CN104316604B - A kind of determination method based on derivative ginsenoside and aglycon thereof - Google Patents
A kind of determination method based on derivative ginsenoside and aglycon thereof Download PDFInfo
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Abstract
The present invention relates to a kind of determination method based on derivative ginsenoside and aglycon thereof, use derivatization reagent 4 '-carboxyrhodamine to carry out derivatization to the free hydroxyl group in ginsenoside and aglycon thereof first, and utilize the mass spectrum enhanced sensitivity analytical approach that the Ultra Performance Liquid Chromatography triple quadrupole bar Mass Spectrometer Method of multiple-reaction monitoring pattern is analyzed.Method of the present invention can be used for analyzing detection 10 kinds of ginsenosides 4 kinds of ginsenoside aglycons, by computational analysis testing result, accurately obtain the content of ginsenoside and aglycon thereof, the method highly sensitive, accuracy is good, selectivity is good, and can improve matrix effect significantly, significant for pharmacology, drug screening, ginseng crude drug and the quality control of ginseng Related product.
Description
Technical field
The present invention relates to analytical chemistry field, be specifically related to a kind of determination method based on derivative ginsenoside and aglycon thereof, especially relate to after one utilizes the activity hydroxy of 4 '-carboxyrhodamine to ginsenoside and aglycon thereof to carry out derivatization and utilize Ultra Performance Liquid Chromatography triple quadrupole bar mass spectroscopy to detect the mass spectrum enhanced sensitivity analytical approach analyzed.
Background technology
Ginsenoside is the main active in ginseng, and much research has been reported ginsenoside and had the effects such as anticancer health-care.Most ginsenoside can be divided into protopanoxadiol (PPD) type and Protopanaxatriol (PPT) type according to the difference of its aglycone structure, and protopanoxadiol (PPD) and Protopanaxatriol (PPT) are also considered to ginsenoside final metabolic product in vivo and embody its pharmacological activity.Ginsenoside, because having superior physiology, pharmacy characteristic, becomes the popular domain of research both at home and abroad.
Can by the molecular structure of highly sensitive detection because ginsenoside and aglycon thereof lack, the detection sensitivity of conventional spectroscopic methodology, chromatography, mass spectroscopy, immunization is difficult to meet microanalysis demand.Document " in high performance liquid chromatography-tandem mass method Simultaneously test Shenmai injection 9 kinds of saponin(es " (Chinese traditional Chinese medicine academic periodical, 32nd volume in 2014,1st phase) and " using high performance liquid chromatography tandem mass spectrum method analyzes Ginsenosides in purplish red ginseng " (Pharmaceutical Analysis magazine, 33rd volume the 5th phase in 2013), have studied chromatograph-mass spectrometer coupling technology for ginsenoside direct-detection, but be only carry out direct-detection for the sample that the saponin contents such as parenteral solution are higher, there is the limitation such as sensitivity is low, matrix interference is serious.Derivative is the effective means improving ginsenoside and the sensitivity of aglycon Mass Spectrometer Method thereof, existing derivating agent utilizes chlorobenzoyl chloride etc. that the hydroxyl on ginsenoside parent nucleus is carried out derivative reaction more, reaction time is long, condition is harsh, existing deriving method uses pyridine to hold concurrently catalyzer as solvent, pyridine high volatility, have pungency, contaminated environment and matrix effect are comparatively obvious, therefore more need to find a kind of more convenient, effective, eco-friendly highly sensitive determination method.
Summary of the invention
Technical matters to be solved by this invention is how accurate and sensitive analysis detects ginsenoside and aglycon thereof.The object of this invention is to provide a kind of determination method based on derivative ginsenoside and aglycon thereof, employ 4 '-carboxyrhodamine first and derivatization is carried out to the activity hydroxy in ginsenoside and aglycon thereof, utilize acetonitrile to instead of traditional high volatility simultaneously, there is irritating pyridine as solvent, solve the problems such as reaction time length.Utilize 4 '-carboxyrhodamine as derivating agent associating Ultra Performance Liquid Chromatography triple quadrupole bar mass spectroscopy, due in rhodamine molecular structure containing a stable quaternary ammonium cation, therefore derivatization method can bring significant mass spectrum enhanced sensitivity Detection results.This method overcomes the problems such as the sensitivity of conventional method is not high, and derivative reaction is gentle quick, Mass Spectrometer Method sensitivity, selectivity and accuracy can be improved, and substantially improve matrix effect, it is significant for pharmacology, drug screening, ginseng crude drug and the quality control of ginseng Related product that the present invention carries out quantitative test to the trace ginsenoside in biological sample and aglycon thereof.
