CN100390538C - Detection method for dammarane type four-ring triterpenoid saponin - Google Patents
Detection method for dammarane type four-ring triterpenoid saponin Download PDFInfo
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Abstract
The present invention discloses a measuring method of dammarane type tetracyclic triterpenoid saponin and secondary aglycone. In the method, a device combining a high-performance liquid chromatogram and a mass spectrum is used, wherein the conditions of the high-performance liquid chromatogram are as follows: a chromatographic column is an ODS C18 column; the temperature of the column is from 20 to 38 DEG C. The conditions of the mass spectrum are as follows: an ion source is a Turbo Ionspray source; a jetting voltage is from 3000 to 5000V; a temperature is from 250 to 300 DEG C; a measurer uses triplicate four-electrode bar tandem mass spectrum measurement and positive ion measurement; a scanning mode is multi-reaction measurement (MRM); CE is from 17 to 36V; DP is from 80 to 110V; for the measurement of ions, collision-induced dissociation is carried out to the quasi-molecular ion peaks of the monomeric saponin of the dammarane type tetracyclic triterpenoid saponin of a first-level whole-scanning mass spectrum to obtain secondary fragment ions. The scanning mode of multi-reaction measurement (MRM) is used for quantitatively analyzing the ion reaction. The blood concentration of the monomeric saponin of the dammarane type tetracyclic triterpenoid saponin and the secondary aglycone can be accurately reflected.
Description
Technical field
The invention provides a kind of sample detection method that contains micro-dammarane type four-ring triterpenoid saponin.
Technical background
Modern study shows that the saponins material in Chinese medicine genseng, American Ginseng, the pseudo-ginseng is an active component, and all contain dammarane type four-ring triterpenoid saponin (Chemistry for Chinese Traditional Medicine, Kuang Hai learns P231-232), its aglycon mainly contains two kinds on 20 (S)-protopanoxadiol types and 20 (S)-Protopanaxatriol types, has β-OH to exist at C3, C12 and C20 position.The difference of protopanoxadiol type and Protopanaxatriol's type only is that the latter also has the α-OH of C6 position, and the position that becomes glycosides is usually in C3, C20 and C6 position.
So far, people have obtained 28 kinds of monomer saponins to the different parts separation of pseudo-ginseng (Panax notoginseng F.H.Chen and himalaicus).Wherein, 13 kinds of Protopanaxatriol's type saponin(es, 15 kinds of protopanoxadiol type saponin(es, Protopanaxatriol's type saponin(e is 3: 1 (1.Masayuki Yoshikawa with the content ratio of protopanoxadiol type saponin(e, et al.Bioactive Saponinsand Glycosides.VIII.Notoginseng (1): New Dammarane-TriterpeneOligoglycosides, Notoginsenosides-A,-B,-C, and-D, from the Dried Root of Panax notoginseng (Burk.) .Chem PharmBull, 1997,45 (6): 1039.
Contain 5 kinds of Protopanaxatriol's type saponin(es in the genseng (Panax ginseng), 8 kinds of protopanoxadiol type saponin(es (Chemistry for Chinese Traditional Medicine, Kuang Hai learns P252-257).
Contain in the American Ginseng (Panax quinquefolium L.) 5 kinds of protopanoxadiol type saponin(es (Cao Min. the recent developments of general introduction American Ginseng chemical constitution study, Jiangsu pharmacy and clinical research, 2004,12 (2): 25).
But because the saponin(e concentration in the blood plasma is too low behind oral this saponins, prior art can't detect, thereby for its bioavilability, the interior mechanism of action of body etc. can't carry out deep evaluation and research.Therefore, if set up a kind of saponin(e in the blood plasma and method of aglycon or secondary aglycon thereof of can detecting behind oral this saponins, will help the development of above-mentioned research.
Summary of the invention
One object of the present invention has been to provide a kind of new method that is used for detecting dammarane type four-ring triterpenoid saponin monomer saponin content in blood plasma, and another object of the present invention has been to provide a kind of new method of quality control that is used for containing dammarane type four-ring triterpenoid saponin monomer saponin preparation.
