CN103033570B - Quality detection method for tripterygii hypoglauci medicinal materials - Google Patents
Quality detection method for tripterygii hypoglauci medicinal materials Download PDFInfo
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Abstract
The invention aims to disclose a quality detection method for a fingerprint spectrum of tripterygii hypoglauci medicinal materials. An acetonitrile-methyl alcohol-0.02% of phosphoric acid solution is used as a moving phase; gradient elution is carried out; and the injection temperature is 20 DEG C. With the adoption of the fingerprint spectrum provided by the invention, not only can the content of triptolide, tripterine and wilforlide be determined, but also the microcosmic components of the tripterygii hypoglauci medicinal materials are analyzed, and a common mode of the fingerprint spectrum is formulated, so that the most advanced level can be reached in qualitative and quantitative aspects.
Description
Technical field
The present invention relates to a kind of quality determining method of Chinese crude drug, particularly relate to the finger print quality detecting method of tripterygium hypoglaucum hutcs medicinal material.
Background technology
Chromatographic fingerprinting detects the polycomponent feature that meets prodrug complex for traditional Chinese medicine quality, in the situation that some chemical constitution it be unclear that, can control comparatively all sidedly the stability of Chinese crude drug.Differentiate and compare with tradition, finger-print can better reflect the total quality information of Chinese medicine (autonomic drug), analyzes by advanced meanses such as Modern Analytical Instrument, convenient and easy.
U.S. FDA and WHO have passed through the regulation of " autonomic drug being allowed to criticize with finger-print judgement listing product to the consistance of a sample ".In recent years, the autonomic drug of Japan and Korea S., De Deng state is studied also widespread use finger-print and is carried out quality testing.As can be seen here, finger-print has become effective control device of controlling autonomic drug, is extensively applied.
Tripterygium hypoglaucum hutcs is the perennial fallen leaves liana of Celastraceae Thunder God Calamus shrub [Tripterygium hypoglaucum (Levl) Hutch], have another name called powder back of the body thunder godvine, tripterygium hypoglaucum hutch, glaucousback threewingnut root-bark etc., the traditional Chinese medical science for desinsection, stimulate the circulation of the blood and cause the muscles and joints to relax, clearing heat and detoxicating, dispel rheumatism etc.The important value of tripterygium hypoglaucum hutcs at aspects such as numerous autoimmune diseases (as lupus erythematosus etc.), tumour, leukaemia and acquired immune deficiency syndrome (AIDS) found in research both at home and abroad in succession.But at present, only Shanghai City medicinal material standard and Guangdong Province's Chinese crude drug standard have the quality determining method that records tripterygium hypoglaucum hutcs to existing method.The quality testing means of tripterygium hypoglaucum hutcs medicinal material are only limited to simple proterties and micro-discriminating, not qualitative or quantitative discriminating, quality determining method and the shortage thereof to main effective constituent.The present invention has set up the discrimination method of the finger-print of tripterygium hypoglaucum hutcs medicinal material, by finger-print and the standard finger-print contrast of medicinal material, stable, controlled to guarantee quality of medicinal material.
Summary of the invention
The object of the invention is to the finger print quality detecting method of open tripterygium hypoglaucum hutcs medicinal material.
The present invention seeks to be achieved through the following technical solutions:
The finger print quality detecting method of tripterygium hypoglaucum hutcs medicinal material of the present invention, the method comprises the steps:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 0.5～1.5 weight portion after sieve No. 2, put in conical flask, add methyl alcohol 60～100 parts by volume, put in 30～50 DEG C of ultrasonic water bath ultrasonic extraction 0.5～1.5 hour, room temperature is placed 0.5～1.5 hour, under 4000 revs/min of conditions centrifugal 5～15 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, transfer and set to 0 .5～1.5 hour in 1～5 DEG C, refrigerator, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.00005～0.00015 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent 1200 type high performance liquid chromatographs; Chromatographic column Symmetry C18 (250 × 4.6mm, 5 μ are m); Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 10～30 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and each 0.00001 parts by volume of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.030～1.050, No. 1,37, peak, No. 0.820～0.840,36, peak, No. 0.560～0.580,32, peak, No. 5 No. 0.140～0.160,24, peaks.
