CN104007205A - Method for detecting pharmaceutical preparation for treating xiaoke disease - Google Patents
Method for detecting pharmaceutical preparation for treating xiaoke disease Download PDFInfo
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Abstract
The invention belongs to the field of detection of quality of traditional Chinese preparations, and particularly relates to a method for detecting a pharmaceutical preparation for treating the xiaoke disease. The pharmaceutical preparation comprises the active pharmaceutical ingredients, by weight, 6000-8000 parts of silkworm feces and 100-200 parts of radix glycyrrhizae. According to the method for detecting the pharmaceutical preparation, the pharmaceutical preparation is detected by measuring the content of 1-Deoxynojirimycin through an HPLC method. The method for detecting the pharmaceutical preparation is rapid, comprehensive and highly targeted, the quality of the preparation can be effectively controlled, and use safety and stability of the preparation can be improved.
Description
Technical field
The invention belongs to Chinese medicine preparation detection field, be specifically related to a kind of detection method of pharmaceutical preparation for the treatment of diabete.
Background technology
At present, in global range, the patient of diabete (diabetes) is about more than 200,000,000, and wherein 85% patient is old-aged diabetic, and the incidence of disease has the trend day by day increasing; China is one of country that diabetic's number is in the world maximum, is only second to the U.S..The mankind's even life of health in diabetes serious threat.
Chinese patent CN101496829A discloses a kind of pharmaceutical composition for the treatment of diabete, the bulk drug of this pharmaceutical composition is mainly made up of silkworm excrement and Radix Glycyrrhizae, silkworm excrement in prescription has dispels rheumatism, invigorate blood circulation effect of analgesic therapy, this pharmaceutical composition has good hypoglycemic effect, and treatment diabetes is had to good curative effect.In the document, also disclose by methods such as TLC method detection cupreol, HPLC method detection ammonium glycyrrhizinate and HPLC method detection Pipecolic Acids this pharmaceutical composition has been carried out to quality control.
But, along with the further investigation to this medicine, clinical test results shows, the active principle of these medicine composite for curing diabetes is mainly the alkaloid containing in silkworm excrement, there is effect of remarkable inhibition a-glucosidase activity, and described alkaloidal main active is 1-DNJ.But existing quality control standard and detection method are all only to measure for the Pipecolic Acid wherein containing, the index not only detecting is comparatively single, control effectively nor be beneficial to the quality to this medicine on the whole.Therefore, set up a kind of quality testing of aforementioned pharmaceutical compositions that can be quick, comprehensive, with strong points and the method for quality control has great importance.
Summary of the invention
For this reason, the detection method of the pharmaceutical composition that is existing treatment diabete to be solved by this invention exist detect index single, be unfavorable for problem that the quality of this medicine is control effectively, thereby a kind of method that can detect the pharmaceutical composition of described treatment diabetes fast, comprehensively, is targetedly proposed.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
The detection method of a kind of pharmaceutical preparation for the treatment of diabete of the present invention, the bulk drug of described pharmaceutical preparation consists of: silkworm excrement 6000-8000 weight portion and Radix Glycyrrhizae 100-200 weight portion;
This detection method comprises the following assay step to 1-DNJ:
Accurately weighed 1-DNJ reference substance 5-15mg, thin up is made the reference substance storing solution of every mL containing 0.1-0.5mg; Precision measures reference substance storing solution described in 1mL, and accurate to add mass concentration be the NaHCO of 1-3%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 1-4mL of solution 1-4mL and 1-3mg/mL, mixes and carries out water-bath 20-40min in 30-60 DEG C, is settled to 20-30mL subsequently, in contrast product solution with the acetonitrile of 30-60%;
Get pharmaceutical preparation porphyrize to be measured, and precision takes 0.5-2.0g, the water 30-60mL that to add with concentrated hydrochloric acid adjust pH be 3-4, stirs, ultrasonic processing 20-30min, and centrifuging and taking supernatant; Residue adds the water that pH value is 3-4 and washs in right amount, centrifugal merging supernatant, and be concentrated into 10-20mL; Concentrate is placed in to cation exchange resin column, first washes with water, collect and discard water elution liquid, then use 0.5M ammoniacal liquor wash-out, and collect ammoniacal liquor eluent, and being concentrated near doing, the 10mL that adds water subsequently dissolves, ultrasonic processing 1-5 minute, and add water and be settled to 20-30mL, test sample storing solution obtained; Precision measures test sample storing solution described in 1mL, and adding successively mass concentration is the NaHCO of 1-3%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1-3mg/mL, mixes and carries out water-bath 40min in 30 DEG C, is settled to 20-30mL subsequently, as need testing solution with the acetonitrile of 30-60%;
According to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, the phosphoric acid solution taking second eyeball as mobile phase A, taking 0.2% carries out gradient elution: 0-8min as Mobile phase B according to following program, and A:B is 25%:75%; 8-40min, A:B is 25%:75% → 50%:50%; 40-45min, A:B is 50%:50%; 45-46min, A:B is 50%:50% → 25%:75%; 46-75min, A:B is 25%:75%; Coutroi velocity 1.0mL/min, 25 DEG C of column temperatures, detect wavelength 263nm, and number of theoretical plate calculates and should be not less than 6000 by 1-DNJ peak;
Accurate described reference substance solution and the each 20 μ L of need testing solution of drawing, injection liquid chromatography, measures.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, describedly the content of 1-DNJ is carried out to determination step comprise:
Accurately weighed 1-DNJ reference substance 10mg, thin up is made the reference substance storing solution of every mL containing 0.2mg; Precision measures reference substance storing solution described in 1mL, and the accurate mass concentration that adds is 2% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 2mL of solution 2mL and 2mg/mL, mixes and carries out water-bath 40min in 50 DEG C, is settled to 25mL subsequently, in contrast product solution with 30% acetonitrile;
Get pharmaceutical preparation porphyrize to be measured, and precision takes 1g, the water 50mL that to add with concentrated hydrochloric acid adjust pH be 3-4, stirs, ultrasonic processing 30min, and centrifuging and taking supernatant; Residue adds the water that pH value is 3-4 and washs in right amount, centrifugal merging supernatant, and be concentrated into 10mL; Concentrate is placed in to cation exchange resin column, first washes with water, collect and discard water elution liquid, then use 0.5M ammoniacal liquor wash-out, and collect ammoniacal liquor eluent, and being concentrated near doing, the 10mL that adds water subsequently dissolves, ultrasonic processing 1 minute, and add water and be settled to 25mL, test sample storing solution obtained; Precision measures test sample storing solution described in 1mL, and adding successively mass concentration is 2% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1mg/mL, mixes and carries out water-bath 40min in 50 DEG C, is settled to 25mL subsequently, as need testing solution with 30% acetonitrile;
