CN1940551A - In-vivo content determination of bisnosaponin compound - Google Patents
In-vivo content determination of bisnosaponin compound Download PDFInfo
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- CN1940551A CN1940551A CN 200510011046 CN200510011046A CN1940551A CN 1940551 A CN1940551 A CN 1940551A CN 200510011046 CN200510011046 CN 200510011046 CN 200510011046 A CN200510011046 A CN 200510011046A CN 1940551 A CN1940551 A CN 1940551A
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Abstract
A method for determining content of metano-saponit compound in human body includes collecting biological sample being used as comparison set before test animals are fed with medicine, collecting biological samples being used as test set after man or test animal are fed with said compound with dosage of 0.005-100mg / kg animal body weight, extracting collected samples in solid phase or liquid phase, carrying out liquid phase chromatograph on extracted samples then carrying out mass spectrometric detection for determining content of said compound in man or animal body.
Description
Technical field
The present invention relates to a kind of in-vivo content determination method of compound, belong to biological technical field.
Background technology
Chinese patent 85108520 discloses two kinds of bisnosaponin compound application aspect gynecologic blood diseases.But distinct issues are that the bisnosaponin compound taking dose is very little, although in-vivo content can't be directly with common---and be high-sensitive method, as mensuration such as high performance liquid chromatogram, gas phase process.Therefore absorption, metabolism and the dynamic characteristic of bisnosaponin compound never are studied and report.In existing research, we confirm that bisnosaponin compound is to bring into play pharmacological action in animal body with original shape.Because its taking dose is minimum, in-vivo content is extremely low, press for a kind of energy fast, simple, disturb for a short time, and can be used for the in-vivo content determination method of human body.
Summary of the invention
One object of the present invention is to provide the in-vivo content determination method of the bisnosaponin compound of a kind of mensuration of the bioavilability that can be used for the various formulations of bisnosaponin compound and the timing of bisnosaponin compound internal metabolism.
In-vivo content determination method of the present invention can be divided into sxemiquantitative and quantitative measurement, and its method is as follows respectively:
The in-vivo content determination method of bisnosaponin compound of the present invention is made up of following steps:
One, collects the biological specimen of the preceding experimental animal of administration, in contrast group;
Two, with bisnosaponin compound administration of human or experimental animal, dosage is the 0.005-100mg/kg the weight of animals, regathers the biological specimen of above-mentioned experimental animal, as test group;
Three, difference Solid-Phase Extraction or liquid-phase extraction treatment step one, two gained samples;
Four, step 3 gained sample being carried out one by one liquid chromatography separates;
Five, be right after step 4 and carry out Mass Spectrometer Method.
The in-vivo content determination method of bisnosaponin compound of the present invention is made up of following steps:
One, collects the biological specimen of the preceding experimental animal of administration, in contrast group;
Two, with bisnosaponin compound administration of human or experimental animal, dosage is the 0.005-100mg/kg the weight of animals, regathers the biological specimen of above-mentioned experimental animal, as test group;
Three, difference Solid-Phase Extraction or liquid-phase extraction treatment step one, two gained samples;
Four, the solution of the internal standard compound matter 0.01-0.001mg/mL of preparation known molecular amount, and this is molten miscible with step 3 gained sample;
Five, precision takes by weighing solution that bisnosaponin compound is made into 0.1-5mg/mL as storing solution, further is made into the standard solution of 1-100ng/mL again;
Six, drawing curve: accurate respectively the absorption marked liquid in the test tube bottom in step 5 gained standard solution 0.1 μ L to 200 μ several gradients of L and the 1-10 μ L, add step 1 gained biological specimen 0.1-10ml after inert gas dries up respectively, three described sample treatments and step 2 gained sample are done parallel processing set by step; Carry out linear regression with bisnosaponin compound concentration in the comparison biological specimen of the peak area of bisnosaponin compound and interior mark peak area, obtain regression equation;
Seven, step 3 gained sample adds the solution 1-10 μ L of the internal standard compound matter 0.01-0.001mg/mL that contains the known molecular amount respectively, and mark concentration is at 1-20ng/mL in making in the step 2 gained sample;
Eight, step 7 gained sample being carried out one by one liquid chromatography separates;
Nine, be right after step 8 and carry out Mass Spectrometer Method;
Ten, with the peak area of bisnosaponin compound and interior mark peak area than the content of obtaining bisnosaponin compound in the substitution working curve.