The finger print quality detecting method of tripterygium hypoglaucum hutcs medicinal material of the present invention, is preferably as follows step:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 1 weight portion after sieve No. 2, put in conical flask, add methyl alcohol 80 parts by volume, put in 40 DEG C of ultrasonic water bath ultrasonic extraction 1 hour, room temperature is placed 1 hour, under 4000 revs/min of conditions centrifugal 10 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, at 1～5 DEG C, refrigerator, place 1 hour, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.0001 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent 1200 type high performance liquid chromatographs; Chromatographic column Symmetry C18 (250 × 4.6mm, 5 μ are m); Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 20 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and each 0.00001 parts by volume of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.044, No. 1,37, peak, No. 0.829,36, peak, No. 0.569,32, peak, No. 5 No. 0.156,24, peaks.
The finger print quality detecting method of tripterygium hypoglaucum hutcs medicinal material of the present invention, can also be preferably as follows step:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 0.8 weight portion after sieve No. 2, put in conical flask, add methyl alcohol 90 parts by volume, put in 45 DEG C of ultrasonic water bath ultrasonic extraction 0.8 hour, room temperature is placed 0.8 hour, under 4000 revs/min of conditions centrifugal 12 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, transfer and set to 0 .6 hour in 1～5 DEG C, refrigerator, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.00008 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent 1200 type high performance liquid chromatographs; Chromatographic column Symmetry C18 (250 × 4.6mm, 5 μ are m); Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 15 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and each 0.00001 parts by volume of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.035, No. 1,37, peak, No. 0.819,36, peak, No. 0.560,32, peak, No. 5 No. 0.148,24, peaks.
The finger print quality detecting method of tripterygium hypoglaucum hutcs medicinal material of the present invention can also be preferably as follows step
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 1.2 weight portions after sieve No. 2, put in conical flask, add methyl alcohol 70 parts by volume, put in 35 DEG C of ultrasonic water bath ultrasonic extraction 1.3 hours, room temperature is placed 1.2 hours, under 4000 revs/min of conditions centrifugal 6 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, at 1～5 DEG C, refrigerator, place 1.2 hours, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.00012 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent 1200 type high performance liquid chromatographs; Chromatographic column Symmetry C18 (250 × 4.6mm, 5 μ are m); Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 25 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and each 0.00001 parts by volume of reference substance solution, injection liquid chromatography, measures, and to obtain final product.Gradient elution mobile phase in the finger print quality detecting method step C of above-mentioned tripterygium hypoglaucum hutcs medicinal material is set in following ratio:
1, gradient 1 mobile phase: A: B: C=30: 70: 0, elution time was 30 minutes;
2, gradient 2 mobile phases: A: B: C=49: 51: 0, elution time 20 minutes;
3, gradient 3 mobile phases: A: B: C=61: 29: 10, elution time was 10 minutes;
4, gradient 4 mobile phases: A: B: C=65: 20: 15, elution time was 15 minutes;
5, gradient 5 mobile phases: A: B: C=92: 8: 0, till being eluted to characteristic peak and occurring completely.
The mobile phase of above-mentioned gradient elution is overall process of continually varying, and wherein A is acetonitrile, and B is methyl alcohol, and C is 0.02% phosphoric acid solution; A+B+C=100%.
The quality determining method of tripterygium hypoglaucum hutcs medicinal material of the present invention, No. 5 peaks are that triptolide, No. 36 peaks are that Celastrol, No. 37 peaks are wilforlide A.Triptolide, Celastrol and wilforlide A content can calculate by the peak area ratio of the peak area of need testing solution and reference substance solution.The pass of foregoing invention weight portion and parts by volume is the relation of g/ml.
The inventive method feature: triptolide, Celastrol and wilforlide A are the main effective constituent of tripterygium hypoglaucum hutcs medicinal material, and the height of its content can directly react the quality of quality of medicinal material; Finger-print of the present invention not only can be measured the content of above-mentioned three kinds of compositions, also the one-tenth of its microcosmic is grouped into and is analyzed, and has formulated finger-print common pattern, has all reached state-of-the-art level from quantitative and qualitative analysis aspect.
