CN1598572A - Quality determination method for external cultivated bezoar - Google Patents
Quality determination method for external cultivated bezoar Download PDFInfo
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- CN1598572A CN1598572A CN 200410041748 CN200410041748A CN1598572A CN 1598572 A CN1598572 A CN 1598572A CN 200410041748 CN200410041748 CN 200410041748 CN 200410041748 A CN200410041748 A CN 200410041748A CN 1598572 A CN1598572 A CN 1598572A
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Abstract
The invention discloses a quality identifying method for bezoar which is implanted in vitro, at first, make standard fingerprint of bile acid and bilirubin of bezoar which is implanted in vitro: 1.preparing tested solution and contrapositive solution, 2. reversed phase of chromatographic column to detect its highperformance liquid chromatogram, 3. analyse the data of highperformance liquid chromatogram of more than 10 groups tested solution, in order to establish standard fingerprint of bile acid and bilirubin. Then establish highperformance liquid chromatogram data of tested sample, which is compared and identify with above standard fingerprint. The invention is used to differentiate and identify the quality of bezoar which is implanted in vitro, which is contained in implanted in vitro bezoar material and preparation, which includes implanted in vitro bezoar. The invention can be used as an effective method to differentiate implanted in vitro bezoar and natural bezoar, artificial bezoar. The excellences of the invention are simple, stability, high precision, good reproductivity, easily to be predominated, the style and quality can be know from the whole character appearance of the chromatogram.
Description
Technical field
The present invention relates to a kind of method of quality control of cow-bezoar, utilize finger-print to differentiate specifically or contain the quality of external cultural calculus bovis in the preparation of external cultural calculus bovis with the outer cultural calculus bovis raw material of control volume.
Background technology
The traditional Chinese medicine fingerprint technology has become a kind of development trend at home and abroad.The traditional Chinese medicine fingerprint technology is a kind of novel standard of the Chinese medicine quality of production, can guarantee the consistance of tcm product; Also be the characteristic spectrum of component colony, can reflect the kind and the quantity of the contained chemical constitution of Chinese medicine comprehensively, and then reflect the quality of Chinese crude drug and products thereof.What finger-print obtained is the chemical constitution information of Chinese medicine Different Individual, express with figure or pattern mode, compare and analyze, can realize quality control to Chinese medicine unknown materials group integral body, being the gordian technique that realizes that the whole correlated quality of multiple composition is estimated, also is the necessary technology means of Chinese medicine innovation, research and development, quality discrimination, evaluation.
Natural ox gallstone is famous and precious rare Chinese crude drug, the puzzlement of rabid ox disease in addition, and its source, output are all very limited, far can not satisfy social needs.Therefore various substitutes occur, as external cultural calculus bovis, calculus bovis factitius etc., but its principal ingredient and medicinal efficacy have gap bigger.The finger-print one of animal drug is to the difficult point that is the research of fingerprint collection of illustrative plates, and present stage is not suitable for this analysis of complex ingredient system of cow-bezoar with the viewpoint of single chemical composition analysis.And solve the effective means of this difficult problem, exactly will be by the traditional Chinese medicine fingerprint technology of having been approved by international community.
Summary of the invention
The technical problem to be solved in the present invention is: by commercially available natural ox gallstone, external cultural calculus bovis, calculus bovis factitius etc. are studied; a kind of research method of external cultural calculus bovis finger-print is provided; be used for external cultural calculus bovis raw material and the quality discrimination, the evaluation that contain the external cultural calculus bovis of preparation of external cultural calculus bovis, can be used as the effective means of external cultural calculus bovis of difference and natural ox gallstone, calculus bovis factitius.