Pennogenin of the present invention (pennogenin) compounds, general structure is:
In the general formula:
R
2＝H，CH
3，CH
2OH，＝CH
2
X＝O，N，S
C
17The position is R-configuration or S-configuration
R
1=by monose, disaccharide, trisaccharide, tetrose, pentasaccharides, six sugar, the straight chain sugar chain of the various types that seven sugar are formed or side chain sugar chain, the sugar composed type of its sugar chain comprise β-D-glucose (β-D-glucose), alpha-D-glucose (α-D-glucose), α-L-rhamnose (α-L-rhamnose), β-D-galactose (β-D-galactose), α-D-galactose (α-D-galactose), β-D-mannose (β-D-mannose), α-D-mannose (β-D-mannose), α-D-arabinose (α-D-aradinose), β-D-arabinose (α-D-aradinose), alpha-D-xylose (α-D-xylose), β-D-wood sugar (α-D-xylose), α-D-ribose (α-D-ribose), β-D-ribose (β-D-ribose), α-D-lyxose (α-D-lyxose), β-D-lyxose (β-D-lyxose), α-L-husband sugar (α-L-fucose).
Described bisnosaponin compound is positioned at the sugared indispensable of the 3rd of steroid backbone, and is not limited to seven sugar.
Comprise 4 bisnosaponin compound that activity is stronger: X=O, R
2=H,
[I]R
1＝-3-O-α-L-rha·py(1→4)-α-L-rha·py(1→4)-[α-L-rha-py(1→2)]-β-D-glc·py
[II]R
1＝-3-O-α-L-rha·py(1→2)-[α-L-rha·py(1→4)]-β-D-glc·py
[III]R
1＝-3-O-α-L-ara·fura-(1→4)-[α-L-rha·py(1→2)]-β-D-glc·py
[IV]R
1＝-3-O-α-L-rha·py(1→2)-β-D-glc·py。
Described Solid-Phase Extraction method is: adopt commercially available solid-phase extraction column, its filler can be C18, C8, CN and bondings such as phenyl be sorptive material such as silica gel or macroreticular resin mutually, its loading amount specification can be the 0.01-1 gram, by solid-phase extraction column operation instructions method pillar is activated, common available methyl alcohol, each 1mL of water crosses the post activation, (A) gets the biological specimen of 1mL liquid then, as blood plasma, serum, urine, bile, internal organs homogenate filtered fluid or the like, upper prop, (B) use 1mL methanol-eluted fractions 1-4 time again, collect meoh eluate, (C) in 40 ℃ of water-baths, dry up with nitrogen stream, residue adds 100-1000 μ L dissolve with methanol, obtains testing sample.
Described liquid-phase extraction method is: (A) in biological specimen blood plasma, serum, urine sample, bile, dry ight soil, internal organs homogenate, add concentration and be 70% or more contain carbon number at the lower alcohol 2-5 below 6 and 6 times to sample volume, soak and extract, the centrifugal precipitation that obtains, repeat to extract precipitation 1-4 time with pure 2-5 times of identical weight again, merge alcoholic solution, concentrating under reduced pressure gets CE below 60 ℃;
B, add suitable quantity of water and make CE dissolving, add chloroform 10-300 times weight and extract in CE, 1-5 time altogether, each time chloroform layer eluent with the washing of 1/10 volume once, the combined chloroform extract layer, decompression and solvent recovery, concentrated extract, supply detection usefulness.
Can add the miscible solution 5-10 μ L that contains internal standard compound matter 0.01-0.001mg/mL in the above-mentioned disposal route, mark concentration is at 1-20ng/mL in making in the testing sample, and internal standard compound matter can be Verapamil [MW=455.2904], and solvent is a methyl alcohol.Interior target act as calibration molecule amount and assay reference.Interior mark can also be for other molecular weight known and material that be of moderate size.
(1), chromatographic separation condition: reverse-phase chromatographic column, as silica gel bonded phase chromatographic columns such as C18, C8, CN.
(2), moving phase: methanol-water (60-95: 5-40) fixed proportion or gradient elution; Flow velocity: 0.2-2mL/min, column temperature: 10-42 ℃.
(3) mass spectrometer condition: electro-spray ionization interface flight time mass spectrum detecting device, mass spectrum workstation; Positive ion detects, spray nozzle voltage: 5000-6000V, and Nozzle potential: 300-400V detects voltage: 1500-2500V, and Jet Pipe Temperature: 110-170 ℃, sample range (m/z): 10-1000; The ion selector channel is [M+Na]
+, [M+K]
+, M represents bisnosaponin compound, [M+Na]
+Be main peak, the ion channel scope can be ± 0.5amu.
Method of the present invention is harmless, can be applied to human body and various animal used as test.