Brief description of the drawings:
Accompanying drawing 1: methanol solvate extraction effect figure
Accompanying drawing 2: ethyl acetate extraction effect figure
Accompanying drawing 3: the comparison diagram of extraction time
Accompanying drawing 4: the comparison diagram that detects wavelength
Accompanying drawing 5: stability comparison diagram
Accompanying drawing 6: repeated comparison diagram
Accompanying drawing 7: the standard finger-print of tripterygium hypoglaucum hutcs medicinal material
Accompanying drawing 8: tripterygium hypoglaucum hutcs medicinal material standard finger-print common characteristic peak
Accompanying drawing 9: below the standard finger-print of tripterygium hypoglaucum hutcs medicinal material and control sample collection of illustrative plates stacking diagram, experiment and embodiment are used for further illustrating but are not limited to the present invention.
Experimental example one: methodology experiment
1, extract the selection of solvent
Get tripterygium hypoglaucum hutcs medicinal powder 1g, add respectively ethyl acetate, the each 80ml of methyl alcohol, measure according to the method described in the embodiment of the present invention 1, result shows that acetic acid ethyl acetate extract impurity is fewer than methyl alcohol, liposoluble constituent is except No. 32 peak extraction ratios are higher than methyl alcohol, and all the other are as suitable in Celastrol, the first-class composition of Triptolide, and water soluble ingredient is that methyl alcohol extraction is comparatively abundant, weigh choices, finally selecting methyl alcohol is to extract solvent.See accompanying drawing one and two.
2, the comparison of extraction time
Get tripterygium hypoglaucum hutcs medicinal powder 1g, successively extract 3 times respectively with methyl alcohol, each 80ml measures in accordance with the law, and result shows that once extraction can be to can meet fingerprint pattern technology requirement.See accompanying drawing three.
3, detect the selection of wavelength
According to bibliographical information, compare 210nm, 220nm, 267nm, tetra-kinds of detection wavelength of 280nm, finally choose the basic 210nm that can show Global Information and detect wavelength for measuring.See accompanying drawing four (1-210nm; 2-220nm; 3-267nm; 4-280nm).
Experimental example two: stability test
Get need testing solution, respectively at 0,1.5,3,4.5,6,7.5,24,48,72 hour different time points sample introduction, gained chromatogram imports similarity software for calculation, calculate included angle cosine similarity, the similarity of each time point is all more than 0.99, collection of illustrative plates is consistent, shows to have good stability, and sees accompanying drawing five.
Experimental example three: replica test
Get tripterygium hypoglaucum hutcs medicinal powder, take in quintuplicate, measure according to the method described in the embodiment of the present invention 1, gained chromatogram imports similarity software for calculation, calculates included angle cosine similarity, and result is all more than 0.99,5 duplicate samples collection of illustrative plates are consistent, show that repeatability is good, see accompanying drawing six.
Experimental example four: the HPLC finger-print common pattern of setting up Root of Tripterygium Hypoglaucum
Collection of illustrative plates is imported to fingerprint map analyzing software, choose 10 batches of tripterygium hypoglaucum hutcs samples, adopt median screening to set up common pattern, 42 characteristic peaks are selected altogether, wherein No. 5 peaks are triptolide triptolide, No. 36 peaks are Celastrol Tripterine, No. 37 peak is wilforlide A wilforlide, sees accompanying drawing seven (in figure chromatographic peak be respectively 1 to No. 42 peak) from left to right, accompanying drawing eight, accompanying drawing nine (in figure, from left to right three chromatographic peaks are respectively triptolide, Celastrol, wilforlide A).
Following embodiment is used for further illustrating but is not limited to the present invention.