The technical problem to be solved in the present invention is achieved through the following technical solutions, and a kind of quality discrimination method of external cultural calculus bovis is characterized in:
(1) foundation of external cultural calculus bovis bile acid constituents finger-print
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis; accurate respectively title is fixed; add the mixed solution of 0～5% aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) respectively with the doubly amount of 20～100ml/g, put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube; centrifugal; supernatant filters with 0.45 μ m filter membrane, discards filtrate just, gets subsequent filtrate; promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 3～10: 90～97,
The preparation of b, reference substance solution
Get cholic acid and the deoxycholic aicd reference substance is an amount of, accurately claim surely, add dissolve with methanol, make the solution that every ml contains the 1mg reference substance respectively,
C, detecting instrument, reagent
High performance liquid chromatograph, detecting device are evaporative light-scattering detector,
Methyl alcohol is chromatographically pure, and formic acid, trifluoroacetic acid are that analysis is pure, and water is ultrapure water,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 0.8～1.2ml/min; Column temperature: 25～30 ℃; Sample size: 10～20 μ l; Moving phase: A is 0.1% VFA, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bile acid constituents standard finger-print data,
(2) foundation of external cultural calculus bovis bilirubinoid ingredients fingerprint
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis; accurate respectively title is fixed; the mixed solution that adds 0～5% aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) respectively with the doubly amount of 20～100ml/g; put ultrasonic Extraction 10～45min in the tool plug conical flask; extract is put in the centrifuge tube; centrifugal; the supernatant that inclines, the same solution centrifugal of residue is washed till colourless, adds dimethyl sulfoxide 10.0ml and puts ultrasonic Extraction 10～45min in the tool plug conical flask; centrifugal; supernatant filters with 0.45 μ m filter membrane, discards filtrate just, gets subsequent filtrate; promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 3～10: 90～97,
The preparation of b, reference substance solution
It is an amount of to get the cholerythrin reference substance, and accurate the title decides, and it is an amount of to add dimethyl sulfoxide, and making concentration is the cholerythrin reference substance solution of 0.5mg/ml,
C, detecting instrument, reagent,
High performance liquid chromatograph,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 0.8～1.2ml/min; Column temperature: 25～30; Detect wavelength: 451nm; Sample size: 1～5 μ l; Moving phase: A is 0.1～5% ammonium acetate water, and B is acetonitrile/dimethyl sulfoxide, and its ratio is 1～5: 5～8,45～55%B isocratic elution; Writing time: 20min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bilirubinoid standard finger-print data,
(3) with external cultural calculus bovis raw material or contain the samples to be measured such as preparation of external cultural calculus bovis, set up the high performance liquid chromatography data with above-mentioned same procedure, compare, differentiate with above-mentioned standard finger-print data.
The technical problem to be solved in the present invention can also come by the following technical programs further to realize that described organic acid adopts formic acid, acetate, trifluoroacetic acid, phosphoric acid, its preferable formic acid; The organic alcohol of described low-carbon (LC) adopts methyl alcohol, ethanol, normal butyl alcohol, isobutyl alcohol, amylalcohol, its particular methanol.
The technical problem to be solved in the present invention can also come to realize that further external cultural calculus bovis bile acid constituents finger-print chromatogram optimum condition is: chromatographic column: reverse-phase chromatographic column by the following technical programs; Flow velocity is 1.0ml/min; Column temperature is 30 ℃; Sample size is 20 μ l; Moving phase: A is 0.1% VFA, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min.
The technical problem to be solved in the present invention can also come to realize that further external cultural calculus bovis bilirubinoid ingredients fingerprint chromatogram optimum condition is: chromatographic column: reverse-phase chromatographic column by the following technical programs; Flow velocity is 1.0ml/min; Column temperature is 30; Detect wavelength: 451nm; Sample size is 1 μ l; Moving phase: A is 1% ammonium acetate water, and B is that acetonitrile and its ratio of dimethyl sulfoxide are 4: 7, can add tetrahydrofuran below 10% in the B system as modifier, the 52%B isocratic elution; Writing time: 20min.
The total peak of standard diagram of the external cultural calculus bovis bile acids ELSD-HPLC finger-print of setting up by method of the present invention is 12.
The standard diagram data of external cultural calculus bovis bile acids ELSD-HPLC finger-print
| Peak number * | ????1 | ??5 | ??6 | ??7 | ??8 | ??9 | ??11 | ??14 | ?16(S) | ??17 | ??18 | ??19 |
| Relative retention time | 0.110 do not apply out | ??0.335 | ??0.367 | ??0.477 | ??0.519 | ??0.611 | ??0.700 | ??0.927 | ?1 | ??1.099 | ??1.617 | ??1.714 |
| Relative peak area ratio | ??0.705 | ??0.0171 | ??0.00726 | ??0.0170 | ??0.00602 | ??0.00892 | ??0.00323 | ??0.00637 | ?1 | ??0.00431 | ??0.0230 | ??0.328 |
S: be reference peak-cholic acid (cholic acid).