According to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, the phosphoric acid solution taking second eyeball as mobile phase A, taking 0.2% carries out gradient elution: 0-8min as Mobile phase B according to following program, and A:B is 25%:75%; 8-40min, A:B is 25%:75% → 50%:50%; 40-45min, A:B is 50%:50%; 45-46min, A:B is 50%:50% → 25%:75%; 46-75min, A:B is 25%:75%; Coutroi velocity 1.0mL/min, 25 DEG C of column temperatures, detect wavelength 263nm, and number of theoretical plate calculates and should be not less than 6000 by 1-DNJ peak;
Accurate described reference substance solution and the each 20 μ L of need testing solution of drawing, injection liquid chromatography, measures.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, in the assay step of above-mentioned 1-DNJ, above-mentioned cation exchange resin column is D001-CC type cation exchange resin column, and resin volume is 50mL, and blade diameter length ratio is 1:8.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, also comprise the step of above-mentioned Zeo-karb being carried out to pre-treatment, pre-treatment specifically comprises the steps: with isopyknic 2M salt acid soak D001-CC type ion exchange resin more than 4 hours; Rinse resin with the 2M hydrochloric acid of 4 times of resin volumes again, be washed to neutrality; With the 2M NaOH solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M hydrochloric acid solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M NaOH solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M hydrochloric acid solution flushing resin of 3 times of resin volumes, be washed to neutrality.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, in the assay step of above-mentioned 1-DNJ, the condition of above-mentioned ultrasonic processing is: power 300W, frequency 50KHz.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, the method also comprises at least one in the step of following discriminating and/or assay:
A, TLC differentiate
Get described pharmaceutical preparation 0.4-0.8g to be measured, add absolute ethyl alcohol 3-8mL, in 60-90 DEG C of water-bath backflow 20-40 minute, leave standstill and get supernatant, put in water-bath and steam to 0.5-2.0mL, as need testing solution;
Separately get cupreol reference substance, add absolute ethyl alcohol and make the reference substance solution containing 0.5-2.0mg in every 1mL;
Test according to thin-layered chromatography, accurate above-mentioned two kinds of need testing solutions and the each 1-3 μ of the reference substance solution L of drawing, put respectively on same silica gel g thin-layer plate, methenyl choloride taking volume ratio as 18-19.8:0.2-2.0: acetone is developping agent, launch, take out, dry, spray, with 5-15% phosphorus molybdenum acid solution, is differentiated in 100-110 DEG C of colour developing;
B, HPLC method are measured the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 5-15mg, the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution taking volume ratio as 50-70:30-50:0.5-1.5 dissolves and is diluted to 50mL, in contrast product solution;
Get pharmaceutical preparation 1-3 gram to be measured, adding volume ratio is methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 50-70:30-50:0.5-1.5, and ultrasonic processing 30 minutes, continues to add described mixed solution to be settled to 25mL after letting cool, centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid taking volume ratio as 50-70:30-50:0.5-1.5 is mobile phase, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 2000 by ammonium glycyrrhetate peak;
Accurate described reference substance solution and the each 5-15 μ of the need testing solution L of drawing respectively, injection liquid chromatography, measures;
C, HPLC method are measured the content of Pipecolic Acid
Accurately weighed Pipecolic Acid reference substance 3-8mg, thin up is settled to 50mL; Precision measures 0.5-2.0mL solution, add water successively 0.5-2.0mL, add mass concentration be 0.8% 2, the sodium bicarbonate solution 1-3mL of 4-dinitrofluorobenzene acetonitrile solution 1-3mL and 0.5mol/L, in 60-90 DEG C of water-bath 0.5-2.0 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, product solution in contrast;
Accurately weighed pharmaceutical preparation 0.3-0.8g to be measured, add water after jolting, in 60-90 DEG C of water-bath, heat 20-40 minute, after letting cool, add water and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 1-3mL, and add successively mass concentration be 0.8% 2, the sodium bicarbonate solution 1-3mL of 4-dinitrofluorobenzene acetonitrile solution 1-3mL and 0.5mol/L, in 60-90 DEG C of water-bath 0.5-2.0 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution taking volume ratio as 15-25:0.1-1:75-85 is mobile phase, and detection wavelength is 390nm; Number of theoretical plate calculates and should be not less than 2500 by Pipecolic Acid peak;
Accurate reference substance solution 4-6 μ L and the need testing solution 5-15 μ L of drawing respectively, injection liquid chromatography, measures.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, the method also comprises at least one in the step of following discriminating and/or assay:
A, TLC differentiate
Get described pharmaceutical preparation 0.5g to be measured, add absolute ethyl alcohol 5mL, reflux 30 minutes in 60 DEG C of water-baths, leave standstill and get supernatant, put in water-bath and steam to 1mL, as need testing solution;
Separately get cupreol reference substance, add absolute ethyl alcohol and make the reference substance solution containing 1mg in every 1mL;
Test according to thin-layered chromatography, accurate above-mentioned two kinds of need testing solutions and the each 2 μ L of reference substance solution of drawing, put respectively on same silica gel g thin-layer plate, methenyl choloride taking volume ratio as 19.5:0.5: acetone is developping agent, launch, take out, dry, spray, with 10% phosphorus molybdenum acid solution, is differentiated in 105 DEG C of colour developings;
B, HPLC method are measured the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 10mg, the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution taking volume ratio as 60:40:1 dissolves and is diluted to 50mL, in contrast product solution;
Get 2.0 grams of pharmaceutical preparations to be measured, adding volume ratio is methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 60:40:1, and ultrasonic processing 30 minutes, continues to add described mixed solution to be settled to 25mL after letting cool, and centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid taking volume ratio as 60:40:1 is mobile phase, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 2000 by ammonium glycyrrhetate peak;
Accurate described reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography, measures;
C, HPLC method are measured the content of Pipecolic Acid
Accurately weighed Pipecolic Acid reference substance 5mg, thin up is settled to 50mL; Precision measures 1mL solution, add water successively 1mL, add mass concentration be 0.8% 2, the sodium bicarbonate solution 2mL of 4-dinitrofluorobenzene acetonitrile solution 2mL and 0.5mol/L, in 60 DEG C of water-baths 1 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, product solution in contrast;
Accurately weighed pharmaceutical preparation 0.4g to be measured, add water after jolting, in 60 DEG C of water-baths, heat 30 minutes, after letting cool, add water and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 2mL, and to add successively mass concentration be 0.8% DNF acetonitrile solution 2mL and the sodium bicarbonate solution 2mL of 0.5mol/L, in 60 DEG C of water-baths 1 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution taking volume ratio as 21:0.5:79 is mobile phase, and detection wavelength is 390nm; Number of theoretical plate calculates and should be not less than 2500 by Pipecolic Acid peak;
Accurate reference substance solution 5 μ L and the need testing solution 10 μ L of drawing respectively, injection liquid chromatography, measures.
