CN1885032A - 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 invention relates to a method for detecting dammarane tetracyclic triterpenoid saponin and secondary aglycone. The inventive method uses high-efficiency liquid color spectrum-mass spectrum coupled device, wherein the conditions of high-efficiency liquid color spectrum comprises: color spectrum column is ODSC18 column; the column temperature is 20-38Deg. C; the conditions of mass spectrum comprise: the ion source is Turbo Ionspray source; the ejecting voltage is 3000-5000V; the Tem is 250-300Deg. C; the detector uses triplicate four-electrode serial mass spectrum detection and positive ion detection; the scanning method uses multi-reaction detection (MRM); CE is 17-36V; DP is 8-110V; the ion detection comprises: processing impact induce dissociation on the quasi-molecule ion peak of said saponin of first-stage scanning mass spectrum, to obtain the second-stage fragment ion; and uses multi-reaction detection (MRM) scanning method to analyze the ion reaction quantitatively, to represent the blood drug density of monomer saponin and secondary aglycone.
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 a1.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:
| Time (min) | Flow velocity (ml/min) | Methyl alcohol | The 10mM ammonium acetate |
| 0 4 4.1 14.0 25 | 0.2-0.4 0.2-0.4 0.2-0.4 0.2-0.4 0.2-0.4 | 40-60％ 40-60％ 70-90％ 70-90％ 40-60％ | 60-40％ 60-40％ 30-10％ 30-10％ 60-40％ |
Detecting device: triple quadrupole bar tandem mass spectrum detects
Column temperature: 20-38 ℃
The mass spectrum condition:
Ion gun: Turbo Ionspray source, injection electric: 3000-5000V, Tem:250-300 ℃,
Positive ion detects
Scan mode is many reaction detection (MRM)
CE：17-36V； DP：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, obtain the secondary fragmention, adopt many reaction detection (MRM) scan mode, 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/z 950.8, and 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/z 203.3, m/z 643.1, m/z 723.5, and m/z 423.
The ginsenoside Rg
1The mass spectral quasi-molecular ion peak of one-level full scan is m/z 374.3, and m/z 396.6, and m/z 405.7, and m/z 423.0, m/z 441.7, and m/z 491.6, and m/z 603.7, and m/z 801.6, m/z 823.8, and m/z 830.7, and m/z 840.6, and m/z 874.9, m/z 878.6, and m/z 915.9, and m/z 940.7, and m/z 962.8, m/z 1006.9, and it is carried out collision induced dissociation, obtain the secondary fragmention and are respectively m/z 203.3, m/z 643.1, and m/z 823.5, and m/z 423.5.
Ginsenoside Rb
1The mass spectral quasi-molecular ion peak of one-level full scan is m/z 1109.9, and m/z 1112.2, and m/z 1127.0, m/z 1129.3, and m/z 1132.8, and m/z 1148.9, m/z 1173.7, and m/z 1217.6, and m/z 1216.1, m/z m/z 1231.7, m/z 1300.6, and it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 144.7, m/z 163.4, and m/z 325.4, and m/z 487.3.
The mass spectral quasi-molecular ion peak of panoxadiol one-level full scan is m/z 425.6, and m/z 443.6, and m/z 444.7, m/z 459.6, and m/z 461.5, and m/z 462.6, m/z 463.6, and m/z 483.6, and m/z 484.4, m/z 499.5, and m/z 524.5, and m/z 534.6, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 99.0, m/z 109.4, m/z 122.0, and m/z 127.1, and m/z 191.5, m/z 203.2, and m/z 206.9, and m/z 217.5, m/z 235.0, and m/z 271.3, and m/z 369.4, m/z 407.6, m/z 425.4, and m/z 443.5, and m/z 461.4.
The mass spectral quasi-molecular ion peak of one-level full scan of panaxatriol is m/z 127.2, and m/z 423.5, and m/z 441.6, and m/z 459.6, m/z 477.5, and m/z 499.6, and m/z 550.7, and it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 99.1, m/z 109.6, and m/z 123.0, and m/z 127.1, m/z 187.6, and m/z 203.1, and m/z 205.2, and m/z 207.2, m/z 217.3, and m/z 405.7, and m/z 423.5, m/z 441.5, and m/z 459.6, and m/z 477.5.