The external cultural calculus bovis bile acids ELSD-HPLC finger-print of measuring with method of the present invention, condition calculates through included angle cosine method or correlation coefficient process, and the similarity of itself and standard diagram should be between 0.95-1.0.
The total peak of standard diagram of the external cultural calculus bovis bilirubinoid HPLC finger-print of setting up by method of the present invention is 12.
The standard diagram data of external cultural calculus bovis bilirubinoid HPLC finger-print
Peak number
*123456789 10 11 (S) 12
Protect relatively
0.154???0.177???0.200???0.225???0.248???0.259???0.293???0.321???0.456???0.786????1???????1.228
Stay the time
Relative peak
Area is than 0.017 0.008 0.013 0.009 0.007 0.012 0.008 0.002 0.043 0.036 1 0.042
Value
The external cultural calculus bovis bilirubinoid HPLC finger-print of measuring with method of the present invention, condition calculates through included angle cosine method or correlation coefficient process, and the similarity of itself and standard diagram is between 0.95-1.0.
Embodiment
Embodiment 1,
(1) external cultural calculus bovis bile acid constituents finger-print Study on standards method
1, the preparation of need testing solution
Get 10 batches of external cultural calculus bovis fine powders or get the preparation fine powder 0.2g that contains external cultural calculus bovis; accurate respectively title is fixed; mixed solution (the v: v=5: 95) 10.0ml that adds 0～5% aqueous formic acid and methyl alcohol respectively; put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube, and is centrifugal; supernatant filters with 0.45 μ m filter membrane; discard filtrate just, get subsequent filtrate, promptly.
2, the preparation of reference substance solution
Get cholic acid (cholic acid) and deoxycholic aicd (deoxycholic acid) reference substance is an amount of, accurately claim surely, add dissolve with methanol, make the solution that every ml contains the 1mg reference substance respectively.
3, detection method (instrument, reagent, chromatographic condition)
(for example Agilent 1100 series contain unit such as binary high-pressure pump, automatic sample handling system, the online degassing, column oven to high performance liquid chromatograph.)
Detecting device is evaporative light-scattering detector (for example Alltech 500ELSD).
Methyl alcohol is chromatographically pure, and formic acid, trifluoroacetic acid are that analysis is pure, and water is ultrapure water.
Chromatographic column: reverse-phase chromatographic column, select C for use
18Post, for example Zorbax SB C
18, 5 μ m, 4.6 * 12.5mm (pre-column)+4.6 * 150mm (analytical column); Flow velocity is 1.0ml/min; Column temperature is 30; Sample size selects 20 μ l; Moving phase: A is 0.1% VFA formic acid, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min.Carry out data analysis promptly.
High performance liquid chromatography to above-mentioned 10 batches of above need testing solutions carries out the data analysis formulation,
External cultural calculus bovis bile acid constituents typical fingerprint collection of illustrative plates under the optimum condition
The standard diagram data of external cultural calculus bovis bile acids ELSD-HPLC finger-print
| Peak number * | 1 | 5 | 6 | 7 | 8 | 9 | 11 | 14 | 16(S) | 17 | 18 | 19 |
| Relative retention time | 0.110 | 0.335 | 0.367 | 0.477 | 0.519 | 0.611 | 0.700 | 0.927 | 1 | 1.099 | 1.617 | 1.714 |
| Relative peak area ratio | 0.705 | 0.0171 | 0.00726 | 0.0170 | 0.00602 | 0.00892 | 0.00323 | 0.00637 | 1 | 0.00431 | 0.0230 | 0.328 |
S: be reference peak-cholic acid (cholic acid).
The total peak of the standard diagram of above-mentioned external cultural calculus bovis bile acids ELSD-HPLC finger-print is 12.The external cultural calculus bovis bile acids ELSD-HPLC finger-print of measuring with above-mentioned condition calculates through included angle cosine method or correlation coefficient process, and the similarity of itself and standard diagram should be between 0.95～1.0.