Technical scheme
Detection method of the present invention adopts high performance liquid chromatography-GC-MS to carry out, and is used to detect dammarane type four-ring triterpenoid saponin, comprising Panax Notoginseng saponin R
1, the ginsenoside Rg
1, ginsenoside Rb
1And the detection method of the secondary aglycon panaxatriol of the secondary aglycon panoxadiol of protopanoxadiol type saponin(e, Protopanaxatriol's type saponin(e.Saponin(e of the present invention is tablet, capsule, granule, oral liquid and other dosage forms of active component from arasaponin and extract thereof with the pseudo-ginseng, also applicable to ginsenoside, gypenoside, tetracyclic triterpene saponinses such as American ginseng saponin.Detection method of the present invention can be used to contain the quality control of above-mentioned dammarane type four-ring triterpenoid saponin pharmaceutical preparation.
Wherein the high-efficient liquid phase chromatogram condition of high performance liquid chromatography-GC-MS of the present invention is: chromatographic column: ODS (octadecyl bonding phase) C18 post, and moving phase:
Detecting device: triple quadrupole bar tandem mass spectrum detects
Column temperature: 20-38 ℃
The mass spectrum condition:
Ion gun: eddy current ionspray source, injection electric: 3000-5000V, temperature: 250-300 ℃,
Positive ion detects
Scan mode is many reaction detection
Collision voltage: 17-36V; Remove family's voltage: 80-110V
Detect ion: the quasi-molecular ion peak to the monomer saponin of the mass spectral dammarane type four-ring triterpenoid saponin of one-level full scan carries out collision induced dissociation, obtains the secondary fragmention, adopts many reaction detection scan mode, and above-mentioned ionic reaction is carried out quantitative test.Wherein, the Panax Notoginseng saponin R of monomer saponin in the dammarane type four-ring triterpenoid saponin for example
1, the ginsenoside Rg
1, ginsenoside Rb
1And the one-level full scan mass spectrum quasi-molecular ion peak of the secondary aglycon panaxatriol of the secondary aglycon panoxadiol of protopanoxadiol type saponin(e, Protopanaxatriol's type saponin(e and carry out the peak value that collision induced dissociation obtains the secondary fragmention and be:
The mass spectral Panax Notoginseng saponin R of one-level full scan
1Quasi-molecular ion peak is m/z 355.6, and m/z 405.8, and m/z 423.6, m/z441.7, m/z 735.7, and m/z 753.9, m/z950.8, m/z 956.0, and m/z 971.8, m/z 1006.9, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z203.3, m/z643.1, m/z723.5, m/z423.
The ginsenoside Rg
1The mass spectral quasi-molecular ion peak of one-level full scan is m/z374.3, m/z396.6, m/z405.7, m/z423.0, m/z441.7, m/z 491.6, and m/z 603.7, m/z801.6, m/z 823.8, m/z830.7, m/z840.6, m/z874.9, m/z 878.6, m/z915.9, and m/z 940.7, and m/z 962.8, m/z1006.9 carries out collision induced dissociation to it, obtains the secondary fragmention and is respectively m/z 203.3, m/z643.1, m/z823.5, m/z423.5.
Ginsenoside Rb
1The mass spectral quasi-molecular ion peak of one-level full scan is m/z1109.9, m/z1112.2, m/z1127.0, m/z1129.3, m/z1132.8, m/z1148.9, m/z1173.7, m/z1217.6, m/z1216.1, m/z m/z1231.7, m/z1300.6 carries out collision induced dissociation to it, obtain the secondary fragmention and be respectively m/z144.7, m/z163.4, m/z325.4, m/z 487.3.
The mass spectral quasi-molecular ion peak of panoxadiol one-level full scan is m/z425.6, m/z443.6, m/z444.7, m/z459.6, m/z461.5, m/z462.6, m/z463.6, m/z483.6, m/z484.4, m/z499.5, m/z524.5, m/z534.6, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z99.0, m/z109.4, m/z122.0, m/z127.1, m/z191.5, m/z203.2, m/z206.9, m/z217.5, m/z235.0, m/z271.3, m/z369.4, m/z407.6, m/z425.4, m/z443.5, m/z461.4.