Embodiment mono-:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing the powder 1g after sieve No. 2, put in conical flask, add methyl alcohol 80ml, put in 40 DEG C of ultrasonic water bath ultrasonic extraction 1 hour, room temperature is placed 1 hour, under 4000 revs/min of conditions centrifugal 10 minutes, take out, incline and get supernatant and put and in Rotary Evaporators, be concentrated near dryly, residue dissolves with methyl alcohol 4ml, at 1～5 DEG C, refrigerator, place 1 hour, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5ml dilution for supernatant, be the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution;
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every 1ml containing 0.0001g triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent 1200 type high performance liquid chromatographs; Chromatographic column Symmetry C18 (250 × 4.6mm, 5 μ are m); Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 20 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and the each 10 μ l of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.044, No. 1,37, peak, No. 0.829,36, peak, No. 0.569,32, peak, No. 5 No. 0.156,24, peaks.
Gradient elution mobile phase in the finger print quality detecting method step C of above-mentioned tripterygium hypoglaucum hutcs medicinal material is set in following ratio:
1, gradient 1 mobile phase: A: B: C=30: 70: 0, elution time was 30 minutes;
2, gradient 2 mobile phases: A: B: C=49: 51: 0, elution time 20 minutes;
3, gradient 3 mobile phases: A: B: C=61: 29: 10, elution time was 10 minutes;
4, gradient 4 mobile phases: A: B: C=65: 20: 15, elution time was 15 minutes;
5, gradient 5 mobile phases: A: B: C=92: 8: 0, till being eluted to characteristic peak and occurring completely.
The mobile phase of above-mentioned gradient elution is overall process of continually varying, and wherein A is acetonitrile, and B is methyl alcohol, and C is 0.02% phosphoric acid solution; A+B+C=100%.
Embodiment bis-:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing the powder 0.8g after sieve No. 2, put in conical flask, add methyl alcohol 90ml, put in 45 DEG C of ultrasonic water bath ultrasonic extraction 0.8 hour, room temperature is placed 0.8 hour, under 4000 revs/min of conditions centrifugal 12 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves with methyl alcohol 4ml, transfer and set to 0 .6 hour in 1～5 DEG C, refrigerator, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5ml dilution for supernatant, it is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every 1ml containing 0.00008g triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent 1200 type high performance liquid chromatographs; Chromatographic column Symmetry C18 (250 × 4.6mm, 5 μ are m); Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 15 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and the each 10 μ l of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.035, No. 1,37, peak, No. 0.819,36, peak, No. 0.560,32, peak, No. 5 No. 0.148,24, peaks.
Gradient elution mobile phase in the finger print quality detecting method step C of above-mentioned tripterygium hypoglaucum hutcs medicinal material is set in following ratio:
1, gradient 1 mobile phase: A: B: C=30: 70: 0, elution time was 30 minutes;
2, gradient 2 mobile phases: A: B: C=49: 51: 0, elution time 20 minutes;
3, gradient 3 mobile phases: A: B: C=61: 29: 10, elution time was 10 minutes;
4, gradient 4 mobile phases: A: B: C=65: 20: 15, elution time was 15 minutes;
5, gradient 5 mobile phases: A: B: C=92: 8: 0, till being eluted to characteristic peak and occurring completely.
The mobile phase of above-mentioned gradient elution is overall process of continually varying, and wherein A is acetonitrile, and B is methyl alcohol, and C is 0.02% phosphoric acid solution; A+B+C=100%.
Embodiment tri-,
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing the powder 1.2g after sieve No. 2, put in conical flask, add methyl alcohol 70ml, put in 35 DEG C of ultrasonic water bath ultrasonic extraction 1.3 hours, room temperature is placed 1.2 hours, under 4000 revs/min of conditions centrifugal 6 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves with methyl alcohol 4ml, at 1～5 DEG C, refrigerator, place 1.2 hours, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5ml dilution for supernatant, it is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every 1ml containing 0.00012g triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent 1200 type high performance liquid chromatographs; Chromatographic column Symmetry C18 (250 × 4.6mm, 5 μ are m); Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 25 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and the each 10 μ l of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.048, No. 1,37, peak, No. 0.835,36, peak, No. 0.578,32, peak, No. 5 No. 0.160,24, peaks.