We have detected 10 batches of external cultural calculus bovises with this condition; lot number is respectively 20030401,20030402,20030403,20030601,20030801,20030802,20030803,20031001,20031002,20031003; similarity is all above 0.99 as a result, See Figure
10 crowdes of external cultural calculus bovis bile acid constituents finger-print stacking diagrams and five batches of non-external cultural calculus bovis contrasts, then similarity then differs greatly, See Figure
The HPLC comparison diagram of external cultural calculus bovis, calculus bovis factitius test sample and bile acid reference substance, blank sample is from top to bottom successively: the deoxycholic aicd reference substance; Calculus bovis factitius; External cultural calculus bovis; The cholic acid reference substance; Blank right
According to
The comparison diagram of 4 batches of natural ox gallstones and external cultural calculus bovis bile acid constituents finger-print
Order from top to bottom is respectively: Calculus Bovis, Brazil's Huang, Indian yellow, natural ox gallstone (species indeterminata), external cultural calculus bovis
(two) external cultural calculus bovis bilirubinoid ingredients fingerprint research method
1, the preparation of need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis, accurate respectively claim fixed.(v: v=5: 95) 10.0ml puts ultrasonic Extraction 10～45min in the tool plug conical flask to add 0～5% organic acid aqueous formic acid of 50 times of amounts (ml/g) and the mixed solution of the organic pure methyl alcohol of low-carbon (LC) respectively, extract is put in the centrifuge tube, centrifugal, the supernatant that inclines, the same solution centrifugal of residue is washed till colourless, add dimethyl sulfoxide 10.0ml and put ultrasonic Extraction 10～45min in the tool plug conical flask, centrifugal, supernatant filters with 0.45 μ m filter membrane, discards filtrate just, get subsequent filtrate, promptly.
2, the preparation of reference substance solution
It is an amount of to get cholerythrin (bilirubin) reference substance, and accurate the title decides, and it is an amount of to add dimethyl sulfoxide, and making concentration is the cholerythrin reference substance solution of 0.5mg/ml.
3, detection method and checking
4, instrument, reagent, chromatographic condition
High performance liquid chromatograph (for example Agilent 1100 serial HPLC chromatographs contain unit such as binary high-pressure pump, DAD detecting device, automatic sample handling system, the online degassing, column oven).
Chromatographic column: reverse-phase chromatographic column, preferably C
8Post (Zorbax SB C for example
185 μ m, 4.6 * 12.5mm (pre-column)+Zorbax Eclipse XDB-C
85 μ m, 4.6 * 150mm (analytical column)); Flow velocity is 1.0ml/min; Column temperature is 30 ℃; Detect wavelength: 451nm; Sample size selects 1 μ l; Moving phase: A is 1% ammonium acetate water, and B is acetonitrile/dimethyl sulfoxide (4: 7), adds 8% tetrahydrofuran in the B system as modifier, the 52%B isocratic elution; Writing time: 20min.Carry out data analysis promptly.
External cultural calculus bovis bilirubinoid HPLC standard finger-print
The standard diagram data of external cultural calculus bovis bilirubinoid HPLC finger-print
Peak number
*123456789 10 11 (S) 12
Keep relatively
0.154?????0.177?????0.200????0.225????0.248????0.259????0.293????0.321????0.456????0.786????1????????1.228
Time
Relative peak face
0.017?????0.008?????0.013????0.009????0.007????0.012????0.008????0.002????0.043????0.036????1????????0.042
Long-pending ratio
The total peak of the standard diagram of above-mentioned external cultural calculus bovis courage bilirubinoid HPLC finger-print is 12.The external cultural calculus bovis bilirubinoid HPLC finger-print of measuring with above-mentioned condition calculates through included angle cosine method or correlation coefficient process, and the similarity of itself and standard diagram should be between 0.95～1.0.
We have detected 10 batches of external cultural calculus bovises with this condition; lot number is respectively 20030401,20030402,20030403,20030601,20030801,20030802,20030803,20031001,20031002,20031003; similarity is all above 0.99 as a result; detect a collection of calculus bovis factitius, similarity is 0.77 as a result.
10 crowdes of cow-bezoar test sample bilirubinoid composition HPLC finger-print stacking diagrams
Annotate: be followed successively by calculus bovis factitius and ten batches of external cultural calculus bovises from top to bottom.
(3) with external cultural calculus bovis raw material or contain the samples to be measured such as preparation of external cultural calculus bovis, set up the high performance liquid chromatography data with above-mentioned same procedure, compare, differentiate with above-mentioned standard finger-print data.