Technical scheme provided by the invention is:
Based on a determination method for derivative ginsenoside and aglycon thereof, it is characterized in that, comprise the following steps:
A adds 4 '-carboxyrhodamine in the extract of ginsenoside and aglycon thereof, the chloro-1-methylpyridinium iodide of 2-and DMAP, acetonitrile, shake up, be placed in water-bath to heat, free hydroxyl group on 4 '-carboxyrhodamine and ginsenoside and aglycon thereof carries out derivative reaction, obtains derivatization product;
Derivatization product in a utilizes Ultra Performance Liquid Chromatography tandem mass spectrometry to carry out analysis and detects by b after membrane filtration;
Described ginsenoside and aglycon thereof have free hydroxyl group in C-20 or C-3 position.
Described analyzing detecting method for analysis thing comprise 10 kinds of ginsenosides and 4 kinds of ginsenoside aglycons, described ginsenoside is protopanaxadiol-type's ginsenoside or Protopanaxatriol's type ginsenoside, described protopanaxadiol-type's ginsenoside is Rg3, Rh2, Rs3 or CompoundK(C-K), described Protopanaxatriol's type ginsenoside is Re, Rf, Rg1, Rg2, Rh1 or F1, described aglycon is protopanaxadiol-type's ginsenoside aglycon or Protopanaxatriol's type ginsenoside aglycon, described protopanaxadiol-type's ginsenoside aglycon is protopanoxadiol (PPD) or panoxadiol (PD), described Protopanaxatriol's type ginsenoside aglycon is Protopanaxatriol (PPT) or panaxatriol (PT).Concrete structure formula is as follows:
The preparation method of the ginsenoside in described step a and the extract of aglycon thereof is ultrasonic wave added-dispersive liquid-liquid microextraction: join in centrifuge tube by the aqueous solution of ginsenoside and aglycon thereof, deionized water constant volume, add extractant and spreading agent again, ultrasonic process puts into supercentrifuge after forming uniform emulsion system, centrifugal 3 minutes, rotating speed is 10000r/min, after centrifugal gained sedimentary facies nitrogen dries up, redissolve in acetonitrile, the extract of obtained ginsenoside and aglycon thereof.
The aqueous solution of above-mentioned ginsenoside and aglycon thereof is the animal blood plasma treating fluid in the researchs such as pharmacology, and the actual sample of the extract of ginseng crude drug and product.
In above-mentioned ultrasonic wave added-dispersive liquid-liquid microextraction technology, extractant is selected from methylene chloride, methenyl choloride, triclene, zellon and chlorobenzene, more preferably methenyl choloride; Spreading agent is selected from acetone, acetonitrile, methyl alcohol and ethanol, more preferably methyl alcohol.
Above-mentioned sonication treatment time is 0-10 minute, and optimized is 2 minutes; In ultrasonic extraction process, pH value regulates between 5-7.
The synthetic method that in described step a, derivating agent 4 '-carboxyrhodamine (CSR) is reported according to Cardoso in 2012 etc. performs (EuropeanJournalofOrganicChemistry, 2012,5810 – 5817), adopting High Performance Liquid Chromatography/Photodiode Array Detection to detect for monitoring synthetic reaction process, can the highest yield be obtained through three microwave radiation technology reaction treatment (each 10min).Thick product adopts semi-preparative liquid chromatography method to carry out purifying, obtains the CRS that purity is 99%, productive rate 45%.
For the ease of controlling the addition of each reagent, above-mentioned 4 '-carboxyrhodamine is dissolved in acetonitrile and obtains 4 '-carboxyrhodamine/acetonitrile solution, in order to obtain the highest derivatization efficiency, the volumetric molar concentration of 4 '-carboxyrhodamine solution is 5-15 times of ginsenoside and aglycon extract volumetric molar concentration thereof, and the volumetric molar concentration of optimized 4 '-carboxyrhodamine solution is 10 times of the concentration of ginsenoside and aglycon extract volumetric molar concentration thereof.
The chloro-1-methylpyridinium iodide (CMPI) of described 2-is dissolved in acetonitrile makes the solution that mass concentration is 1-10%, and its volumetric molar concentration is 500-10000 times of 4 '-carboxyrhodamine volumetric molar concentration, is more preferably 7000 times.