The relevant prescription of the pharmaceutical preparation for the treatment of diabete of the present invention and preparation method and disclosed detection method refer to the disclosed content of Chinese patent CN101496829A.
The bulk drug of pharmaceutical preparation of the present invention consists of: silkworm excrement 7150 weight portions and Radix Glycyrrhizae 137.5 weight portions; Or silkworm excrement 6150 weight portions and Radix Glycyrrhizae 187.5 weight portions; Or silkworm excrement 7850 weight portions and Radix Glycyrrhizae 117.5 weight portions.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, said medicine preparation is prepared by the following method: the silkworm excrement of getting selected weight portion adds 2-6 doubly to measure 50-70% ethanol, and dipping spends the night, slowly be heated to boil, backflow 1-3 time, each 0.5-1.5 hour, filter, merging filtrate, reclaims ethanol, being concentrated into relative density is 0.95-1.10, adds 1 times of water gaging to continue to be heated to boil, cooling, placement is spent the night, get supernatant, be concentrated into relative density and be the thick paste A of 1.00-1.15, for subsequent use; The Radix Glycyrrhizae of separately getting selected weight portion adds 8-15 times of water gaging and decocts 1-3 time, each 1-3 hour, and merging filtrate, placing spends the night makes precipitation, gets the thick paste B that supernatant concentration to relative density is 1.00-1.15, for subsequent use; Merge above-mentioned thick paste A and B, add conventional auxiliary material, make clinical acceptable formulation according to common process.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, said medicine preparation is prepared by the following method: the silkworm excrement of getting selected weight portion adds 4 times of amount 60% ethanol, and dipping spends the night, slowly be heated to boil, reflux twice, each 1 hour, filter, merging filtrate, reclaims ethanol, being concentrated into relative density is 1.01-1.06, adds 1 times of water gaging to continue to be heated to boil, cooling, placement is spent the night, get supernatant, be concentrated into the thick paste that relative density is 1.06-1.15, for subsequent use; The Radix Glycyrrhizae of separately getting selected weight portion adds 10 times of water gagings and decocts 2 times, and each 2 hours, merging filtrate, placing spends the night makes precipitation, gets supernatant concentration to relative density and is 1.04-1.15 thick paste, for subsequent use; Merge above-mentioned two thick pastes, add conventional auxiliary material, make clinical acceptable formulation according to common process.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, the pharmaceutical preparation of above-mentioned treatment diabete is tablet, capsule, pill, granule, honey refining pill, sustained release preparation, quick releasing formulation, controlled release preparation, oral liquid or ejection preparation.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, by the content of HPLC method mensuration 1-DNJ.This detection method is quick, comprehensive, with strong points, is conducive to the quality of this medicine to control effectively, and contributes to improve the safety and stability of this drug use.
Brief description of the drawings
For content of the present invention is more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein
Fig. 1 is the mould reference substance chromatogram of the wild buttocks of 1-deoxidation in the embodiment of the present invention 2;
Fig. 2 is the negative chromatogram of the scarce silkworm excrement in the embodiment of the present invention 2;
Fig. 3 is the capsule sample chromatogram that the embodiment 1 in the embodiment of the present invention 2 prepares gained;
Fig. 4 is the reagent blank chromatogram in the embodiment of the present invention 2;
Fig. 5 is the 1-DNJ canonical plotting in the embodiment of the present invention 2.
Embodiment
The preparation of embodiment 1 capsule
[prescription] silkworm excrement 7.15kg and Radix Glycyrrhizae 137.5g.
[method for making] above 2 tastes, get silkworm excrement and add 4 times of amount 60% ethanol, and dipping spends the night, slowly be heated to boil, reflux twice, each 1 hour, filter, merging filtrate, reclaims ethanol, being concentrated into relative density is 1.01-1.06 (55 DEG C), adds 1 times of water gaging to continue to be heated to boil, cooling, placement is spent the night, get supernatant, be concentrated into relative density and be the thick paste of 1.06-1.15 (55 DEG C of surveys), for subsequent use.Another extracting Radix Glycyrrhizae decocts 2 times with 10 times of water gagings, and each 2 hours, merging filtrate, placing spends the night makes precipitation, gets supernatant concentration to relative density and is 1.04-1.15 (55 DEG C of surveys) thick paste, for subsequent use.Merge two thick pastes, add according to a conventional method conventional auxiliary material to make 1000, to obtain final product.