The mass spectral quasi-molecular ion peak of one-level full scan of panaxatriol is m/z127.2, m/z423.5, m/z441.6, m/z459.6, m/z477.5, m/z499.6, m/z550.7 carries out collision induced dissociation to it, obtain the secondary fragmention and be respectively m/z99.1, m/z109.6, m/z123.0, m/z127.1, m/z187.6, m/z203.1, m/z205.2, m/z207.2, m/z217.3, m/z405.7, m/z423.5, m/z441.5, m/z459.6, m/z477.5.
The disposal route of plasma sample:
Precision is measured blood plasma 0.5ml, adds the organic solvent protein precipitation of 2-5 times of volume, behind vortex and the high speed centrifugation, gets the supernatant sample introduction, and sampling volume is 20 μ l.
Disposal route can also be that precision is measured blood plasma 0.5ml, the 8000-15000r/min rotating speed is centrifugal, draw solid phase extraction column on the supernatant, adopt the 2ml redistilled water, after the drip washing of 2ml 15-25% methyl alcohol, with 2ml chromatogram methanol-eluted fractions, collect eluent, 50-70 ℃ of following nitrogen dries up, and redissolves with the 50-70% methanol solution, get the supernatant sample introduction behind the high speed centrifugation, sampling volume is 20 μ l.
The disposal route of plasma sample can be preferably:
A. precision is measured blood plasma 0.5ml, adds the organic solvent protein precipitation of 3 or 4 times of volumes, behind vortex and the high speed centrifugation, gets the supernatant sample introduction, and sampling volume is 20 μ l; Or
B. precision is measured blood plasma 0.5ml, the 10000r/min rotating speed is centrifugal, draw solid phase extraction column on the supernatant, adopt the 2ml redistilled water, after the 2ml 20% methyl alcohol drip washing, with 2ml chromatogram methanol-eluted fractions, collect eluent, 60 ℃ of following nitrogen dry up, and redissolve with 50% methanol solution, get the supernatant sample introduction behind the high speed centrifugation, sampling volume is 20 μ l.
Above-mentioned high performance liquid chromatogram (HPLC)-mass spectrum (MS) analytic system optimum condition is: chromatographic column: Kromasil C18 post (50mm * 2.1mm, 5 μ m), moving phase adopts methyl alcohol, the 10mM ammonium acetate carries out gradient elution, 0 moment methyl alcohol, the volume ratio of 10mM ammonium acetate is 50%: 50%, methyl alcohol during with the 4th minute, the volume ratio of 10mM ammonium acetate is 50%: 50%, methyl alcohol in the time of the 4.1st minute, the volume ratio of 10mM ammonium acetate is 90%: 10%, methyl alcohol in the time of the 14.0th minute, the volume ratio of 10mM ammonium acetate is 90%: 10%, methyl alcohol in the time of the 25th minute, the volume ratio of 10mM ammonium acetate is 50%: 50%, and flow velocity is 0.25 (ml/min); Mass spectrum condition intermediate ion source: eddy current ionspray source, injection electric: 4000V, positive ion detects, and temperature is 300 ℃.
Beneficial effect
High performance liquid chromatogram (HPLC)-mass spectrum (MS) analytic system single injected sampling that the present invention sets up can determine the concentration of dammarane type four-ring triterpenoid saponin monomer saponin simultaneously exactly, thereby can reflect the blood concentration of dammarane type four-ring triterpenoid saponin monomer saponin exactly.This method is suitable for containing the detection of the blood concentration of dammarane type four-ring triterpenoid saponin monomer saponin in the animal of dammarane type four-ring triterpenoid saponin monomer saponin medicine and the clinical testing, to estimating biosome the absorption and the mechanism of action of dammarane type four-ring triterpenoid saponin monomer saponin is had important effect.This method can reflect Panax Notoginseng saponin R especially exactly
1, the ginsenoside Rg
1, ginsenoside Rb
1, protopanoxadiol type saponin(e the blood concentration of secondary aglycon panaxatriol of secondary aglycon panoxadiol, Protopanaxatriol's type saponin(e.This method also is more suitable for Panax Notoginseng saponin R in the animal of the medicine that contains arasaponin and clinical testing
1, the ginsenoside Rg
1, ginsenoside Rb
1, protopanoxadiol type saponin(e the detection of blood concentration of secondary aglycon panaxatriol of secondary aglycon panoxadiol, Protopanaxatriol's type saponin(e, to estimating biosome the absorption and the mechanism of action of arasaponin is had important effect.Detection method of the present invention has good sensitivity and linear relationship, can satisfy trace (10
-9G/ml) the analysis requirement of sample.Test the requirement that various monomers can both reach the recovery by average recovery simultaneously.