Described DMAP (DMAP) is dissolved in acetonitrile makes the solution that mass concentration is 12-25%, and its volumetric molar concentration is 4 '-carboxyrhodamine volumetric molar concentration 100-1000 times, is more preferably 500 times.
Described derivative reaction heats 10-60 minute for being placed in 25-60 ° of C water-bath.The reaction time more optimized and temperature are in the water-bath of 45 ° of C, heat 30min carry out.
In above-mentioned derivative reaction, the reason of and molecular dynamics sterically hindered due to derivative reaction, the ginsenoside of a part 4 '-carboxyrhodamine and a part or its aglycon react generation one and replace derivatization product, the hydroxyl reaction of 4 '-carboxyrhodamine preferentially and on C-20 position; If C-20 position is without free hydroxyl group, the free hydroxyl group that will again have precedence on C-3 position carries out derivative reaction, due in rhodamine molecular structure containing a stable quaternary ammonium cation, specific generation can contain the report ion of rhodamine structure in mass spectrum multiple-reaction monitoring pattern, bring significant mass spectrum enhanced sensitivity Detection results.Fig. 1 is seen for the derivative reaction schematic diagram of PPD and PD.
In described step b, derivatization product utilizes Ultra Performance Liquid Chromatography-mass spectroscopy to carry out a point agent and analyses detection after 0.45 μm of membrane filtration.
Described Ultra Performance Liquid Chromatography tandem mass spectrometry is Ultra Performance Liquid Chromatography triple quadrupole bar mass spectrometer system: the serial Ultra Performance Liquid Chromatography of Agilent 1290, is equipped with Agilent 6460 triple quadrupole bar tandem mass spectrum system composition.Chromatographic resolution uses Agilent SBC18 post (2.1mm × 50mm, 1.8 μm), and adopt mobile phase linear gradient elution method, sampling volume is 2 μ L, and column temperature is constant in 30 ° of C.
Above-mentioned mobile phase is acid acetonitrile/water solution (containing 0.1% formic acid), and mobile phase is made up of mobile phase A and Mobile phase B, and mobile phase A is that 5% acetonitrile contains 0.1% formic acid, and Mobile phase B is that acetonitrile contains 0.1% formic acid.
During above-mentioned linear gradient elution method 0 minute, the volume ratio of mobile phase A and Mobile phase B is 60:40; During 5min, the volume ratio of mobile phase A and Mobile phase B is 35:65.
The chloro-1-methylpyridinium iodide (CMPI) of 2-described in the present invention, DMAP (DMAP), methenyl choloride, the chemical reagent such as acetone are pure for analyzing, and acetonitrile is that high performance liquid chromatography is pure.
The analyzing detecting method of ginsenoside provided by the invention and aglycon thereof, additionally providing a kind of optimized Mass Spectrometry Conditions is: dry gas temperature 300 ° of C, flow velocity 10L/min, sprayer air pressure 40psi, sheath temperature degree 280 ° of C, flow velocity 11L/min, capillary voltage 3.5kV.
The Ultra Performance Liquid Chromatography triple quadrupole bar mass spectroscopy adopted in the present invention can carry out qualitative and quantitative analysis.For ginseng aglycon PPD and PPT that content in blood plasma is minimum, the process of brief description mass spectrometry, quantitative test.Standard solution containing PPD and PPT or plasma sample solution, obtain CSR derivant after treatment, for ensuing mass spectrophotometry.
The first, adopt the autoscan function of chromatograph-mas spectrometer device, scanning obtains the parent ion of the CSR derivant of PPD and PPT, respectively corresponding [M-H
2o]
+(m/z is respectively 865.3) and [M-2H
2o]
+(m/z865.4), and within the scope of 0-300V, optimize cracked voltage (Fragmentor) parameter of two kinds of parent ions, the cracked voltage of the best of PPD and PPT derivant is respectively 260V and 280V.Second, adopt the autoscan function of chromatograph-mas spectrometer device, the highest product ion of the CSR derivant abundance of PPD and PPT is chosen in scanning, the primary product ion of the two is m/z551.0(quantitative amount of product ion) and the qualitative product ion of m/z623.1(), and within the scope of 0-100ev, optimize impact energy, to obtaining maximum mass spectral sensitivity.In a word, the mass spectrum multiple-reaction monitoring parameter of the CSR derivant of PPD and PPT is: PPD-CSR, quota ion to 865.3 → 551.0, cracked voltage 260V, impact energy 75ev; Qualitative ion pair 865.3 → 623.1, cracked voltage 260V, impact energy 83ev.PPT-CSR, quota ion to 865.4 → 551.0, cracked voltage 280V, impact energy 77ev; Qualitative ion pair 865.4 → 623.1, cracked voltage 280V, impact energy 88ev.In blood plasma, mass spectrum many reaction detection ion flow graph of PPD and PPT is shown in Fig. 2.