Embodiment 2HPLC method is measured the content of 1-DNJ
1 instrument and reagent
Agilent-1200 high performance liquid chromatograph comprises: vacuum degassing machine, quaternary gradient pump, automatic sampler, diode array detector (DAD), Agilent-Chemistation data handling system (Agilent company); Superpure water machine (MilliQ-Gradient type); Ultrasonic generator (Kunshan KQ-300E type).
1-DNJ reference substance (being purchased from SIGMA company), fluorenes methoxy dicarbonyl chloride (being purchased from SIGMA company), acetonitrile is chromatographically pure, and water is ultrapure water, and it is pure that all the other reagent are analysis.
2 methods and result
The preparation of 2.1 reference substance solution, need testing solution and 1-DNJ negative sample solution
2.1.1 the preparation of reference substance solution
Get the about 10mg of 1-DNJ reference substance, accurately weighed, put in 50mL measuring bottle, be diluted with water to scale, shake up, make the reference substance solution of every mL containing 0.2mg.Precision measures 1mL, to 25mL measuring bottle, adds 2%NaHCO
3solution 2mL, jolting 10 seconds, precision adds fluorenes methoxy dicarbonyl chloride acetonitrile solution (FMOC-Cl) 2mL of 2mg/mL, jolting 10 seconds, 50 DEG C of water-bath 40min, are settled to 25mL with 30% acetonitrile, to obtain final product.
2.1.2 the preparation of need testing solution
The pre-treatment of D001-CC type Zeo-karb: use in turn isopyknic 2M salt acid soak D001-CC type ion exchange resin more than 4 hours, then rinse resin with the 2M hydrochloric acid of 4 times of resin volumes, be washed to neutrality; With the 2M NaOH solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M hydrochloric acid solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M NaOH solution flushing resin of 5 times of resin volumes, be washed to neutrality; Rinse resin with the 2M hydrochloric acid solution of 3 times of resin volumes again, be washed to neutrality, for subsequent use.
Get the content porphyrize that embodiment 1 prepares the capsule of gained, get about 1g, accurately weighed, put in 100mL beaker, the water 50mL that to add with concentrated hydrochloric acid adjust pH be 3-4, stirs, ultrasonic processing (power 300W, frequency 50KHz) 30min, centrifugal 10min (4000 turn/min), divides and gets supernatant, residue adds the water that pH value is 3-4 and washs in right amount, centrifugal 10min (4000 turn/min), merges supernatant, is concentrated into about 10mL.Put D001-CC type cation exchange resin column (resin volume 50mL, footpath (2.5cm) high ratio: 1:8, H of processed mistake
+), first use 60mL water elution, discard eluent, then use 0.5M ammoniacal liquor 600mL wash-out (elution speed is 2.5mL/min), collect ammoniacal liquor eluent, be concentrated near doing, the 10mL that adds water, ultrasonic processing (power 300W, frequency 50KHz) 1 minute, transfer in 25mL volumetric flask, add water to scale, shake up.Precision measures 1mL, to 25mL measuring bottle, adds 2%NaHCO
3solution 1mL, jolting 10 seconds, precision adds fluorenes methoxy dicarbonyl chloride acetonitrile solution (FMOC-Cl) 4mL of 1mL/min, jolting 10 seconds, 50 DEG C of water-bath 40min, are settled to 25mL with 30% acetonitrile, to obtain final product.
2.1.3 the preparation of negative sample solution
Press the prescription extracting Radix Glycyrrhizae medicinal material of embodiment 1, do not contain the negative sample solution of silkworm excrement by preparation method's preparation of 2.1 listed need testing solutions.
2.1.4 blank sample solution preparation
According to the preparation method of 2.1 listed need testing solutions, preparation does not contain the blank sample solution of described test sample.
2.1.5 blank test
With octadecylsilane chemically bonded silica be filling agent; Select Agilent Hypersil ODS-C18 chromatographic column (4.0mm × 250mm, 5 μ m), diode array detector (detect wavelength 263nm); Mobile phase: second eyeball-0.2% phosphoric acid solution; Flow velocity: 1.0mL/min, sample size 20 μ L, number of theoretical plate (calculating by 1-DNJ peak) should be not less than 6000.Gradient elution is as follows:
| Time | Acetonitrile | 0.2% phosphoric acid solution |
| 0 | 25 | 75 |
| 8 | 25 | 75 |
| 40 | 50 | 50 |
| 45 | 50 | 50 |
| 46 | 25 | 75 |
| 75 | 25 | 75 |
Draw respectively 1-DNJ reference substance solution, negative sample solution, need testing solution and blank reagent solution, injecting high performance liquid chromatograph according to HPLC chromatographic condition measures, result is shown in respectively Fig. 1,2,3,4, result shows under this chromatographic condition, 1-DNJ peak separates better with other impurity peaks, negative sample chromatogram with the corresponding retention time of reference substance chromatogram place, without chromatographic peak occur, side in the not mensuration of disturbed one-DNJ of licorice medicinal materials.
2.2 system suitability
By testing under 2.1 listed test conditions, the parameter such as number of theoretical plate, degree of separation of sample and reference substance is tested, the results are shown in Table 1.
Table 1 system suitability measurement result
| Composition | Number of theoretical plate (Plates) | Degree of separation (Resolution) |
| 1-DNJ sample | 22858 | 9.10 |
| Reference substance | 22137 | 9.40 |
The investigation of 2.3 derivatization conditions
2.3.1 investigate 2%NaHCO
3solution usage, the impact of fluorenes methoxy dicarbonyl chloride acetonitrile solution consumption on derivative reaction
Precision measures 1-DNJ reference substance solution (0.2mg/mL) 1.0mL, each 9 parts, put respectively in 25mL measuring bottle, 9 duplicate samples are divided into 3 groups, every group respectively precision add 2%NaHCO
3solution 0.5mL, 1mL, 1.5mL, jolting 10 "; at 1,2, No. 3 sample of every group successively accurate fluorenes methoxy dicarbonyl chloride acetonitrile solution (FMOC-Cl) 2mL, 4mL, the 5mL that adds 2mg/mL respectively; jolting 10 seconds; 30 DEG C of water-bath 40min; be settled to 25mL with 50% acetonitrile, carry out liquid phase analysis by 2.1 listed chromatographic conditions, the results are shown in Table 2.Result shows to adopt 2%NaHCO
3solution 1mL, 2mg/mL fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL, can make derivative reaction complete.