The present invention is described in further detail for following embodiment, but should not be construed as limitation of the present invention.
Blood concentration detects test in the beasle dog body of embodiment 1 oral commercially available notoginsenoside sheet (XUESAITONG PIAN)
1. medication: beasle dog fasting 12h before the test, press arasaponin 90mg/kg dosed administration early morning.Get blank blood before taking medicine, in the back 0.5,1,2,3,4 of taking medicine,, 6,8,10,12,16,24,36,48h gets foreleg venous blood 3ml, anticoagulant heparin, immediately in the centrifugal 5min of 3000rpm, it is standby in-20 ℃ of preservations to isolate blood plasma.
2. the processing of plasma sample: precision is measured blood plasma 0.5ml, the 10000r/min rotating speed is centrifugal, draw solid phase extraction column on the supernatant, adopt the 2ml redistilled water, after the 2ml 20% methyl alcohol drip washing, with 2ml chromatogram methanol-eluted fractions, collect eluent, 60 ℃ of following nitrogen dry up, and redissolve with 50% methanol solution, get the supernatant sample introduction behind the high speed centrifugation, sampling volume is 20 μ l.
3. analysis condition:
High-efficient liquid phase chromatogram condition
Chromatographic column: Kromasil C18 post (50mm * 2.1mm, 5 μ m)
Moving phase:
Detecting device: triple quadrupole bar tandem mass spectrum detects
Column temperature: 22 ℃
The mass spectrum condition
Ion gun: eddy current ionspray source, injection electric: 4000V, positive ion detects, and temperature: 300 ℃ of scan modes are many reaction detection
Collision voltage: 17-36V; Remove family's voltage: 80-110V
Detect ion:
The mass spectral Panax Notoginseng saponin R of one-level full scan
1, the ginsenoside Rg
1, ginsenoside Rb
1, panoxadiol, panaxatriol quasi-molecular ion peak be respectively m/z950.8, m/z801.6, m/z1127.0, m/z461.5 and m/z477.5, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z423, m/z423.5, m/z487.3, m/z127.1 and m/z127.1, adopt many reaction detection (MRM) scan mode, to carrying out quantitative test with above-mentioned ionic reaction, retention time is respectively 2.13min, 2.85min, 9.06min, 12.47min, 9.51min.Internal standard compound is the Ge Lieli urea, and its secondary fragmention retention time is 6.21min.
With concentration is horizontal ordinate, with the ratio of the peak area of internal standard compound be ordinate, carry out regressing calculation by weighted least-squares method, the typical curve equation, calculate plasma concentration.
4. the foundation of typical curve
Precision is measured blank plasma 0.5ml, puts in the centrifuge tube, the accurate respectively Panax Notoginseng saponin R that adds
1, the ginsenoside Rg
1, ginsenoside Rb
1, panoxadiol and panaxatriol the standard solution of variable concentrations, vortex mixed 1min adds 2ml methanol extraction albumen, behind vortex and the high speed centrifugation, gets the supernatant sample introduction, sampling volume is 20 μ l, the record chromatogram.
Typical curve is with Panax Notoginseng saponin R in the blood plasma
1, the ginsenoside Rg
1, ginsenoside Rb
1, panoxadiol and panaxatriol concentration be horizontal ordinate, with the ratio of the peak area of internal standard compound be ordinate, carry out regressing calculation by weighted least-squares method, the gained linear regression equation is typical curve.