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: Turbo 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:
| Time (min) | Flow velocity (ml/min) | Methyl alcohol | The 10mM ammonium acetate |
| 0 4 4.1 14.0 25 | 0.250 0.250 0.250 0.250 0.250 | 50％ 50％ 90％ 90％ 50％ | 50％ 50％ 10％ 10％ 50％ |
Detecting device: triple quadrupole bar tandem mass spectrum detects
Column temperature: 22 ℃
The mass spectrum condition
Ion gun: Turbo Ionspray source, injection electric: 4000V, positive ion detects, Tem:300 ℃
Scan mode is many reaction detection (MRM)
CE：17-36V；DP：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/z 950.8, m/z 801.6, m/z 1127.0, m/z 461.5 and m/z 477.5, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 423, m/z 423.5, m/z 487.3, m/z 127.1 and m/z 127.1, 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/m1)
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/m1 (10
-9G/m1)
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, panoxadiol and panaxatriol the standard solution of high, medium and low three kinds of concentration, by above-mentioned experimental example 1 plasma treatment method operation, the record chromatogram, other gets same concentration standard product direct injected, writes down chromatogram.Relative recovery is calculated in three parts of replicate determinations.
Relative recovery=(amount of recording after the processing/addition) * 100%
The result is as follows:
Panax Notoginseng saponin R
1
| Concentration ng/ml | 2.8ng/ml | 14ng/ml | 140ng/ml |
| Relative recovery (%) | 95.6 | 96.3 | 102 |
The ginsenoside Rg
1
| Concentration ng/ml | 3.8ng/ml | 19ng/ml | 190ng/ml |
| Relative recovery (%) | 99.8 | 94.0 | 105 |
Ginsenoside Rb
1
| Concentration ng/ml | 18ng/ml | 45ng/ml | 180ng/ml |
| Relative recovery (%) | 111 | 95.4 | 101.5 |
Panoxadiol
| Concentration ng/ml | 12.4ng/ml | 63.6ng/ml | 254ng/ml |
| Relative recovery (%) | 98.17 | 103.83 | 96.88 |
Panaxatriol
| Concentration ng/ml | 0.53ng/ml | 5.3ng/ml | 26.5ng/ml |
| Relative recovery (%) | 101.12 | 96.74 | 104 |
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:
| Time (min) | Flow velocity (ml/min) | Methyl alcohol | The 10mM ammonium acetate |
| 0 4 4.1 14.0 25 | 0.30 0.30 0.30 0.30 0.30 | 50％ 50％ 90％ 90％ 50％ | 50％ 50％ 10％ 10％ 50％ |
Detecting device: triple quadrupole bar tandem mass spectrum detects
Column temperature: 25 ℃
The mass spectrum condition
Ion gun: Turbo Ionspray source, injection electric: 4000V, positive ion detects, Tem:280 ℃
Be many reaction detection (MRM)
CE：17-36V； DP：80-110V
Detect ion:
The mass spectral ginsenoside Rb of one-level full scan
1, panoxadiol, panaxatriol quasi-molecular ion peak be respectively m/z 1183, m/z 461.1 and m/z 477.6, selectivity is carried out collision induced dissociation to it, obtain the secondary fragmention and be respectively m/z 487.2, m/z 127.1 and m/z 127.1, adopt many reaction detection (MRM) 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, 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.