The different derivatization condition of table 2 1-DNJ peak area measurement result
| NO. | Derivatization reagent consumption | 1-DNJ peak area |
| 1 | NaHCO 3Solution 0.5mL, FMOC-Cl2mL | 962.5897 |
| 2 | NaHCO 3Solution 0.5mL, FMOC-Cl4mL | 925.4458 |
| 3 | NaHCO 3Solution 0.5mL, FMOC-Cl5mL | 913.5612 |
| 4 | NaHCO 3Solution 1.0mL, FMOC-Cl2mL | 833.7783 |
| 5 | NaHCO 3Solution 1.0mL, FMOC-Cl4mL | 985.2354 |
| 6 | NaHCO 3Solution 1.0mL, FMOC-Cl5mL | 988.4532 |
| 7 | NaHCO 3Solution 1.5mL, FMOC-Cl2mL | 918.0567 |
| 8 | NaHCO 3Solution 1.5mL, FMOC-Cl4mL | 939.0082 |
| 9 | NaHCO 3Solution 1.5mL, FMOC-Cl5mL | 977.2541 |
2.3.2 investigate the impact on derivative reaction of temperature of reaction and reaction time
Precision measures 1-DNJ reference substance solution (0.2mg/mL) 1.0mL, and each 9 parts, put respectively in 25mL measuring bottle, precision adds 2%NaHCO respectively
3solution 1mL, jolting 10 seconds, accurate fluorenes methoxy dicarbonyl chloride acetonitrile solution (FMOC-Cl) 4mL that adds 2mg/mL respectively again, jolting 10 seconds, is divided into 3 groups by 9 duplicate samples, and 3 duplicate samples of every group are placed in respectively 20 DEG C, 30 DEG C, 40 DEG C water-baths, 1,2, No. 3 example reaction time of every group is followed successively by 20min, 30min, 40min, be settled to 25mL with 50% acetonitrile, carry out liquid phase analysis by 2.1 listed chromatographic conditions respectively, the results are shown in Table 3.Result shows to adopt 30 DEG C of water-bath 40min, can make derivative reaction complete.
Table 3 differential responses temperature and reaction time 1-DNJ peak area measurement result
| NO. | Temperature of reaction and reaction time | 1-DNJ peak area |
| 1 | 20 DEG C of water-bath 20min | 568.4563 |
| 2 | 20 DEG C of water-bath 30min | 598.6548 |
| 3 | 20 DEG C of water-bath 40min | 603.5564 |
| 4 | 30 DEG C of water-bath 20min | 925.6541 |
| 5 | 30 DEG C of water-bath 30min | 978.5011 |
| 6 | 30 DEG C of water-bath 40min | 989.5642 |
| 7 | 40 DEG C of water-bath 20min | 922.3122 |
| 8 | 40 DEG C of water-bath 30min | 985.4631 |
| 9 | 40 DEG C of water-bath 40min | 990.2879 |
The selection of 2.4 extracting method
2.4.1 extract the amount of solvent and the selection of extraction time
Adopt 50mL water, 100mL water, the capsule 's content that the embodiment 1 that ultrasonic 30min, ultrasonic 60min, backflow 60min are 1002001 to lot number prepares gained extracts, and measures 1-DNJ content results in table 4.Result shows to extract sample with 50mL water, ultrasonic 30min, just can sample extraction is complete.
Table 4 Different Extraction Method 1-DNJ assay result
2.4.2 eluting solvent volume is investigated
Adopt 0.5M ammoniacal liquor 400mL, 500mL, 600mL, 700mL wash-out respectively, the capsule 's content that the embodiment 1 that is 1002001 to lot number prepares gained extracts, and measures 1-DNJ content results in table 5.Result shows that use 0.5M ammoniacal liquor 600mL wash-out can be by complete 1-DNJ wash-out.
Table 5 different volumes eluting solvent 1-DNJ assay result
| NO. | Eluting solvent volume | 1-DNJ content (mg/g) |
| 1 | 0.5M ammoniacal liquor 400mL | 3.03 |
| 2 | 0.5M ammoniacal liquor 500mL | 3.25 |
| 3 | 0.5M ammoniacal liquor 600mL | 3.46 |
| 4 | 0.5M ammoniacal liquor 700mL | 3.43 |
2.4.3 elution speed is investigated
Adopt 0.5M ammoniacal liquor 600mL wash-out, elution speed is respectively 1mL/min, 2.5mL/min, 5mL/min, 8mL/min, the embodiment 1 that is 1002001 to lot number prepares the capsule 's content of gained and investigates, and measures 1-DNJ content, the results are shown in Table 6.Result shows by the elution speed result of 2.5mL/min better.
The different elution speed 1-DNJ of table 6 assay result
| NO. | Eluting solvent volume | 1-DNJ content (mg/g) |
| 1 | 1mL/min | 3.52 |
| 2 | 2.5mL/min | 3.49 |
| 3 | 5mL/min | 3.12 |
| 4 | 8mL/min | 2.93 |
2.5 linear relationships are investigated
It is appropriate that precision takes 1-DNJ reference substance, be dissolved in water, make the reference substance solution (I) of every 1mL containing 0.420mg, accurate reference substance solution (I) 2mL that draws, to 10mL measuring bottle, be diluted with water to scale, make the reference substance solution (II) of every 1mL containing 0.084mg.