The present invention can be used for the mensuration of the bioavilability of the various formulations of bisnosaponin compound.
The present invention can be used for the timing of bisnosaponin compound internal metabolism.
The detectability of this method can reach 0.2ng/mL, and range of linearity 2-3 the order of magnitude, correlation coefficient r in a few days reach day to day precision less than 15% more than 0.99, and absolute recovery is more than 80%, relative recovery 92%-115%.
The invention has the advantages that (1) is highly sensitive, the sample process process promptly is a concentration process, and its detecting device is a mass detector, and far above the UV-detector of general liquid and gas, detectability can reach 0.2ng/mL; (2) selectivity is good, strong interference immunity, and biological specimen extremely difficult the separation, adopts mass spectrum selectivity ion detection mode, with [M+Na] because various organic impurities are arranged
+[M+K]
+Two peaks are the ion selector channel, effectively get rid of and disturb; (3) sample preparation is simple; (4) lacking detection time, is tens minutes only, is applicable to mass detection; (5) harmless, radioactive method, is used for human body and is restricted though sensitivity is very high owing to introduce radioelement.The inventive method especially is fit to do the bioavilability mensuration of the various formulations of bisnosaponin compound.
Embodiment
1, the mensuration of bisnosaponin compound bioavilability: getting blood on one's body from the volunteer will, measure the content of pennogenin compound IV in the blood plasma, calculate area AUC under the drug-time curve, compare with area AUC under the drug-time curve that the intramuscular injection of equivalent pennogenin produces, calculate relative bioavailability (%).The comparison of the bioavilability of untreated pennogenin, the various preparations of pennogenin:
Liquid phase chromatogram condition: Waters 2690 HPLC (U.S. Waters company) contain quaternary gradient pump, online vacuum degassing machine, automatic sampler, column oven, carbon-18 reverse-phase chromatographic column KromasilC18 (5 μ m, 4.6 * 250mm).Moving phase: methanol-water (80: 20), (90: 10), (85: 15) gradient elution; Flow velocity: 0.8mL/min, column temperature: 30 ℃.Diode array detector detects wavelength 203nm.
Mass spectrometer condition: Mariner
TM5410 mass spectrometers (U.S. Applied Biosystem company), electro-spray ionization interface flight time mass spectrum detecting device, mass spectrum workstation: Mariner
TMWorkstation 4.0 editions.The mass spectrometer parameter: positive ion detects, spray nozzle voltage: 5500V, Nozzle potential: 340V, detect voltage: 2200V, Jet Pipe Temperature: 140 ℃, sample range (m/z): 10-1000.
The ion selector channel is [M+Na]
+, [M+K]
+, M represents bisnosaponin compound, [M+Na]
+Be main peak, the ion channel scope can be ± 0.5amu, and promptly 761.4,777.4.
In mark: get an amount of Verapamil with dissolve with methanol and be diluted to 0.2mg/mL, with the preceding 0.01mg/mL that is diluted to again as storing solution.
Standard substance: precision takes by weighing methanol solution that bisnosaponin compound IV is made into 1mg/mL as storing solution, further is made into 10ng/mL again.
Working curve: mark liquid in test tube at the bottom of the tool plug tip in the accurate respectively above-mentioned 10ng/mL standard substance 0.3 μ L of absorption, 0.5 μ L, 1 μ L, 2 μ L, 5 μ L, 10 μ L, 20 μ L, 50 μ L, 100 μ L and the 5 μ L, respectively add biological specimen blood plasma 1.0ml after nitrogen dries up, adopt solid phase extraction to handle by sample treatment mentioned above; Carry out linear regression with bisnosaponin compound concentration in the comparison biological specimen of the peak area of bisnosaponin compound and interior mark peak area.
The precision and the recovery: get respectively and mark liquid in 0.3 μ L, 1 μ L, 5 μ L, 20 μ L and the 5 μ L in test tube at the bottom of the tool plug tip, respectively add biological specimen blood plasma 1.0ml after nitrogen dries up, adopt solid phase extraction to handle by sample treatment mentioned above; Each concentration re-treatment in a few days mensuration gets withinday precision 6 times, handles mensuration continuously and gets day to day precision in three days.With bisnosaponin compound peak area and interior mark peak area than substitution equation of linear regression, calculate relative recovery.In addition the bisnosaponin compound standard items are made into the methanol solution of respective concentration, direct injected as standard, compares the ratio of two groups of peak areas with interior mark peak area, calculates absolute recovery.
The result: this method detection speed is fast, only needs 15 minutes just can finish one-time detection.