Described ginsenoside and aglycon standard items thereof are purchased from Chinese medicine and biological products assay institute.
In order to verify the applicability of the coupling technique that the present invention sets up, for the range of linearity, detection limit, quantitative limit, enrichment factor, accuracy, precision, repeatability, the recovery and matrix effect have all carried out detailed investigation.For the above-mentioned ginsenoside in biological sample or aglycon, its range of linearity is at 0.1 – 100ngmL
-1, regression coefficient R
2all be greater than 0.98, demonstrate good linear, detection limit is respectively 0.010-0.105ngmL
-1, quantitative limit is respectively 0.030-0.350ngmL
-1.1 is the results are shown in Table for the analytical approach of PPT and the PPD checking recovery, matrix effect, reappearance, preci-sion and accuracy result.Result shows, the analytical approach set up can be advantageously applied to and detect ginsenoside in biological sample and aglycon thereof.
Table 1
Ginsenoside in ultrasonic wave added in the present invention-dispersive liquid-liquid microextraction technology Extraction and enrichment biological sample and aglycon thereof, have the advantages such as simple, quick, efficient, green; Utilize 4 '-carboxyrhodamine to carry out derivative reaction mild condition to ginsenoside and aglycon thereof quick, and due in rhodamine molecular structure containing a stable quaternary ammonium cation, therefore derivatization method can bring significant mass spectrum enhanced sensitivity Detection results.
The mass spectrum enhanced sensitivity analytical approach of the ginsenoside based on derivatization of the present invention and aglycon thereof, 4 '-carboxyrhodamine is used to carry out derivatization as derivatization reagent to the ginsenoside of activity hydroxy functional group and aglycon thereof and utilize the analysis of Ultra Performance Liquid Chromatography triple quadrupole bar mass spectrographic mass spectrum enhanced sensitivity first, derivative reaction mild condition is quick, only can complete derivative reaction with 30 minutes.Detection method is highly sensitive, accuracy and selectivity good, matrix effect is little, and detection limit is respectively 0.010-0.105ngmL
-1, quantitative limit is respectively 0.030-0.350ngmL
-1, completely can practical requirement for the Detection results of ginsenoside and aglycon thereof in the biological samples such as rat plasma, ginseng crude drug and product.
Accompanying drawing explanation
Fig. 1 is for PPD and PD, with derivatization reagent CSR respectively at the derivatization reaction schematic diagram of reaction site C-20, C-3.
Fig. 2 is mass spectrum many reaction detection ion flow graph of PPD and PPT in blood plasma.
Embodiment
For ease of understanding summary of the invention, below in conjunction with specific embodiment, the present invention is further described:
Embodiment 1
The detection of ginsenoside aglycon PD and PT in ginseng crude drug
1. ultrasonic wave added-the dispersive liquid-liquid microextraction of ginsenoside aglycon PD and PT
Take 1.0g ginseng crude drug powder (Beijing, Changchun, Jinan pharmacy of Tongrentang buys), extraction process and acid hydrolysis is carried out according to literature method, the ginsenoside in medicinal material is made to be converted into PD and PT(physical and chemical inspection-chemical fascicle, 2010, 46 (5): 482-484), carry out micro-extraction: draw the above-mentioned sample solution of 5ml in point end centrifuge tube, add 500 μ L ethanol as spreading agent, 150 μ L chloroforms are as extractant, add 4.35mL pure water again and make cumulative volume 10mL, ultrasonic assistant under room temperature-dispersive liquid-liquid microextraction 3min, centrifugal 2min (12000rpm), drawing lower floor's organic phase nitrogen blows to dry, add 200 μ L acetonitriles to redissolve, obtained concentration is 2.0 × 10
-5the ginsenoside of mol/L and the extract of aglycon thereof.