Embodiment 2, a kind of quality discrimination method of external cultural calculus bovis,
(1) foundation of external cultural calculus bovis bile acid constituents finger-print
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis, accurate respectively claim fixed.Add the 5% organic acid acetic acid aqueous solution of 100 times of amounts (ml/g) and the mixed solution of the organic pure ethanol of low-carbon (LC) respectively, put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube, centrifugal, supernatant filters with 0.45 μ m filter membrane, discards filtrate just, get subsequent filtrate, promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 5: 95,
The preparation of b, reference substance solution
Get cholic acid and the deoxycholic aicd reference substance is an amount of, accurately claim surely, add dissolve with methanol, make the solution that every ml contains the 1mg reference substance respectively,
C, detecting instrument, reagent
High performance liquid chromatograph, detecting device are evaporative light-scattering detector,
Methyl alcohol is chromatographically pure, and formic acid, trifluoroacetic acid are that analysis is pure, and water is ultrapure water,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 0.8ml/min; Column temperature: 25 ℃; Sample size: 10 μ l; Moving phase: A is 0.1% VFA, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bile acid constituents standard finger-print,
(2) foundation of external cultural calculus bovis bilirubinoid ingredients fingerprint
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis, accurate respectively claim fixed.Add 5% aqueous solutions of organic acids of 90 times of amounts (ml/g) and the mixed solution 10.0ml of the organic alcohol of low-carbon (LC) respectively and put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube, and is centrifugal, and supernatant inclines, the same solution centrifugal of residue is washed till colourless, add dimethyl sulfoxide 10.0ml and put ultrasonic Extraction 10～45min in the tool plug conical flask, centrifugal, supernatant filters with 0.45 μ m filter membrane, discard filtrate just, get subsequent filtrate, promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 3: 90,
The preparation of b, reference substance solution
It is an amount of to get the cholerythrin reference substance, and accurate the title decides, and it is an amount of to add dimethyl sulfoxide, and making concentration is the cholerythrin reference substance solution of 0.5mg/ml,
C, detecting instrument, reagent,
High performance liquid chromatograph,
D, chromatographic condition
7. chromatographic column: reverse-phase chromatographic column; Flow velocity: 1.2ml/min; Column temperature: 28; Detect wavelength: 451nm; Sample size: 5 μ l; Moving phase: A is 0.1% ammonium acetate water, and B is acetonitrile/dimethyl sulfoxide, and its ratio is 3: 6, adds 5% tetrahydrofuran in the B system as modifier, the 45%B isocratic elution; Writing time: 20min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bilirubinoid standard finger-print,
(3) with external cultural calculus bovis raw material or contain the samples to be measured such as preparation of external cultural calculus bovis, set up the high performance liquid chromatography data with above-mentioned same procedure, compare, differentiate with above-mentioned standard finger-print data.
Embodiment 3
A kind of quality discrimination method of external cultural calculus bovis,
(1) foundation of external cultural calculus bovis bile acid constituents finger-print
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis, accurate respectively claim fixed.Add the 2% organic acid trifluoroacetic acid aqueous solution of 20 times of amounts (ml/g) and the mixed solution 10.0ml of the organic pure normal butyl alcohol of low-carbon (LC) respectively and put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube, centrifugal, supernatant filters with 0.45 μ m filter membrane, discard filtrate just, get subsequent filtrate, promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 10: 97,
The preparation of b, reference substance solution
Get cholic acid and the deoxycholic aicd reference substance is an amount of, accurately claim surely, add dissolve with methanol, make the solution that every ml contains the 1mg reference substance respectively,
C, detecting instrument, reagent
High performance liquid chromatograph, detecting device are evaporative light-scattering detector,
Methyl alcohol is chromatographically pure, and formic acid, trifluoroacetic acid are that analysis is pure, and water is ultrapure water,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 1.2ml/min; Column temperature: 25 ℃; Sample size: 10 μ l; Moving phase: A is 0.1% VFA, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bile acid constituents standard finger-print,
(2) foundation of external cultural calculus bovis bilirubinoid ingredients fingerprint
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis, accurate respectively claim fixed.Add 0～5% aqueous solutions of organic acids of 80 times of amounts (ml/g) and the mixed solution 10.0ml of the organic alcohol of low-carbon (LC) respectively and put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube, and is centrifugal, and supernatant inclines, the same solution centrifugal of residue is washed till colourless, add dimethyl sulfoxide 10.0ml and put ultrasonic Extraction 10～45min in the tool plug conical flask, centrifugal, supernatant filters with 0.45 μ m filter membrane, discard filtrate just, get subsequent filtrate, promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 8: 92,
The preparation of b, reference substance solution
It is an amount of to get the cholerythrin reference substance, and accurate the title decides, and it is an amount of to add dimethyl sulfoxide, and making concentration is the cholerythrin reference substance solution of 0.5mg/ml,
C, detecting instrument, reagent: high performance liquid chromatograph,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 1.2ml/min; Column temperature: 30; Detect wavelength: 451nm; Sample size: 2 μ l; Moving phase: A is 3% ammonium acetate water, and B is acetonitrile/dimethyl sulfoxide, and its ratio is 5: 8, the 55%B isocratic elution; Writing time: 20min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bilirubinoid standard finger-print,
(3) with external cultural calculus bovis raw material or contain the samples to be measured such as preparation of external cultural calculus bovis, set up the high performance liquid chromatography data with above-mentioned same procedure, compare, differentiate with above-mentioned standard finger-print data.