The mensuration of table 1 bisnosaponin compound bioavilability
| Auxiliary material | Relative bioavailability (%) (n=3) |
| Unprocessed | 7.1±4.8 |
| PEG6000 (dripping pill) | 22.9±10.2 |
| PEG600 (soft capsule) | 27.5±13.4 |
| Lipidosome injection | 95.7±6.2 |
| Preparation capable of permeating skin | 16.3±8.6 |
| The anus bolt | 18.9±7.8 |
| The bisnosaponin compound sodium salt | 30.5±13.6 |
| The suction-type aerosol | 45.7±18.5 |
Also can measure other preparation in human or animal's bioavilability on one's body with method.
Embodiment 2
Bisnosaponin compound metabolism time study: behind the oral administration, got blood plasma once in per four hours, the solid phase extraction extraction, the LC-MS method is analyzed.The result is as follows:
| Sample time | The pennogenin compound IV |
| 4h | + |
| 8h | ++ |
| 12h | + |
| 16h | + |
| 20h | + |
| 24h | ± |
Can obviously detect pennogenin compound IV [M+Na] on the 4th, 8 hour after the administration
+Mass spectra peak (m/z761.4), after the administration the 24th hour, the original shape concentration was extremely low, near detecting lower limit, just can detect mass spectra peak after one times of the enrichment once more.Also fail to detect the mass spectra peak of PS-M1 and PS-M2 metabolic product in the blood plasma once more after the enrichment.
The result shows that pennogenin compound peak time is long, and 1-2 hour is peaking behind the general drug administration, and the pennogenin compound needs about 8 hours, illustrates that its bio-absorbable is not good, and bioavilability is low; And the half life period is shorter, reaches behind the peak promptly to drain substantially the same day to finish.
Claims (15)
1, a kind of in-vivo content determination method of bisnosaponin compound is characterized in that being made up of following steps:
One, collects the biological specimen of the preceding experimental animal of administration, in contrast group;
Two, with bisnosaponin compound administration of human or experimental animal, dosage is the 0.005-100mg/kg the weight of animals, regathers the biological specimen of above-mentioned experimental animal, as test group;
Three, difference Solid-Phase Extraction or liquid-phase extraction treatment step one, two gained samples;
Four, step 3 gained sample being carried out one by one liquid chromatography separates;
Five, be right after step 4 and carry out Mass Spectrometer Method.
2, a kind of in-vivo content determination method of bisnosaponin compound is characterized in that being made up of following steps:
One, collects the biological specimen of the preceding experimental animal of administration, in contrast group;
Two, with bisnosaponin compound administration of human or experimental animal, dosage is the 0.005-100mg/kg the weight of animals, regathers the biological specimen of above-mentioned experimental animal, as test group;
Three, difference Solid-Phase Extraction or liquid-phase extraction treatment step one, two gained samples;
Four, the solution of the internal standard compound matter 0.01-0.001mg/mL of preparation known molecular amount, and this is molten miscible with step 3 gained sample;
Five, precision takes by weighing solution that bisnosaponin compound is made into 0.1-5mg/mL as storing solution, further is made into the standard solution of 1-100ng/mL again;
Six, drawing curve: accurate respectively the absorption marked liquid in the test tube bottom in step 5 gained standard solution 0.1 μ L to 200 μ several gradients of L and the 1-10 μ L, add step 1 gained biological specimen 0.1-10ml after inert gas dries up respectively, three described sample treatments and step 2 gained sample are done parallel processing set by step; Carry out linear regression with bisnosaponin compound concentration in the comparison biological specimen of the peak area of bisnosaponin compound and interior mark peak area, obtain regression equation;
Seven, step 3 gained sample adds the solution 1-10 μ L of the internal standard compound matter 0.01-0.001mg/mL that contains the known molecular amount respectively, and mark concentration is at 1-20ng/mL in making in the step 2 gained sample;
Eight, step 7 gained sample being carried out one by one liquid chromatography separates;
Nine, be right after step 8 and carry out Mass Spectrometer Method;
Ten, with the peak area of bisnosaponin compound and interior mark peak area than the content of obtaining bisnosaponin compound in the substitution working curve.