2. the derivative reaction of ginsenoside aglycon PD and PT extract
In above-mentioned ginsenoside aglycon extract, add 400 μ L concentration is 2.0 × 10
-44 '-carboxyrhodamine/the acetonitrile solution of mol/L, the DMAP/ acetonitrile solution of 200 μ L concentration to be the CMPI/ acetonitrile solution of 5w% and 200 μ L concentration be 20w%, shakes up after sealing, is placed in 45 ° of C water-baths and reacts 30min.Obtained derivatization product.
3. Ultra Performance Liquid Chromatography triple quadrupole bar mass spectroscopy detects
Derivatization reaction mixture carries out UHPLC-MS/MS(MRM after 0.45 μm of membrane filtration) analyze and detect.Chromatographic resolution: sampling volume is 2 μ L, column temperature is constant in 30 ° of C.Linear gradient elution program 8min completes, and flow velocity is 0.2mL/min.Mobile phase A is that 5% acetonitrile/water contains 0.1% formic acid, and Mobile phase B is that acetonitrile contains 0.1% formic acid.Adopt linear gradient elution method, 0min mobile phase consists of 80%A+20%B, 10%A+90%B during 8min.Positive ion mode, adopts multiple-reaction monitoring pattern.Mass Spectrometry Conditions is: dry gas temperature 300 ° of C, flow velocity 10L/min, sprayer air pressure 40psi, sheath temperature degree 280 ° of C, flow velocity 11L/min, capillary voltage 3.5kV.Grope method according to mass spectrum multiple-reaction monitoring experiment parameter in embodiment 2, obtain the mass spectrum multiple-reaction monitoring experiment condition of PD and PT in ginseng crude drug: PD-CSR derivant, quota ion to 885.4 → 443.1, cracked voltage 280V, impact energy 75ev; Qualitative ion pair 885.4 → 399.1, cracked voltage 280V, impact energy 80ev; PT-CSR derivant, quota ion to 885.4 → 443.1, cracked voltage 280V, impact energy 73ev; Qualitative ion pair 885.4 → 399.1, cracked voltage 280V, impact energy 78ev.Obtain the detection limit of 2 kinds of ginsenoside aglycons (PD and PT) in different pharmacy ginseng crude drug according to the experiment condition of embodiment 3, quantitative limit, the range of linearity, linearly dependent coefficient the results are shown in Table 2.
Table 2
Comparing embodiment 1
The procedure of this embodiment is identical with embodiment 1, just in derivative reaction process, adopts chlorobenzoyl chloride to be derivatization reagent.
Comparing embodiment 2
The procedure of this embodiment is identical with embodiment 1, just in derivative reaction process, adopts 3,5-dinitrobenzoyl chloride to be derivatization reagent.
Following table 3 gives the data result of embodiment 1 with comparing embodiment 1,2 gained
Table 3
Embodiment 2
The detection of ginsenoside and aglycon thereof in rat plasma
1. ultrasonic wave added-the dispersive liquid-liquid microextraction of ginsenoside and aglycon thereof
Get 50 μ L contain ginsenoside and aglycon thereof and Deproteinated rat plasma in centrifuge tube, with deionized water constant volume to 2mL(PH value 5-7), inject the mixed solution containing 80 μ L methenyl cholorides and 300 μ L methyl alcohol fast.After ultrasound wave process 2min, form uniform emulsion system.Solution is put into supercentrifuge, centrifugal 3min, rotating speed is 10000r/m.Centrifugal rear organic phase is deposited on bottom centrifuge tube, and draw sedimentary facies with micro syringe and be transferred in another bottle, nitrogen dries up and redissolves in appropriate acetonitrile afterwards, and obtained concentration is 2.0 × 10
-5the ginsenoside of mol/L and the extract of aglycon thereof.
2. the derivative reaction of ginsenoside and aglycon extract thereof
In step 1, fill in the extract of ginsenoside and aglycon thereof that to add 200 μ L concentration be 3.0 × 10
-44 '-carboxyrhodamine/the acetonitrile solution of mol/L, the DMAP/ acetonitrile solution of 150 μ L concentration to be the CMPI/ acetonitrile solution of 10w% and 150 μ L concentration be 10w%, shakes up after sealing, is placed in 45 ° of C water-baths and reacts 30min.Obtained derivatization product.