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, a kind of detection method of 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: Turbo Ionspray source, injection electric: 3000-5000V, Tem:250-300 ℃, positive ion detects, and scan mode is many reaction detection (MRM), CE:17-36V, DP: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 (MRM) 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, 10mM ammonium acetate to carry out gradient elution in this method, the volume ratio of 0 moment methyl alcohol, 10mM ammonium acetate is 50%: 50%, methyl alcohol, 10mMNH during with the 4th minute
4The volume ratio of Oac is 50%: 50%, the volume ratio of methyl alcohol, 10mM ammonium acetate is 90%: 10% in the time of the 4.1st minute, the volume ratio of methyl alcohol, 10mM ammonium acetate is 90%: 10% in the time of the 14.0th minute, the volume ratio of methyl alcohol, 10mM ammonium acetate is 50%: 50% in the time of the 25th minute, flow velocity is 0.25 (ml/min), chromatographic column: ODS C18 post (50mm * 2.1mm, 5 μ m); Mass spectrum condition intermediate ion source: Turbo 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,, it is characterized in that described dammarane type four-ring triterpenoid saponin is the monomer saponin in the arasaponin as the detection method of claim 1,2 or 3 described dammarane type four-ring triterpenoid saponins.
5, the detection method of the dammarane type four-ring triterpenoid saponin described in claim 4 is characterized in that described dammarane type four-ring triterpenoid saponin Panax Notoginseng saponin R
1, the ginsenoside Rg
1, ginsenoside Rb
1, the secondary aglycon panoxadiol of protopanoxadiol type saponin(e, the secondary aglycon panaxatriol of Protopanaxatriol's type saponin(e.
6, the detection method of the dammarane type four-ring triterpenoid saponin described in claim 5 is characterized in that detecting in the mass spectrum condition ion: 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/z 441.7, and m/z 735.7, and m/z 753.9, m/z 950.8, and 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/z 203.3, m/z 643.1, m/z 723.5, and m/z 423;
The ginsenoside Rg
1The mass spectral quasi-molecular ion peak of one-level full scan is m/z 374.3, and m/z 396.6, and m/z 405.7, and m/z 423.0, m/z 441.7, and m/z 491.6, and m/z 603.7, and m/z 801.6, m/z 823.8, and m/z 830.7, and m/z 840.6, and m/z 874.9, m/z 878.6, and m/z 915.9, and m/z 940.7, and m/z 962.8, m/z 1006.9, and it is carried out collision induced dissociation, obtain the secondary fragmention and are respectively m/z 203.3, m/z 643.1, and m/z 823.5, and m/z 423.5;
Ginsenoside Rb
1The mass spectral quasi-molecular ion peak of one-level full scan is m/z 1109.9, and m/z 1112.2, and m/z 1127.0, m/z 1129.3, and m/z 1132.8, and m/z 1148.9, m/z 1173.7, and m/z 1217.6, and m/z 1216.1, m/z m/z 1231.7, m/z 1300.6, and it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 144.7, m/z 163.4, and m/z 325.4, and m/z 487.3;
The mass spectral quasi-molecular ion peak of panoxadiol one-level full scan is m/z 425.6, and m/z 443.6, and m/z 444.7, m/z 459.6, and m/z 461.5, and m/z 462.6, m/z 463.6, and m/z 483.6, and m/z 484.4, m/z 499.5, and m/z 524.5, and m/z 534.6, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 99.0, m/z 109.4, m/z 122.0, and m/z 127.1, and m/z 191.5, m/z 203.2, and m/z 206.9, and m/z 217.5, m/z 235.0, and m/z 271.3, and m/z 369.4, m/z 407.6, m/z 425.4, and m/z 443.5, and m/z 461.4;
The mass spectral quasi-molecular ion peak of one-level full scan of panaxatriol is m/z 127.2, and m/z 423.5, and m/z 441.6, and m/z 459.6, m/z 477.5, and m/z 499.6, and m/z 550.7, and it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 99.1, m/z 109.6, and m/z 123.0, and m/z 127.1, m/z 187.6, and m/z 203.1, and m/z 205.2, and m/z 207.2, m/z 217.3, and m/z 405.7, and m/z 423.5, m/z 441.5, and m/z 459.6, and m/z 477.5.
7, the detection method of the dammarane type four-ring triterpenoid saponin described in claim 6 is characterized in that detecting in the mass spectrum condition 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/z 950.8, m/z 801.6, m/z 1127.0, m/z 461.5 and m/z 477.5, it is carried out collision induced dissociation, obtain the secondary fragmention and be respectively m/z 423, m/z 423.5, m/z 487.3, m/z 127.1 and m/z 127.1.
8, a kind of 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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