Embodiment 4
A kind of quality discrimination method of external cultural calculus bovis,
(1) foundation of external cultural calculus bovis bile acid constituents finger-print
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis, accurate respectively claim fixed.Add 0～5% organic acid phosphate aqueous solution of 100 times of amounts (ml/g) and the mixed solution 10.0ml of the organic pure isobutyl alcohol of low-carbon (LC) respectively and put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube, centrifugal, supernatant filters with 0.45 μ m filter membrane, discard filtrate just, get subsequent filtrate, promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 3: 97,
The preparation of b, reference substance solution
Get cholic acid and the deoxycholic aicd reference substance is an amount of, accurately claim surely, add dissolve with methanol, make the solution that every ml contains the 1mg reference substance respectively,
C, detecting instrument, reagent
High performance liquid chromatograph, detecting device are evaporative light-scattering detector, and methyl alcohol is chromatographically pure, and formic acid, trifluoroacetic acid are that analysis is pure, and water is ultrapure water,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 1ml/min; Column temperature: 27 ℃; Sample size: 18 μ l; Moving phase: A is 0.1% VFA, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bile acid constituents standard finger-print,
(2) foundation of external cultural calculus bovis bilirubinoid ingredients fingerprint
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis, accurate respectively claim fixed.Add the 5% organic acid water beetle acid solution of 50 times of amounts (ml/g) and the mixed solution 10.0ml of the organic pure amylalcohol of low-carbon (LC) respectively and put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube, and is centrifugal, and supernatant inclines, the same solution centrifugal of residue is washed till colourless, add dimethyl sulfoxide 10.0ml and put ultrasonic Extraction 10～45min in the tool plug conical flask, centrifugal, supernatant filters with 0.45 μ m filter membrane, discard filtrate just, get subsequent filtrate, promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 3～10: 90～97,
The preparation of b, reference substance solution
It is an amount of to get the cholerythrin reference substance, and accurate the title decides, and it is an amount of to add dimethyl sulfoxide, and making concentration is the cholerythrin reference substance solution of 0.5mg/ml,
C, detecting instrument, reagent, high performance liquid chromatograph,
D, chromatographic condition
8. chromatographic column: reverse-phase chromatographic column; Flow velocity: 1ml/min; Column temperature: 30; Detect wavelength: 451nm; Sample size: 4 μ l; Moving phase: A is 2% ammonium acetate water, and B is acetonitrile/dimethyl sulfoxide, and its ratio is 1: 5, adds 9% tetrahydrofuran in the B system as modifier, the 50%B isocratic elution; Writing time: 20min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bilirubinoid standard finger-print,
(3) with external cultural calculus bovis raw material or contain the samples to be measured such as preparation of external cultural calculus bovis, set up the high performance liquid chromatography data with above-mentioned same procedure, compare, differentiate with above-mentioned standard finger-print data.
Claims (6)
1. the quality discrimination method of an external cultural calculus bovis is characterized in that,
(1) foundation of external cultural calculus bovis bile acid constituents finger-print
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis; accurate respectively title is fixed; add the mixed solution of 0～5% aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) respectively with the doubly amount of 20～100ml/g, put ultrasonic Extraction 10～45min in the tool plug conical flask, extract is put in the centrifuge tube; centrifugal; supernatant filters with 0.45 μ m filter membrane, discards filtrate just, gets subsequent filtrate; promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 3～10: 90～97,
The preparation of b, reference substance solution
Get cholic acid and the deoxycholic aicd reference substance is an amount of, accurately claim surely, add dissolve with methanol, make the solution that every ml contains the 1mg reference substance respectively,
C, detecting instrument, reagent
High performance liquid chromatograph, detecting device are evaporative light-scattering detector,
Methyl alcohol is chromatographically pure, and formic acid, trifluoroacetic acid are that analysis is pure, and water is ultrapure water,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 0.8～1.2ml/min; Column temperature: 25～30 ℃; Sample size: 10～20 μ l; Moving phase: A is 0.1% VFA, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min,
E, the high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis work out external cultural calculus bovis bile acid constituents standard finger-print data.