By 2.1 listed reference substance solution preparation methods, make respectively reference substance solution (I) #, (II) #.Accurate reference substance solution (II) #10 μ L, the 20 μ L of drawing, reference substance solution (I) #5 μ L, 10 μ L, 15 μ L, 20 μ L, measure with 2.1 listed chromatographic conditions, the results are shown in Table 7.Taking reference substance sample size, (μ is g) as horizontal ordinate, and peak area integrated value (AUm) is ordinate, and drawing standard curve, the results are shown in Figure 5.1-DNJ regression equation Y=229.83X+10.471, correlation coefficient r=1.0000, show that 1-DNJ is good linear relationship within the scope of 0.84 μ g～8.4 μ g.
Table 71-DNJ linear relationship investigation table
| NO. | (μ g) for sample size | 1-DNJ peak area |
| 1 | 0.84 | 198.93598 |
| 2 | 1.68 | 398.97518 |
| 3 | 2.10 | 492.98572 |
| 4 | 4.20 | 979.0094 |
| 5 | 6.30 | 1461.07654 |
| 6 | 8.40 | 1937.37366 |
2.6 precision test
The accurate reference substance solution 1mL of every 1mL containing 1-DNJ 0.365mg that draw, to 25mL measuring bottle, adds 2%NaHCO
3solution 1mL, jolting 10 seconds, precision adds fluorenes methoxy dicarbonyl chloride acetonitrile solution (FMOC-Cl) 4mL of 2mg/mL, jolting 10 seconds, 30 DEG C of water-bath 40min, be settled to 25mL with 50% acetonitrile, the accurate 20 μ L that draw, repeat injection liquid chromatography 5 times continuously, measure peak area by 2.1 listed chromatographic conditions, ask relative standard deviation, the results are shown in Table 8.
Table 81-DNJ Precision test result
2.7 stability test
Get lot number and be the capsule sample solution that 1002001 embodiment 1 prepares gained, at latter 0 hour, 1 hour, 6 hours, 14 hours, 20 hours, 24 hours sample introductions of preparation, analyze by 2.1 listed chromatographic conditions respectively, the results are shown in Table 9.Result shows that the RSD of 1-DNJ peak area in 0～24 hour is 1.36%.Show that sample places in 24 hours stable.
Table 91-DNJ stability test result
2.8 replica test
Get the capsule sample that same lot number (1002001) embodiment 1 prepares gained, carry out 5 replicate determinations by 2.1 listed content assaying methods, the results are shown in Table 10.The RSD of 1-DNJ content is 0.64%, shows that the repeatability of the method is good.
Table 101-DNJ replica test result
2.9 recovery test
The same a collection of embodiment 1 that gets known content prepares the capsule sample of gained (lot number: 1002001) 6 parts, every part of about 0.5g, employing application of sample reclaims, according to the form below precision adds a certain amount of 1-DNJ reference substance respectively, measure by the preparation method of need testing solution and 2.1 listed chromatographic conditions, calculate recovery rate, the results are shown in Table 11.The recovery that shows this law conforms with the regulations, and method is feasible.
Table 111-DNJ determination of recovery rates result
2.10 sample size is measured
The capsule sample content assaying method of respectively following 20 crowdes of embodiment 1 being prepared to gained is measured, and the results are shown in Table 12.
Table 1220 crowd embodiment 1 prepares the capsule sample assay result of gained
According to prescription, every embodiment 1 prepares the capsule of gained containing 7.15g silkworm excrement raw medicinal herbs, the 1-DNJ content being recorded by following 6 batches of silkworm excrements, prepares the assay result of the capsule of gained in conjunction with 20 crowdes of embodiment 1, intend every of regulation this product containing silkworm excrement with 1-DNJ (C
6h
13nO
4) meter, must not be less than 0.50mg.
Embodiment 3HPLC method is measured the content of 1-DNJ
Accurately weighed 1-DNJ reference substance 5mg, thin up is made the reference substance storing solution of every mL containing 0.1mg; Precision measures reference substance storing solution described in 1mL, and the accurate mass concentration that adds is 1% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 1mL of solution 1mL and 1mg/mL, mixes and carries out water-bath 20min in 30 DEG C, is settled to 20mL subsequently, in contrast product solution with 30% acetonitrile.
Get the content porphyrize that embodiment 1 prepares the capsule of gained, and precision takes 0.5g, the water 30mL that to add with concentrated hydrochloric acid adjust pH be 3, stirs, ultrasonic processing 20min, and centrifuging and taking supernatant; It is that 3 water washs in right amount that residue adds pH value, centrifugal merging supernatant, and be concentrated into 10mL; Concentrate is placed in to cation exchange resin column, first washes with water, collect and discard water elution liquid, then use 0.5M ammoniacal liquor wash-out, and collect ammoniacal liquor eluent, and being concentrated near doing, the 10mL that adds water subsequently dissolves, ultrasonic processing 1 minute, and add water and be settled to 20mL, test sample storing solution obtained; Precision measures test sample storing solution described in 1mL, and adding successively mass concentration is 1% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1mg/mL, mixes and carries out water-bath 40min in 30 DEG C, is settled to 20mL subsequently, as need testing solution with 30% acetonitrile.
According to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, the phosphoric acid solution taking second eyeball as mobile phase A, taking 0.2% carries out gradient elution: 0-8min as Mobile phase B according to following program, and A:B is 25%:75%; 8-40min, A:B is 25%:75% → 50%:50%; 40-45min, A:B is 50%:50%; 45-46min, A:B is 50%:50% → 25%:75%; 46-75min, A:B is 25%:75%; Coutroi velocity 1.0mL/min, 25 DEG C of column temperatures, detect wavelength 263nm, and number of theoretical plate calculates and should be not less than 6000 by 1-DNJ peak.
Accurate described reference substance solution and the each 20 μ L of need testing solution of drawing, injection liquid chromatography, measures.
