid, former catechu of 8 kinds of phenolic acid of mobile phase pair of high performance liquid chromatography afterwards
Aldehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A) carry out separation detection.Same mixing is marked interior on the same day
Quasi- product continuous sample introduction 6 times.
Day to day precision
Accurate [HMIM] PF for weighing 0.2%w/v6, the n-butanol of the SDS and 3.0%w/v of 1.0%w/v are in vial
In, it is separately added into pure water and is mixed with, ultrasonic 30min forms the microemulsion of transparent and stable.Then microemulsion is adjusted with phosphoric acid
PH to 2.5.Finally, microemulsion is filtered by 0.2 μm of nylon leaching film, obtained filtrate carries out ultrasound degassing 20min
It can be used as hybrid standard product (Sodium Danshensu, protocatechuic acid, former catechu of 8 kinds of phenolic acid of mobile phase pair of high performance liquid chromatography afterwards
Aldehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A) carry out separation detection.Same hybrid standard product are continued repeatedly
Sample introduction 3 days, 2 times a day.
In a few days, day to day precision experimental result is summarized as follows table 1：
In table 1 day, day to day precision
Repeatability is investigated
Accurate [HMIM] PF for weighing 0.2%w/v6, the n-butanol of the SDS and 3.0%w/v of 1.0%w/v are in vial
In, it is separately added into pure water and is mixed with, ultrasonic 30min forms the microemulsion of transparent and stable.Then microemulsion is adjusted with phosphoric acid
PH to 2.5.Finally, microemulsion is filtered by 0.2 μm of nylon leaching film, obtained filtrate carries out ultrasound degassing 20min
It can be used as hybrid standard product (Sodium Danshensu, protocatechuic acid, former catechu of 8 kinds of phenolic acid of mobile phase pair of high performance liquid chromatography afterwards
Aldehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A) carry out separation detection.It repeats to do three groups by above-mentioned steps,
It is investigated as micro emulsion mobile phase repeatability.
0.15g Danshen Roots accurately are weighed in conical flask, and 50mL methanol-waters (80 are added:20v/v), ultrasonic 30min.
5mL solution is taken to add methanol dilution to 10mL, 13000rpm centrifugations 5min.Take supernatant liquor dress sample analysis.It is parallel by above-mentioned steps
Three groups are done, is investigated as extracting method repeatability.
Medicinal material assay
5 times of methanol dilution of danshen injections, 13000rpm centrifuge 5min.Take supernatant liquor dress sample analysis.
0.15g Danshen Roots accurately are weighed in conical flask, and 50mL methanol-waters (80 are added:20v/v), ultrasonic 30min.
5mL solution is taken to add methanol dilution to 10mL, 13000rpm centrifugations 5min.Take supernatant liquor dress sample analysis.
Fig. 7 A are the chromatogram of danshen injections dilution, and Fig. 7 B are the chromatogram of red rooted salvia extracting solution.
Take Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A
Appropriate reference substance, it is accurately weighed, it is respectively placed in brown measuring bottle, adds methanol that reference substances of every lmL containing 500 μ g is made respectively molten
Liquid.Reference substance solution obtained is configured to a series of mixed reference substance solution of various concentrations, by same condition MELC
Detection, obtains the chromatogram of mixed reference substance solution, with a concentration of abscissa of reference substance, the peak of chromatographic peak in corresponding chromatogram
Area is ordinate, makes the standard curve of this 8 kinds of standard items respectively.According in red sage root extract or danshen injections chromatogram
The content of each ingredient in extracting solution is calculated in the standard curve of the peak area of each chromatographic peak and each ingredient.And then it can be corresponding
Content of the active ingredient extracted that converts relative to red rooted salvia powder.
The standard curve and detection limit and quantitative limit of 6 kinds of ingredients are as shown in table 2 below：
2. standard curve of table, detection limit and quantitative limit
The rate of recovery is tested
Take 5 times of danshen injections methanol dilution, parallel to do three groups, one group adds mixing reference substance to a concentration of 1 μ g/mL,
Another group adds mixing reference substance to a concentration of 50 μ g/mL, and remaining one group is not added with mixing reference substance.13000rpm centrifuges 5min.It takes
Supernatant liquor fills sample analysis.
0.15g Danshen Roots accurately are weighed in conical flask, and 50mL methanol-waters (80 are added:20v/v), ultrasonic 30min.
5mL solution is taken to add methanol dilution to 10mL.Parallel to do three groups, one group adds mixing reference substance to a concentration of 1 μ g/mL, and another group adds
Reference substance is mixed to a concentration of 50 μ g/mL, remaining one group is not added with mixing reference substance.13000rpm centrifuges 5min.Take supernatant liquor
Fill sample analysis.