3,, it is characterized in that described bisnosaponin compound general structure is according to the in-vivo content determination method of claim 1,2 described bisnosaponin compounds:
In the general formula:
R
2＝H，CH
3，CH
2OH，＝CH
2
X＝O，N，S
C
17The position is R-configuration or S-configuration
R
1=by monose, disaccharide, trisaccharide, tetrose, pentasaccharides, six sugar, the straight chain sugar chain of the various types that seven sugar are formed or side chain sugar chain, the sugar composed type of its sugar chain comprise β-D-glucose (β-D-glucose), alpha-D-glucose (α-D-glucose), α-L-rhamnose (α-L-rhamnose), β-D-galactose (β-D-galactose), α-D-galactose (α-D-galactose), β-D-mannose (β-D-mannose), α-D-mannose (β-D-mannose), α-D-arabinose (α-D-aradinose), β-D-arabinose (α-D-aradinose), alpha-D-xylose (α-D-xylose), β-D-wood sugar (α-D-xylose), α-D-ribose (α-D-ribose), β-D-ribose (β-D-ribose), α-D-lyxose (α-D-lyxose), β-D-lyxose (β-D-lyxose), α-L-husband sugar (α-L-fucose).
4, according to the described bisnosaponin compound of claim 3, it is characterized in that X=O in the described general formula, R
2=H, R
1=-3-O-α-L-rhapy (1 → 4)-α-L-rhapy (1 → 4)-[α-L-rha-py (1 → 2)]-β-D-glcpy.
5, according to the described bisnosaponin compound of claim 3, it is characterized in that X=O in the described general formula, R
2=H, R
1=-3-O-α-L-rhapy (1 → 2)-[α-L-rhapy (1 → 4)]-β-D-glcpy.
6, according to the described bisnosaponin compound of claim 3, it is characterized in that X=O in the described general formula, R
2=H, R
1=-3-O-α-L-arafura-(1 → 4)-[α-L-rhapy (1 → 2)]-β-D-glcpy.
7, according to the described bisnosaponin compound of claim 3, it is characterized in that X=O in the described general formula, R
2=H, R
1=-3-O-α-L-rhapy (1 → 2)-β-D-glcpy.
8, according to the in-vivo content determination method of claim 1,2 described bisnosaponin compounds, it is characterized in that its filler of extraction material used in the described Solid-Phase Extraction is bondings such as C18, C8, CN and phenyl sorptive materials such as silica gel or macroreticular resin mutually, its loading amount specification is the 0.01-10 gram.
9, according to the in-vivo content determination method of claim 1,2 described bisnosaponin compounds, it is characterized in that described Solid-Phase Extraction step is: the biological specimen upper prop of 1mL liquid is got in solid-phase extraction column activation back (A), (B) use 1mL methanol-eluted fractions 1-4 time again, collect meoh eluate, (C) dry up with nitrogen stream in water-soluble in 40 ℃, residue adds 100-1000 μ L dissolve with methanol.
10, according to the in-vivo content determination method of claim 1,2 described bisnosaponin compounds, it is characterized in that described liquid-phase extraction step is: A in biological specimen, add concentration be more than 70% contain carbon number at the lower alcohol 2-5 below 6 and 6 doubly to sample volume, soak and extract, the centrifugal precipitation that obtains, repeat to extract precipitation 1-4 time with pure 2-5 times of identical weight again, merge alcoholic solution, concentrating under reduced pressure gets CE below 60 ℃; B, add suitable quantity of water and make CE dissolving, add chloroform 10-300 times weight and extract in CE, 1-5 time altogether, each time chloroform layer eluent is washed once with 1/10 volume, the combined chloroform extract layer, and decompression and solvent recovery must concentrated extract.
11,, it is characterized in that using reverse-phase chromatographic column in the described chromatographic separation process according to the in-vivo content determination method of claim 1,2 described bisnosaponin compounds.
12,, it is characterized in that the moving phase of using in the described chromatographic separation process is methanol-water (60-95: 5-40) by fixed proportion or gradient elution according to the in-vivo content determination method of claim 1,2 described bisnosaponin compounds.
13,, it is characterized in that described Mass Spectrometer Method process intermediate ion selector channel is [M+Na] according to the in-vivo content determination method of claim 1,2 described bisnosaponin compounds
+, [M+K]
+, M is a bisnosaponin compound, the ion channel scope is ± 0.5amu.
14, claim 1,2 described content assaying methods can be used for the mensuration of the bioavilability of the various formulations of bisnosaponin compound.
15, claim 1,2 described content assaying methods can be used for the timing of bisnosaponin compound internal metabolism.
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Cited By (1)
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|---|---|---|---|---|
| CN102539220A (en) * | 2012-01-21 | 2012-07-04 | 华中农业大学 | Analysis method for metabolin of A-type proanthocyanidins |
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| CN102539220A (en) * | 2012-01-21 | 2012-07-04 | 华中农业大学 | Analysis method for metabolin of A-type proanthocyanidins |
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