3. Ultra Performance Liquid Chromatography triple quadrupole bar mass spectroscopy detects
Derivatization reaction mixture carries out UHPLC-MS/MS(MRM after 0.45 μm of membrane filtration) analyze and detect.Chromatographic resolution: sampling volume is 2 μ L, column temperature is constant in 30 ° of C.Linear gradient elution program 5min completes, and flow velocity is 0.2mL/min.Mobile phase A is that 5% acetonitrile/water contains 0.1% formic acid, and Mobile phase B is that acetonitrile contains 0.1% formic acid.Adopt linear gradient elution method, 0min mobile phase consists of 60%A+30%B, 35%A+65%B during 5min.Positive ion mode, adopts multiple-reaction monitoring pattern.Mass Spectrometry Conditions is: dry gas temperature 300 ° of C, flow velocity 10L/min, sprayer air pressure 40psi, sheath temperature degree 280 ° of C, flow velocity 11L/min, capillary voltage 3.5kV.
Embodiment 3
The detection of ginsenoside and aglycon thereof in rat plasma
1. ultrasonic wave added-the dispersive liquid-liquid microextraction of ginsenoside and aglycon thereof
Get 50 μ L contain ginsenoside and aglycon thereof and Deproteinated rat plasma in centrifuge tube, with deionized water constant volume to 2
ML(PH value 5-7), inject the mixed solution containing 100 μ L methenyl cholorides and 350 μ L methyl alcohol fast.After ultrasound wave process 2min, form uniform emulsion system.Solution is put into supercentrifuge, centrifugal 3min, rotating speed is 10000r/m.Centrifugal rear organic phase is deposited on bottom centrifuge tube, and draw sedimentary facies with micro syringe and be transferred in another bottle, nitrogen dries up and redissolves in appropriate acetonitrile afterwards, and obtained concentration is 2.0 × 10
-5the ginsenoside of mol/L and the extract of aglycon thereof.
2. the derivative reaction of ginsenoside and aglycon extract thereof
In above-mentioned steps 1, fill in the bottle of the extract of ginsenoside and aglycon thereof that to add 600 μ L concentration be 1.0 × 10
-4mol/L4 '-carboxyrhodamine/acetonitrile solution, the 200 μ L concentration DMAP/ acetonitrile solution that to be the CMPI/ acetonitrile solution of 10w% and 200 μ L concentration be for 25w%, shakes up after sealing, is placed in 45 ° of C water-baths and reacts 30min.Obtained derivatization product.
3. Ultra Performance Liquid Chromatography triple quadrupole bar mass spectroscopy detects
In detection analysis, device therefor and each parameter are with embodiment 2.
Following table 4 gives the detection limit, quantitative limit, the range of linearity, the linearly dependent coefficient that obtain 10 kinds of ginsenosides (Rg3, Rh2, Rs3, CompoundK, Re, Rf, Rg1, Rg2, Rh1 and F1) and 2 kinds of aglycons (PPD and PPT) according to the experiment condition of embodiment 2 and embodiment 3.
Table 4
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not by the restriction of embodiment; other is any do not deviate from Spirit Essence of the present invention and principle under make change, modification, combination, substitute, simplify and all should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (4)
1., based on a determination method for derivative ginsenoside and aglycon thereof, it is characterized in that, comprise the following steps:
A adds 4 '-carboxyrhodamine in the extract of ginsenoside and aglycon thereof, the chloro-1-methylpyridinium iodide of 2-and DMAP, acetonitrile, shake up, be placed in water-bath to heat, free hydroxyl group on 4 '-carboxyrhodamine and ginsenoside and aglycon thereof carries out derivative reaction, obtains derivatization product;
The preparation method of the extract of described ginsenoside and aglycon thereof is ultrasonic wave added-dispersive liquid-liquid microextraction: join in centrifuge tube by the aqueous solution of ginsenoside and aglycon thereof, deionized water constant volume, add extractant and spreading agent again, after ultrasonic process forms uniform emulsion system, centrifugal, remove supernatant and obtain sedimentary facies, after sedimentary facies nitrogen dries up, be dissolved in acetonitrile, the extract of obtained ginsenoside and aglycon thereof;
Described derivative reaction heats 10-60 minute for being placed in 25-60 DEG C of water-bath;
The volumetric molar concentration of described 4 '-carboxyrhodamine is 5-15 times of ginsenoside and aglycon extract volumetric molar concentration thereof, the volumetric molar concentration of the chloro-1-methylpyridinium iodide of 2-is 500-10000 times of 4 '-carboxyrhodamine volumetric molar concentration, and the volumetric molar concentration of DMAP is 4 '-carboxyrhodamine volumetric molar concentration 100-1000 times;
Derivatization product in a utilizes Ultra Performance Liquid Chromatography tandem mass spectrometry to carry out analysis and detects by b after membrane filtration;
Chromatography column in described Ultra Performance Liquid Chromatography is SBC18 post, and adopt mobile phase linear gradient elution method, sampling volume is 2 μ L, and column temperature is constant in 30 DEG C;
Described mobile phase is made up of mobile phase A and Mobile phase B, and mobile phase A is that the acetonitrile/water solution of 5% volume fraction contains 0.1% formic acid, and Mobile phase B is that acetonitrile contains 0.1% formic acid; When 0 minute, the volume ratio of mobile phase A and Mobile phase B is 60:40; During 5min, the volume ratio of mobile phase A and Mobile phase B is 35:65;
Described mass spectroscopy is for utilizing triple quadrupole bar mass spectroscopy, and Mass Spectrometry Conditions is: dry gas temperature 300 ° of C, flow velocity 10L/min, sprayer air pressure 40psi, sheath temperature degree 280 ° of C, flow velocity 11L/min, capillary voltage 3.5kV;
Described ginsenoside and aglycon thereof have free hydroxyl group in C-20 or C-3 position.