Repeatability, assay, rate of recovery experimental result are summarized as follows table 3：
3 repeatability of table, assay, the rate of recovery
The results show that the method for the present invention is reproducible, the rate of recovery is high, and accuracy is good.
Reference examples：
The accurate ethyl acetate for weighing 0.2%w/v, the n-butanol of the SDS and 3.0%w/v of 1.0%w/v in vial,
It is separately added into pure water to be mixed with, ultrasonic 30min forms the microemulsion of transparent and stable.Then the pH of microemulsion is adjusted with phosphoric acid
To 2.5.Finally, microemulsion is filtered by 0.2 μm of nylon leaching film, after obtained filtrate carries out ultrasound degassing 20min
The hybrid standard product of 8 kinds of phenolic acid of mobile phase pair as high performance liquid chromatography carry out separation detection.Its chromatogram is shown in Fig. 8.There is Fig. 8
As it can be seen that selecting the oil phase that traditional ethyl acetate makees microemulsion that cannot obtain preferable separating effect.The ion that this method uses
Liquid not only environmental protection but also can obtain preferable separating effect as oil phase.
Claims (6)
1. the novel separation method of a kind of Chinese medicine, for the method for the liposoluble ingredient in quick separating detection Radix Salviae Miltiorrhizae, the phenol
Acrylic component is in Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A
One or more, the described method comprises the following steps：
(1) the accurate n-butanol for weighing the oil phase of 0.1-0.4%w/v, the SDS of 0.5-2.0%w/v and 2.0-5.0%w/v with it is pure
Water mixes, and ultrasonic 30min forms the microemulsion of transparent and stable.Then the pH of microemulsion is adjusted with the ammonium hydroxide of phosphoric acid or 10%.Most
Afterwards, microemulsion is filtered by 0.2 μm of nylon leaching film, obtained filtrate can be used as after carrying out ultrasound degassing 20min
8 kinds of phenolic acid of mobile phase pair of high performance liquid chromatography hybrid standard product (Sodium Danshensu, protocatechuic acid, protocatechualdehyde, caffeic acid,
Alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A) or Radix Salviae Miltiorrhizae sample extraction solution or danshen injections carry out separation detection.
(2) it is configured to a series of reference substance solution of various concentrations with Sodium Danshensu reference substance, is carried out with optimal MELC conditions
Detection obtains the chromatogram of Sodium Danshensu reference substance, with a concentration of abscissa of Sodium Danshensu reference substance solution, with Sodium Danshensu
The peak area of chromatographic peak is that ordinate makes Sodium Danshensu standard curve in MELC chromatograms, makes former catechu in the same way
The standard curve of acid, protocatechualdehyde, caffeic acid, alkannic acid, Rosmarinic acid, tanshin polyphenolic acid B, salviandic acid A.According to red sage root extract or
Each ingredient in extracting solution is calculated in the standard curve of the peak area of each chromatographic peak and each ingredient in danshen injections chromatogram
Content.And then content of the active ingredient extracted relative to red rooted salvia powder that can accordingly convert.
2. the method as described in claim 1, it is characterised in that in the step (1), the oil phase is 1- hexyl -3- methyl miaows
Azoles hexafluorophosphate ([HMIM] PF6).
3. the method as described in claim 1, it is characterised in that in the step (1), the ratio of the oil phase is 0.2%w/v.
4. the method as described in claim 1, it is characterised in that in the step (1), the ratio of the SDS is 1.0%w/v.
5. the method as described in claim 1, it is characterised in that in the step (1), the ratio of the n-butanol is 3.0%w/
v。
6. the method as described in claim 1, it is characterised in that in the step (1), the pH is 2.5.
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| CN113185406A (en) * | 2021-04-16 | 2021-07-30 | 山东省分析测试中心 | Efficient preparation method of phenolic acid active ingredients in salvia yunnanensis |
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| WO2007070983A1 (en) * | 2005-12-22 | 2007-06-28 | Apollo Life Sciences Limited | Transdermal delivery of pharmaceutical agents |
| CN103439425A (en) * | 2013-08-28 | 2013-12-11 | 中国人民解放军第四军医大学 | Screening method of active pharmaceutical ingredient |
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| WO2007070983A1 (en) * | 2005-12-22 | 2007-06-28 | Apollo Life Sciences Limited | Transdermal delivery of pharmaceutical agents |
| CN103439425A (en) * | 2013-08-28 | 2013-12-11 | 中国人民解放军第四军医大学 | Screening method of active pharmaceutical ingredient |
| CN105486787A (en) * | 2015-12-04 | 2016-04-13 | 广东药学院 | Microemulsion liquid chromatography method for determination of fingerprint of active ingredient in the root of red-rooted salvia and application thereof |
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| CN113185406A (en) * | 2021-04-16 | 2021-07-30 | 山东省分析测试中心 | Efficient preparation method of phenolic acid active ingredients in salvia yunnanensis |
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