Embodiment 4HPLC method is measured the content of 1-DNJ
Accurately weighed 1-DNJ reference substance 15mg, thin up is made the reference substance storing solution of every mL containing 0.5mg; Precision measures reference substance storing solution described in 1mL, and the accurate mass concentration that adds is 3% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 4mL and 3mg/mL, mixes and carries out water-bath 40min in 60 DEG C, is settled to 30mL subsequently, in contrast product solution with 60% acetonitrile.
Get the content porphyrize that embodiment 1 prepares the capsule of gained, and precision takes 2.0g, the water 60mL that to add with concentrated hydrochloric acid adjust pH be 4, stirs, ultrasonic processing 30min, and centrifuging and taking supernatant; It is that 4 water washs in right amount that residue adds pH value, centrifugal merging supernatant, and be concentrated into 20mL; Concentrate is placed in to cation exchange resin column, first washes with water, collect and discard water elution liquid, then use 0.5M ammoniacal liquor wash-out, and collect ammoniacal liquor eluent, and being concentrated near doing, the 10mL that adds water subsequently dissolves, ultrasonic processing 5 minutes, and add water and be settled to 30mL, test sample storing solution obtained; Precision measures test sample storing solution described in 1mL, and adding successively mass concentration is 3% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 3mg/mL, mixes and carries out water-bath 40min in 30 DEG C, is settled to 30mL subsequently, as need testing solution with 60% acetonitrile.
According to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, the phosphoric acid solution taking second eyeball as mobile phase A, taking 0.2% carries out gradient elution: 0-8min as Mobile phase B according to following program, and A:B is 25%:75%; 8-40min, A:B is 25%:75% → 50%:50%; 40-45min, A:B is 50%:50%; 45-46min, A:B is 50%:50% → 25%:75%; 46-75min, A:B is 25%:75%; Coutroi velocity 1.0mL/min, 25 DEG C of column temperatures, detect wavelength 263nm, and number of theoretical plate calculates and should be not less than 6000 by 1-DNJ peak.
Accurate described reference substance solution and the each 20 μ L of need testing solution of drawing, injection liquid chromatography, measures.
Embodiment 5TLC differentiates
Get the about 0.5g of content that embodiment 1 prepares the capsule of gained, add absolute ethyl alcohol 5mL, 60 DEG C of water-baths reflux 30 minutes, place, and get supernatant, put in water-bath and steam to about 1mL, as need testing solution.
Separately get cupreol reference substance, add absolute ethyl alcohol and make the solution containing 1mg in every 1mL, product solution in contrast.
Test according to thin-layered chromatography (annex VI B of Chinese Pharmacopoeia version in 2005), draw the each 2 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, taking methenyl choloride: acetone (volume ratio 19.5:0.5) is as developping agent, launch, take out, dry, spray, with 10% phosphorus molybdenum acid solution, is heated to spot colour developing in 105 DEG C clear.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.
Embodiment 6HPLC method is measured the content of mono-ammonium glycyrrhizinate
Extracting Radix Glycyrrhizae acid mono-ammonium reference substance 10mg, accurately weighed, put in 50mL measuring bottle, dissolve and be diluted to scale with mobile phase, shake up, obtain (every 1mL contains mono-ammonium glycyrrhizinate 0.2mg, and amounting to glycyrrhizic acid is 0.1959mg).
Get 2.0 grams of the contents that embodiment 1 prepares the capsule of gained, put in the measuring bottle of 25mL, add the 20mL that makes an appointment that flows, ultrasonic processing (power 250W, frequency 50KHz) 30 minutes, take out, let cool, add mobile phase to scale, shake up, centrifugal, get supernatant, to obtain final product.
Taking octadecylsilane chemically bonded silica as filling agent; Taking methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid (volume ratio 60:40:1) as mobile phase; Detection wavelength is 250nm.Number of theoretical plate calculates and should be not less than 2000 by ammonium glycyrrhetate peak.
Accurate reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography, test sample should present the chromatographic peak identical with reference substance retention time.
Embodiment 7HPLC method is measured the content of Pipecolic Acid
Get the about 5mg of Pipecolic Acid reference substance, accurately weighed, put in 50mL measuring bottle, be diluted with water to scale, shake up; Precision measures 1mL, puts in 10mL tool plug test tube, and 1mL adds water, add 0.8%2,4-dinitrofluorobenzene acetonitrile solution 2mL and 0.5mol/L sodium bicarbonate solution 2mL, in 60 DEG C of water-baths 1 hour, take out, let cool, move in 10mL measuring bottle, with 0.2mol/L phosphate buffer (pH7.0) point several washing container, washing lotion is incorporated in measuring bottle, add above-mentioned phosphate buffer and be diluted to scale, shake up, to obtain final product.
Get the about 0.4g of content that embodiment 1 prepares the capsule of gained, accurately weighed, put in 10mL tool plug test tube, add water appropriate, after shake well, in 60 DEG C of water-baths, heat 30 minutes, take out, let cool, move in 10mL measuring bottle, with water fraction time washing container, washing lotion is incorporated in measuring bottle, be diluted with water to scale, shake up.Centrifugal (12000 revs/min) 10 minutes, precision measures supernatant 2mL, puts in 10mL tool plug test tube, add 0.8%2,4-dinitrofluorobenzene acetonitrile solution 2mL and 0.5mol/L sodium bicarbonate solution 2mL, in 60 DEG C of water-baths 1 hour, take out, let cool, move in 10mL measuring bottle, with 0.2mol/L phosphate buffer (pH7.0) point several washing container, washing lotion is incorporated in measuring bottle, add above-mentioned phosphate buffer and be diluted to scale, shake up, to obtain final product.
Taking octadecylsilane chemically bonded silica as filling agent; Taking acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution (21:0.5:79) as mobile phase; Detection wavelength is 390nm.Number of theoretical plate calculates and should be not less than 2500 by Pipecolic Acid peak.
Accurate reference substance solution 5 μ L and the need testing solution 10 μ L of drawing respectively, injection liquid chromatography, measures, and to obtain final product.