(2) foundation of external cultural calculus bovis bilirubinoid ingredients fingerprint
The preparation of a, need testing solution
Get more than 10 batches external cultural calculus bovis fine powder or get the preparation fine powder that contains external cultural calculus bovis; accurate respectively title is fixed; the mixed solution that adds 0～5% aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) respectively with the doubly amount of 20～100ml/g; put ultrasonic Extraction 10～45min in the tool plug conical flask; extract is put in the centrifuge tube; centrifugal; the supernatant that inclines, the same solution centrifugal of residue is washed till colourless, adds dimethyl sulfoxide 10.0ml and puts ultrasonic Extraction 10～45min in the tool plug conical flask; centrifugal; supernatant filters with 0.45 μ m filter membrane, discards filtrate just, gets subsequent filtrate; promptly
Wherein the volume ratio of aqueous solutions of organic acids and the organic alcohol of low-carbon (LC) is 3～10: 90～97,
The preparation of b, reference substance solution
It is an amount of to get the cholerythrin reference substance, and accurate the title decides, and it is an amount of to add dimethyl sulfoxide, and making concentration is the cholerythrin reference substance solution of 0.5mg/ml,
C, detecting instrument, reagent,
High performance liquid chromatograph,
D, chromatographic condition
Chromatographic column: reverse-phase chromatographic column; Flow velocity: 0.8～1.2ml/min; Column temperature: 25～30; Detect wavelength: 451nm; Sample size: 1～5 μ l; Moving phase: A is 0.1～5% ammonium acetate water, and B is acetonitrile/dimethyl sulfoxide, and its ratio is 1～5: 5～8,45～55%B isocratic elution; Writing time: 20min,
The high performance liquid chromatography of above-mentioned 10 batches of above need testing solutions is carried out data analysis works out external cultural calculus bovis bilirubinoid standard finger-print data,
(3) with external cultural calculus bovis raw material or contain the samples to be measured such as preparation of external cultural calculus bovis, set up the high performance liquid chromatography data with above-mentioned same procedure, compare, differentiate with above-mentioned standard finger-print data.
2. the quality discrimination method of external cultural calculus bovis according to claim 1 is characterized in that, described organic acid adopts formic acid, acetate, trifluoroacetic acid, phosphoric acid; The organic alcohol of described low-carbon (LC) adopts methyl alcohol, ethanol, normal butyl alcohol, isobutyl alcohol, amylalcohol.
3. the quality discrimination method of external cultural calculus bovis according to claim 1 and 2 is characterized in that, described organic acid is a formic acid; The organic alcohol of described low-carbon (LC) is methyl alcohol.
4. the quality discrimination method of external cultural calculus bovis according to claim 1 is characterized in that, external cultural calculus bovis bile acid constituents finger-print chromatographic condition is chromatographic column: reverse-phase chromatographic column; Flow velocity is 1.0ml/min; Column temperature is 30 ℃; Sample size is 20 μ l; Moving phase: A is 0.1% VFA, and B is the arbitrary proportion of methyl alcohol and acetonitrile, begins to arrive 90%B in the linear gradient mode through 70min from 70%B; Writing time: 70min.The standard finger-print data of working out are:
The standard diagram data of external cultural calculus bovis bile acids ELSD-HPLC finger-print
Peak number
* 1 5 6 7 8 9 ??11 ??14 ??16(s) ??17 ??18 ??19
Relative retention time 0.110 0.335 0.367 0.477 0.519 0.611 ??0.700 ??0.927 ??1 ??1.099 ??1.617 ??1.714
Relative peak area ratio 0.705 0.0171 0.00726 0.0170 0.00602 0.00892 ??0.00323 ??0.00637 ??1 ??0.00431 ??0.0230 ??0.328
S: be reference peak-cholic acid (cholic acid).