The result is as follows:
(1) Panax Notoginseng saponin R
1Typical curve
The range of linearity: 2.8-140ng/ml
Detection is limited to 0.5ng/ml
Y: detect secondary quasi-molecular ions area/interior mark peak area
X: concentration ng/ml (10
-9G/ml)
R: related coefficient
Equation is y=0.0024x+0.0068
R＝0.9967
(2) ginsenoside Rg
1Typical curve
The range of linearity: 3.8-190ng/ml,
Detection is limited to 0.8ng/ml
Y: detect secondary quasi-molecular ions area/interior mark peak area
X: concentration ng/ml (10
-9G/ml)
R: related coefficient
Equation is y=0.001x+0.0015
R＝0.9991
(3) ginsenoside Rb
1Typical curve
The range of linearity: 18-900ng/ml
Detection is limited to 1ng/ml
Y: detect secondary quasi-molecular ions area/interior mark peak area
X: concentration ng/ml (10
-9G/ml)
R: related coefficient
Equation is y=0.001x+0.0197
R＝0.9982
(4) panoxadiol typical curve
The range of linearity: 1.0ng/ml-1000ng/ml
Detectability: 0.5ng/ml
Y: detect secondary quasi-molecular ions area/interior mark peak area
X: concentration ng/ml (10
-9G/ml)
R: related coefficient
Equation is y=0.0201x+0.8045
R＝0.9951
(5) panaxatriol typical curve
Range of linearity 0.53-26.5ng/ml
Detectability: 0.05ng/ml
Y: detect secondary quasi-molecular ions area/interior mark peak area
X: concentration ng/ml (10
-9G/ml)
R: related coefficient
Equation is y=0.0083x+0.0147
R＝0.9969
Above result shows that the method for being set up has good sensitivity and linear relationship, can satisfy trace (10
-9G/ml) the analysis requirement of sample.
5. the method recovery is investigated
Precision is measured blank plasma 0.5ml, the accurate respectively Panax Notoginseng saponin R that adds
1, the ginsenoside Rg
1, ginsenoside Rb
1.The standard solution of high, medium and low three kinds of concentration of panoxadiol and panaxatriol, by above-mentioned experimental example 1 plasma treatment method operation, the record chromatogram, other gets same concentration standard product direct injected, the record chromatogram.Relative recovery is calculated in three parts of replicate determinations.
Relative recovery=(amount of recording after the processing/addition) * 100% result is as follows:
Panax Notoginseng saponin R
1
The ginsenoside Rg
1
Ginsenoside Rb
1
Panoxadiol
Panaxatriol
The result meets the requirement of analytical approach.
6. plasma concentration result such as following table:
Panax Notoginseng saponin R
1
| Time (h) | 0 | 0.5 | 1 | 2 | 4 | 6 | 8 | 12 | 16 | 24 |
| Concentration (ng/ml) | 0.000 | 0.627 | 0.947 | 0.172 | 21.200 | 20.400 | 4.640 | 1.273 | 1.599 | 0.829 |
The ginsenoside Rg
1
| Time (h) | 0 | 0.5 | 1 | 2 | 4 | 6 |
| Concentration (ng/ml) | 0.000 | 2.540 | 3.060 | 15.800 | 70.100 | 44.300 |
| Time (h) | 8 | 12 | 16 | 24 | 36 | 48 |
| Concentration (ng/ml) | 12.100 | 1.210 | 1.838 | 1.970 | 1.467 | 0.000 |
Ginsenoside Rb
1
| Time (h) | 0 | 0.5 | 1 | 2 | 4 | 6 | 8 | 12 | 16 | 24 | 36 | 48 |
| Concentration (ng/ml) | 0 | 0 | 0 | 73.6 | 505 | 1050 | 762 | 806 | 284 | 618 | 495 | 384 |
Panoxadiol:
| Time (h) | 0 | 0.5 | 1 | 2 | 4 | 6 |
| Concentration (ng/ml) | 0.000 | 9.881 | 0.838 | 0.540 | 1.357 | 0.439 |
| Time (h) | 8 | 12 | 16 | 24 | 36 | 48 |
| Concentration (ng/ml) | 0.735 | 0.941 | 0.405 | 23.749 | 0.994 | 0.685 |
Panaxatriol:
| Time (h) | 0 | 1 | 4 | 6 | 8 | 10 | 24 | 36 | 48 |
| Concentration (ng/ml) | 0.083 | 0.450 | 0.263 | 0.305 | 0.267 | 0.238 | 0.317 | 0.335 | 0.347 |
The detection of blood concentration test in the beasle dog body behind the embodiment 2 oral American ginseng capsules
Medication: beasle dog fasting 12h before the test, press American ginseng saponin 100mg/kg dosed administration early morning.Get blank blood before taking medicine, in the back 0.5,1,2,3,4 of taking medicine,, 6,8,10,12,16,24,36,48h gets foreleg venous blood 3ml, anticoagulant heparin, immediately in the centrifugal 5min of 3000rpm, it is standby in-20 ℃ of preservations to isolate blood plasma.