2. determination method according to claim 1, it is characterized in that: described ginsenoside is protopanaxadiol-type's ginsenoside or Protopanaxatriol's type ginsenoside, described protopanaxadiol-type's ginsenoside is Rg3, Rh2, Rs3 or CompoundK, described Protopanaxatriol's type ginsenoside is Re, Rf, Rg1, Rg2, Rh1 or F1, described aglycon is protopanaxadiol-type's ginsenoside aglycon or Protopanaxatriol's type ginsenoside aglycon, described protopanaxadiol-type's ginsenoside aglycon is protopanoxadiol or panoxadiol, described Protopanaxatriol's type ginsenoside aglycon is Protopanaxatriol or panaxatriol.
3. determination method according to claim 1, is characterized in that: extractant is selected from methylene chloride, methenyl choloride, triclene, zellon or chlorobenzene; Spreading agent is selected from acetone, acetonitrile, methyl alcohol or ethanol.
4. determination method according to claim 1, it is characterized in that: the volumetric molar concentration of 4 '-carboxyrhodamine is 10 times of ginsenoside and aglycon extract volumetric molar concentration thereof, the volumetric molar concentration of the chloro-1-methylpyridinium iodide of 2-is 7000 times of 4 '-carboxyrhodamine volumetric molar concentration, and the volumetric molar concentration of DMAP is 500 times of 4 '-carboxyrhodamine volumetric molar concentration.
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| CN106872627B (en) * | 2015-12-13 | 2019-04-05 | 复旦大学 | A kind of LC-MS detection method of protopanoxadiol |
| CN106645482A (en) * | 2016-12-26 | 2017-05-10 | 河北省药品检验研究院 | Method for determining content of four ginsenoside components in 27-flavor Dingkun pill |
| CN110095524B (en) * | 2019-05-15 | 2022-04-22 | 山东省分析测试中心 | Triterpene saponin mass spectrum structure analysis method |
| CN112305141B (en) * | 2020-10-22 | 2022-12-06 | 天津中医药大学 | Method for constructing ginsenoside virtual database and method for identifying ginsenoside |
| CN114646691A (en) * | 2020-12-18 | 2022-06-21 | 中国科学院上海药物研究所 | Analysis method for simultaneously quantifying 15 ginsenoside components of Danqi Tongmai tablets in rat plasma |
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Non-Patent Citations (4)
| Title |
|---|
| Antitrypanosomal Properties of Panax ginseng C.A. Meyer: New Possibilities for a Remarkable Traditional Drug;Florian Herrmann 等;《PHYTOTHERAPY RESEARCH》;20121231;1-13 * |
| UPLC_HPLC测定血栓通注射液中5个皂苷类成分含量的比较研究;王常顺 等;《药物分析杂志》;20131231;第33卷(第9期);1617-1620 * |
| 人参_附子配伍比例对人参皂苷成分溶出影响研究;汤响林 等;《中草药》;20130131;第44卷(第1期);36-41 * |
| 高效液相色谱衍生化法测定红参及生脉注射液中人参二醇和人参三醇的含量;李章万 等;《华西药学杂志》;19991231;第14卷(第4期);271-273 * |
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