Gradient elution mobile phase in the finger print quality detecting method step C of above-mentioned tripterygium hypoglaucum hutcs medicinal material is set in following ratio:
1, gradient 1 mobile phase: A: B: C=30: 70: 0, elution time was 30 minutes;
2, gradient 2 mobile phases: A: B: C=49: 51: 0, elution time 20 minutes;
3, gradient 3 mobile phases: A: B: C=61: 29: 10, elution time was 10 minutes;
4, gradient 4 mobile phases: A: B: C=65: 20: 15, elution time was 15 minutes;
5, gradient 5 mobile phases: A: B: C=92: 8: 0, till being eluted to characteristic peak and occurring completely.
The mobile phase of above-mentioned gradient elution is overall process of continually varying, and wherein A is acetonitrile, and B is methyl alcohol, and C is 0.02% phosphoric acid solution; A+B+C=100%.
Claims (5)
1. a quality determining method for tripterygium hypoglaucum hutcs medicinal material, is characterized in that the method comprises the steps:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 0.5～1.5 weight portion after sieve No. 2, put in conical flask, add methyl alcohol 60～100 parts by volume, put in 30～50 DEG C of ultrasonic water bath ultrasonic extraction 0.5～1.5 hour, room temperature is placed 0.5～1.5 hour, under 4000 revs/min of conditions centrifugal 5～15 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, transfer and set to 0 .5～1.5 hour in 1～5 DEG C, refrigerator, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.00005～0.00015 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent1200 type high performance liquid chromatograph; Specification is 250 × 4.6mm, the chromatographic column Symmetry C18 of 5 μ m; Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 10～30 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and the each 10 μ L of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, and each peak relative retention time is: peak 1.030～1.050, No. 1,37, peak, No. 0.820～0.840,36, peak, No. 0.560～0.580,32, peak, No. 5 No. 0.140～0.160,24, peaks; Wherein No. 5 peaks are that triptolide, No. 37 peaks are wilforlide A, and No. 36 peak is with reference to peak Celastrol chromatographic peak;
The pass of foregoing invention weight portion and parts by volume is the relation of g/ml.
2. the quality determining method of tripterygium hypoglaucum hutcs medicinal material as claimed in claim 1, is characterized in that the method comprises the steps:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 1 weight portion after sieve No. 2, put in conical flask, add methyl alcohol 80 parts by volume, put in 40 DEG C of ultrasonic water bath ultrasonic extraction 1 hour, room temperature is placed 1 hour, under 4000 revs/min of conditions centrifugal 10 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, at 1～5 DEG C, refrigerator, place 1 hour, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.0001 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent1200 type high performance liquid chromatograph; Specification is 250 × 4.6mm, the chromatographic column Symmetry C18 of 5 μ m; Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 20 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and the each 10 μ L of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.044, No. 1,37, peak, No. 0.829,36, peak, No. 0.569,32, peak, No. 5 No. 0.156,24, peaks;
The pass of foregoing invention weight portion and parts by volume is the relation of g/ml.
3. the quality determining method of tripterygium hypoglaucum hutcs medicinal material as claimed in claim 1, is characterized in that the method comprises the steps:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 0.8 weight portion after sieve No. 2, put in conical flask, add methyl alcohol 90 parts by volume, put in 45 DEG C of ultrasonic water bath ultrasonic extraction 0.8 hour, room temperature is placed 0.8 hour, under 4000 revs/min of conditions centrifugal 12 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, transfer and set to 0 .6 hour in 1～5 DEG C, refrigerator, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.00008 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent1200 type high performance liquid chromatograph; Specification is 250 × 4.6mm, the chromatographic column Symmetry C18 of 5 μ m; Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 15 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and the each 10 μ L of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.035, No. 1,37, peak, No. 0.819,36, peak, No. 0.560,32, peak, No. 5 No. 0.148,24, peaks;
The pass of foregoing invention weight portion and parts by volume is the relation of g/ml.