The total peak of the standard diagram of above-mentioned external cultural calculus bovis bile acids finger-print is 12,
The external cultural calculus bovis bile acids finger-print of measuring with above-mentioned condition calculates through included angle cosine method or correlation coefficient process, and the similarity of itself and standard diagram is between 0.95～1.0.
5. the quality discrimination method of external cultural calculus bovis according to claim 1 is characterized in that, external cultural calculus bovis bilirubinoid ingredients fingerprint chromatographic condition is chromatographic column: reverse-phase chromatographic column; Flow velocity is 1.0ml/min; Column temperature is 30; Detect wavelength: 451nm; Sample size is 1 μ l; Moving phase: A is 1% ammonium acetate water, and B is acetonitrile and dimethyl sulfoxide, and its ratio is 4: 7, the 52%B isocratic elution; Writing time: 20min.The standard finger-print data of working out are:
The standard diagram data of external cultural calculus bovis bilirubinoid finger-print
Peak number
*123456789 10 11 (S) 12
Keep relatively
0.154??0.177??0.200??0.225??0.248??0.259??0.293??0.321??0.456??0.786???1??????1.228
Time
Relative peak face
0.017??0.008??0.013??0.009??0.007??0.012??0.008??0.002??0.043??0.036???1??????0.042
Long-pending ratio
The total peak of the standard diagram of above-mentioned external cultural calculus bovis courage bilirubinoid finger-print is 12,
The external cultural calculus bovis bilirubinoid finger-print of measuring with above-mentioned condition calculates through included angle cosine method or correlation coefficient process, and the similarity of itself and standard diagram should be between 0.95～1.0.
6. the quality discrimination method of external cultural calculus bovis according to claim 5 is characterized in that, can add tetrahydrofuran below 10% in the B system as modifier.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1877322B (en) * | 2005-06-10 | 2010-08-11 | 上海华拓医药科技发展有限公司 | High-efficiency liquid chromatography method for detecting stachydrine content in motherwort |
| CN101995441A (en) * | 2010-02-10 | 2011-03-30 | 中国科学院大连化学物理研究所 | Kit for testing 3-sulfate glycochenodeoxycholic acids and glycochenodeoxycholic acids in blood |
| CN101566607B (en) * | 2009-06-03 | 2011-09-28 | 川渝中烟工业公司 | Method for measuring organic acid radicals in cigarette paper |
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| CN107328875A (en) * | 2017-06-28 | 2017-11-07 | 合肥今越制药有限公司 | A kind of finger-print for calculus bovis factitius bulk drug quality control |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1877322B (en) * | 2005-06-10 | 2010-08-11 | 上海华拓医药科技发展有限公司 | High-efficiency liquid chromatography method for detecting stachydrine content in motherwort |
| CN101566607B (en) * | 2009-06-03 | 2011-09-28 | 川渝中烟工业公司 | Method for measuring organic acid radicals in cigarette paper |
| CN101995441A (en) * | 2010-02-10 | 2011-03-30 | 中国科学院大连化学物理研究所 | Kit for testing 3-sulfate glycochenodeoxycholic acids and glycochenodeoxycholic acids in blood |
| CN106841492A (en) * | 2017-03-30 | 2017-06-13 | 杭州佰辰医学检验所有限公司 | Five kinds of methods of sequestered bile acid in high performance liquid chromatography tandem mass spectrum detection serum |
| CN107328875A (en) * | 2017-06-28 | 2017-11-07 | 合肥今越制药有限公司 | A kind of finger-print for calculus bovis factitius bulk drug quality control |
| CN107881243A (en) * | 2017-10-31 | 2018-04-06 | 中国食品药品检定研究院 | The method and purposes of the fluorescence quantitative PCR detection differentiated for the cow-bezoar true and false |
| CN107881244A (en) * | 2017-10-31 | 2018-04-06 | 中国食品药品检定研究院 | The probe primer and detection method and purposes of the fluorescence quantitative PCR detection differentiated for the cow-bezoar true and false |
| CN107881243B (en) * | 2017-10-31 | 2022-03-18 | 中国食品药品检定研究院 | Fluorescent quantitative PCR detection method for authenticity identification of bezoar and application thereof |
| CN113237960A (en) * | 2020-11-18 | 2021-08-10 | 上海和黄药业有限公司 | Method for detecting main drug effect components of calculus bovis factitius in pharmaceutical preparation |
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