The processing of plasma sample: precision is measured blood plasma 0.5ml, puts in the centrifuge tube, adds 2ml methanol extraction albumen, behind vortex and the high speed centrifugation, gets the supernatant sample introduction, and sampling volume is 20 μ l, the record chromatogram.
Analysis condition:
High-efficient liquid phase chromatogram condition
Chromatographic column: SGE C18 post (50mm * 2.1mm, 5 μ m)
Moving phase:
Detecting device: triple quadrupole bar tandem mass spectrum detects
Column temperature: 25 ℃
The mass spectrum condition
Ion gun: eddy current ionspray source, injection electric: 4000V, positive ion detects, temperature: 280 ℃
Be many reaction detection
Collision voltage: 17-36V; Remove family's voltage: 80-110V
Detect ion:
The mass spectral ginsenoside Rb of one-level full scan
1, panoxadiol, panaxatriol quasi-molecular ion peak be respectively m/z1183, m/z461.1 and m/z477.6, selectivity is carried out collision induced dissociation to it, obtain the secondary fragmention and be respectively m/z487.2, m/z127.1 and m/z127.1, adopt many reaction detection scan mode, to carrying out quantitative test with above-mentioned ionic reaction, retention time is respectively 1.93min, 11.52min, 8.51min internal standard compound is the Ge Lieli urea, its secondary fragmention retention time is 6.21min.
With concentration is horizontal ordinate, with the ratio of the peak area of internal standard compound be ordinate, carry out regressing calculation by weighted least-squares method, the typical curve equation, calculate plasma concentration.
Plasma concentration result such as following table:
Ginsenoside Rb
1
| Time (h) | 0 | 0.5 | 1 | 2 | 4 | 6 | 8 | 12 | 16 | 24 | 36 | 48 |
| Concentration (ng/ml) | 0 | 0 | 0 | 53.6 | 430 | 850 | 742 | 776 | 263 | 586 | 460 | 338 |
Panoxadiol:
| Time (h) | 0 | 0.5 | 1 | 2 | 4 | 6 |
| Concentration (ng/ml) | 0.000 | 7.332 | 0.632 | 0.520 | 1.096 | 0.236 |
| Time (h) | 8 | 12 | 16 | 24 | 36 | 48 |
| Concentration (ng/ml) | 0.523 | 0.736 | 0.502 | 19.360 | 0.763 | 0.362 |
Panaxatriol:
| Time (h) | 0 | 1 | 4 | 6 | 8 | 10 | 24 | 36 | 48 |
| Concentration (ng/ml) | 0.046 | 0.630 | 0.123 | 0.230 | 0.126 | 0.136 | 0.265 | 0.156 | 0.158 |
Claims (9)
1. the detection method of a dammarane type four-ring triterpenoid saponin, it is characterized in that this method adopts high performance liquid chromatography-GC-MS, wherein high-efficient liquid phase chromatogram condition is: chromatographic column: ODS C18 post, moving phase: adopt methyl alcohol, the 10mM ammonium acetate carries out gradient elution, 0 moment methyl alcohol, the volume ratio of 10mM ammonium acetate is 40-60%: 60-40%, methyl alcohol during with the 4th minute, the volume ratio of 10mM ammonium acetate is 40-60%: 60-40%, methyl alcohol in the time of the 4.1st minute, the volume ratio of 10mM ammonium acetate is 70-90%: 30-10%, methyl alcohol in the time of the 14.0th minute, the volume ratio of 10mM ammonium acetate is 70-90%: 30-10%, methyl alcohol in the time of the 25th minute, the volume ratio of 10mM ammonium acetate is 40-60%: 60-40%, flow velocity is 0.2-0.4 (ml/min), detecting device: triple quadrupole bar tandem mass spectrum detects, column temperature: 20-38 ℃; Mass spectrum condition: ion gun: eddy current ionspray source, injection electric: 3000-5000V, temperature: 250-300 ℃, positive ion detects, and scan mode is many reaction detection, collision voltage: 17-36V, remove family's voltage: 80-110V, detect ion: the quasi-molecular ion peak to the mass spectral dammarane type four-ring triterpenoid saponin monomer saponin of one-level full scan carries out collision induced dissociation, obtains the secondary fragmention, adopt many reaction detection scan mode, above-mentioned ionic reaction is carried out quantitative test.