4. the quality determining method of tripterygium hypoglaucum hutcs medicinal material as claimed in claim 1, is characterized in that the method comprises the steps:
A, need testing solution preparation
Take tripterygium hypoglaucum hutcs medicinal material through pulverizing powder 1.2 weight portions after sieve No. 2, put in conical flask, add methyl alcohol 70 parts by volume, put in 35 DEG C of ultrasonic water bath ultrasonic extraction 1.3 hours, room temperature is placed 1.2 hours, under 4000 revs/min of conditions centrifugal 6 minutes, take out, incline get supernatant put in Rotary Evaporators, be concentrated near dry, residue dissolves by methyl alcohol 4 parts by volume, at 1～5 DEG C, refrigerator, place 1.2 hours, take out, under 10000 revs/min of conditions centrifugal 5 minutes, methyl alcohol 5 parts by volume dilutions for supernatant, dilution is the filtering with microporous membrane of 0.45 μ m with aperture, get subsequent filtrate as need testing solution,
B, reference substance solution preparation
Get triptolide, Celastrol, three kinds of reference substances of wilforlide A, be made into the solution of every parts by volume containing 0.00012 weight portion triptolide, Celastrol or wilforlide A, product solution in contrast with methyl alcohol respectively;
C, chromatographic condition
Chromatograph Agilent1200 type high performance liquid chromatograph; Specification is 250 × 4.6mm, the chromatographic column Symmetry C18 of 5 μ m; Mobile phase: acetonitrile-methyl alcohol-0.02% phosphoric acid solution gradient elution; 25 DEG C of column temperatures; Flow velocity 1.0ml/ minute; Detect wavelength 210nm;
D, determination method
Draw need testing solution and the each 10 μ L of reference substance solution, injection liquid chromatography, measures, and to obtain final product; Tripterygium hypoglaucum hutcs medicinal material standard finger-print has 42 characteristic peaks, common characteristic peak is No. 5 peak, No. 24 peaks, No. 32 peaks, No. 36 peaks, Wu Ge peak, No. 37 peaks according to peak sequence, wherein No. 36 peaks are with reference to peak Celastrol chromatographic peak, and each peak relative retention time is: peak 1.048, No. 1,37, peak, No. 0.835,36, peak, No. 0.578,32, peak, No. 5 No. 0.160,24, peaks;
The pass of foregoing invention weight portion and parts by volume is the relation of g/ml.
5. the quality determining method of the tripterygium hypoglaucum hutcs medicinal material as described in as arbitrary in claim 1-4, is characterized in that: in the gradient elution in the fingerprint pattern quality determination method step C of tripterygium hypoglaucum hutcs medicinal material, mobile phase is set in following ratio:
Gradient 1 mobile phase: A:B:C=30:70:0, elution time is 30 minutes;
Gradient 2 mobile phases: A:B:C=49:51:0, elution time 20 minutes;
Gradient 3 mobile phases: A:B:C=61:29:10, elution time is 10 minutes;
Gradient 4 mobile phases: A:B:C=65:20:15, elution time is 15 minutes;
Gradient 5 mobile phases: A:B:C=92:8:0, till being eluted to characteristic peak and occurring completely;
The mobile phase of above-mentioned gradient elution is overall process of continually varying, and wherein A is acetonitrile, and B is methyl alcohol, and C is 0.02% phosphoric acid solution; A+B+C=100%.
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| CN106153750A (en) * | 2015-04-10 | 2016-11-23 | 浙江海洋学院 | One utilizes the method for wilforine content in ion liquid abstraction/HPLC detection tripterygium hypoglaucum hutcs rhizome |
| CN104991020B (en) * | 2015-06-29 | 2017-03-15 | 公安部物证鉴定中心 | The Liquid Chromatography-Tandem Mass Spectrometry method of inspection of wilforlide A, Tripterygium wilfordii lactone ketone, triptolide and tripterine |
| CN110567984A (en) * | 2019-09-30 | 2019-12-13 | 重庆市药研院制药有限公司 | detection method for evaluating quality of medicinal material of herba Humuli Scandentis |
| CN115407005A (en) * | 2022-09-30 | 2022-11-29 | 重庆市药研院制药有限公司 | Method for detecting internal and external components of root piece body of torch flower |
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