2. the detection method of dammarane type four-ring triterpenoid saponin as claimed in claim 1, it is characterized in that moving phase adopts methyl alcohol in this method, the 10mM ammonium acetate carries out gradient elution, 0 moment methyl alcohol, the volume ratio of 10mM ammonium acetate is 50%: 50%, methyl alcohol during with the 4th minute, the volume ratio of 10mM ammonium acetate is 50%: 50%, methyl alcohol in the time of the 4.1st minute, the volume ratio of 10mM ammonium acetate is 90%: 10%, methyl alcohol in the time of the 14.0th minute, the volume ratio of 10mM ammonium acetate is 90%: 10%, methyl alcohol in the time of the 25th minute, the volume ratio of 10mM ammonium acetate is 50%: 50%, flow velocity is 0.25 (ml/min), chromatographic column: ODS C18 post (50mm * 2.1mm, 5 μ m); Mass spectrum condition intermediate ion source: eddy current ionspray source, injection electric: 4000V, positive ion detects, and temperature is 300 ℃.
3. the detection method of dammarane type four-ring triterpenoid saponin as claimed in claim 1 or 2, the disposal route that it is characterized in that plasma sample in this method comprises any of following method:
A. precision is measured blood plasma 0.5ml, adds the organic solvent protein precipitation of 2-5 times of volume, behind vortex and the high speed centrifugation, gets the supernatant sample introduction, and sampling volume is 20 μ l;
B. precision is measured blood plasma 0.5ml, the 8000-15000r/min rotating speed is centrifugal, draw solid phase extraction column on the supernatant, adopt the 2ml redistilled water, after the drip washing of 2ml 15-25% methyl alcohol, with 2ml chromatogram methanol-eluted fractions, collect eluent, 50-70 ℃ of following nitrogen dries up, and redissolves with the 50-70% methanol solution, get the supernatant sample introduction behind the high speed centrifugation, sampling volume is 20 μ l.
4. the detection method of dammarane type four-ring triterpenoid saponin as claimed in claim 3 is characterized in that described dammarane type four-ring triterpenoid saponin is the monomer saponin in the arasaponin.
5. the detection method of the dammarane type four-ring triterpenoid saponin described in claim 4 is characterized in that monomer saponin in the described arasaponin is any one in the secondary aglycon panaxatriol of the secondary aglycon panoxadiol of notoginsenoside R, ginsenoside Rg1, ginsenoside Rb1, protopanoxadiol type saponin(e or Protopanaxatriol's type saponin(e.
6. the detection method of the dammarane type four-ring triterpenoid saponin described in claim 5, it is characterized in that detecting in the mass spectrum condition ion: the mass spectral notoginsenoside R quasi-molecular ion peak of one-level full scan is m/z 355.6, m/z 405.8, m/z 423.6, m/z 441.7, m/z 735.7, and m/z 753.9, m/z950.8, m/z 956.0, m/z 971.8, and m/z 1006.9, and it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z203.3, m/z643.1, m/z723.5, m/z423;
The mass spectral quasi-molecular ion peak of ginsenoside Rg1's one-level full scan is m/z374.3, m/z396.6, m/z405.7, m/z423.0, m/z441.7, m/z 491.6, and m/z 603.7, m/z801.6, m/z 823.8, m/z830.7, m/z840.6, m/z874.9, m/z 878.6, m/z915.9, and m/z 940.7, and m/z 962.8, m/z1006.9 carries out collision induced dissociation to it, obtains the secondary fragmention and is respectively m/z 203.3, m/z643.1, m/z823.5, m/z423.5;
The mass spectral quasi-molecular ion peak of ginsenoside Rb1's one-level full scan is m/z1109.9, m/z1112.2, m/z1127.0, m/z1129.3, m/z1132.8, m/z1148.9, m/z1173.7, m/z1217.6, m/z1216.1, m/z m/z1231.7, m/z1300.6 carries out collision induced dissociation to it, obtain the secondary fragmention and be respectively m/z144.7, m/z163.4, m/z325.4, m/z 487.3;
The mass spectral quasi-molecular ion peak of panoxadiol one-level full scan is m/z425.6, m/z443.6, m/z444.7, m/z459.6, m/z461.5, m/z462.6, m/z463.6, m/z483.6, m/z484.4, m/z499.5, m/z524.5, m/z534.6, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z99.0, m/z109.4, m/z122.0, m/z127.1, m/z191.5, m/z203.2, m/z206.9, m/z217.5, m/z235.0, m/z271.3, m/z369.4, m/z407.6, m/z425.4, m/z443.5, m/z461.4;
The mass spectral quasi-molecular ion peak of one-level full scan of panaxatriol is m/z127.2, m/z423.5, m/z441.6, m/z459.6, m/z477.5, m/z499.6, m/z550.7 carries out collision induced dissociation to it, obtain the secondary fragmention and be respectively m/z99.1, m/z109.6, m/z123.0, m/z127.1, m/z187.6, m/z203.1, m/z205.2, m/z207.2, m/z217.3, m/z405.7, m/z423.5, m/z441.5, m/z459.6, m/z477.5.
7. the detection method of the dammarane type four-ring triterpenoid saponin described in claim 6, it is characterized in that detecting in the mass spectrum condition ion: the quasi-molecular ion peak of the mass spectral notoginsenoside R of one-level full scan, ginsenoside Rg1, ginsenoside Rb1, panoxadiol, panaxatriol is respectively m/z950.8, m/z801.6, m/z1127.0, m/z461.5 and m/z477.5, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z423, m/z423.5, m/z487.3, m/z127.1 and m/z127.1.
8. method of quality control that contains the dammarane type four-ring triterpenoid saponin pharmaceutical preparation is characterized in that this method uses the detection method of above-mentioned dammarane type four-ring triterpenoid saponin described in claim 7.
9. the method for quality control that contains the dammarane type four-ring triterpenoid saponin pharmaceutical preparation as claimed in claim 8 is characterized in that containing that the dammarane type four-ring triterpenoid saponin pharmaceutical preparation is meant arasaponin or its extract or be the various preparations of active component or ginsenoside, gypenoside, American ginseng saponin with the pseudo-ginseng.
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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 |
| CN106918652A (en) * | 2015-12-26 | 2017-07-04 | 复旦大学 | A kind of protopanoxadiol human body metabolism product detection method |
| CN107727772B (en) * | 2017-11-21 | 2020-04-10 | 匡海学 | Method for qualitatively and quantitatively detecting triterpenoid saponin in traditional Chinese medicine by using electrospray protonation cracking-mass spectrum multiple reaction detection mode |
| CN108445126B (en) * | 2018-02-02 | 2020-08-14 | 广州白云山和记黄埔中药有限公司 | Detection method of gypenoside extract |
| CN108918733B (en) * | 2018-07-28 | 2021-05-11 | 夏永刚 | Method for predicting collision energy required by quantitative determination of content of triterpenoid saponins in traditional Chinese medicine based on electrospray protonation cracking |
| CN109765308A (en) * | 2018-12-25 | 2019-05-17 | 浙江大学 | A kind of UPLC-UV-QTOF-MS/MS method of quantitative detection Seed of Camellia Sinensis triterpenoid saponin |
| CN110095524B (en) * | 2019-05-15 | 2022-04-22 | 山东省分析测试中心 | Triterpene saponin mass spectrum structure analysis method |
| CN110849993B (en) * | 2019-11-21 | 2022-07-05 | 华侨大学 | Structure presumption method for oil-tea camellia seed oil saponin compound |
| CN111337612B (en) * | 2020-04-17 | 2022-07-12 | 合肥诺明药物安全研究有限公司 | Quantitative analysis method for HYA13369 concentration in blood plasma and tissues |
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