The capsule that embodiment 1 prepares gained contains silkworm excrement with Pipecolic Acid (C
6h
11nO
2) meter, must not be less than 0.20mg.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.And the apparent variation of being extended out thus or variation are still among the protection domain in the invention.
Claims (10)
1. a detection method for the treatment of the pharmaceutical preparation of diabete, is characterized in that, the bulk drug of described pharmaceutical preparation consists of: silkworm excrement 6000-8000 weight portion and Radix Glycyrrhizae 100-200 weight portion;
This detection method comprises the following assay step to 1-DNJ:
Accurately weighed 1-DNJ reference substance 5-15mg, thin up is made the reference substance storing solution of every mL containing 0.1-0.5mg; Precision measures reference substance storing solution described in 1mL, and accurate to add mass concentration be the NaHCO of 1-3%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 1-4mL of solution 1-4mL and 1-3mg/mL, mixes and carries out water-bath 20-40min in 30-60 DEG C, is settled to 20-30mL subsequently, in contrast product solution with the acetonitrile of 30-60%;
Get pharmaceutical preparation porphyrize to be measured, and precision takes 0.5-2.0g, the water 30-60mL that to add with concentrated hydrochloric acid adjust pH be 3-4, stirs, ultrasonic processing 20-30min, and centrifuging and taking supernatant; Residue adds the water that pH value is 3-4 and washs in right amount, centrifugal merging supernatant, and be concentrated into 10-20mL; Concentrate is placed in to cation exchange resin column, first washes with water, collect and discard water elution liquid, then use 0.5M ammoniacal liquor wash-out, and collect ammoniacal liquor eluent, and being concentrated near doing, the 10mL that adds water subsequently dissolves, ultrasonic processing 1-5 minute, and add water and be settled to 20-30mL, test sample storing solution obtained; Precision measures test sample storing solution described in 1mL, and adding successively mass concentration is the NaHCO of 1-3%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1-3mg/mL, mixes and carries out water-bath 40min in 30 DEG C, is settled to 20-30mL subsequently, as need testing solution with the acetonitrile of 30-60%;
According to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, the phosphoric acid solution taking second eyeball as mobile phase A, taking 0.2% carries out gradient elution: 0-8min as Mobile phase B according to following program, and A:B is 25%:75%; 8-40min, A:B is 25%:75% → 50%:50%; 40-45min, A:B is 50%:50%; 45-46min, A:B is 50%:50% → 25%:75%; 46-75min, A:B is 25%:75%; Coutroi velocity 1.0mL/min, 25 DEG C of column temperatures, detect wavelength 263nm, and number of theoretical plate calculates and should be not less than 6000 by 1-DNJ peak;
Accurate described reference substance solution and the each 20 μ L of need testing solution of drawing, injection liquid chromatography, measures.
2. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 1, is characterized in that, the described step that the content of 1-DNJ is measured comprises:
Accurately weighed 1-DNJ reference substance 10mg, thin up is made the reference substance storing solution of every mL containing 0.2mg; Precision measures reference substance storing solution described in 1mL, and the accurate mass concentration that adds is 2% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 2mL of solution 2mL and 2mg/mL, mixes and carries out water-bath 40min in 50 DEG C, is settled to 25mL subsequently, in contrast product solution with 30% acetonitrile;
Get pharmaceutical preparation porphyrize to be measured, and precision takes 1g, the water 50mL that to add with concentrated hydrochloric acid adjust pH be 3-4, stirs, ultrasonic processing 30min, and centrifuging and taking supernatant; Residue adds the water that pH value is 3-4 and washs in right amount, centrifugal merging supernatant, and be concentrated into 10mL; Concentrate is placed in to cation exchange resin column, first washes with water, collect and discard water elution liquid, then use 0.5M ammoniacal liquor wash-out, and collect ammoniacal liquor eluent, and being concentrated near doing, the 10mL that adds water subsequently dissolves, ultrasonic processing 1 minute, and add water and be settled to 25mL, test sample storing solution obtained; Precision measures test sample storing solution described in 1mL, and adding successively mass concentration is 2% NaHCO
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1mg/mL, mixes and carries out water-bath 40min in 50 DEG C, is settled to 25mL subsequently, as need testing solution with 30% acetonitrile;
According to high performance liquid chromatography, taking octadecylsilane chemically bonded silica as filling agent, the phosphoric acid solution taking second eyeball as mobile phase A, taking 0.2% carries out gradient elution: 0-8min as Mobile phase B according to following program, and A:B is 25%:75%; 8-40min, A:B is 25%:75% → 50%:50%; 40-45min, A:B is 50%:50%; 45-46min, A:B is 50%:50% → 25%:75%; 46-75min, A:B is 25%:75%; Coutroi velocity 1.0mL/min, 25 DEG C of column temperatures, detect wavelength 263nm, and number of theoretical plate calculates and should be not less than 6000 by 1-DNJ peak;
Accurate described reference substance solution and the each 20 μ L of need testing solution of drawing, injection liquid chromatography, measures.
3. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 1 and 2, it is characterized in that, in the assay step of described 1-DNJ, described cation exchange resin column is D001-CC type cation exchange resin column, resin volume is 50mL, and blade diameter length ratio is 1:8.
4. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 3, it is characterized in that, also comprise the step of described Zeo-karb being carried out to pre-treatment, described pre-treatment specifically comprises the steps: with isopyknic 2M salt acid soak D001-CC type ion exchange resin more than 4 hours; Rinse resin with the 2M hydrochloric acid of 4 times of resin volumes again, be washed to neutrality; With the 2M NaOH solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M hydrochloric acid solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M NaOH solution flushing resin of 5 times of resin volumes, be washed to neutrality; With the 2M hydrochloric acid solution flushing resin of 3 times of resin volumes, be washed to neutrality.
5. according to the detection method of the pharmaceutical preparation of the arbitrary described treatment diabete of claim 1-4, it is characterized in that, the method also comprises at least one in the step of following discriminating and/or assay:
A, TLC differentiate
Get described pharmaceutical preparation 0.4-0.8g to be measured, add absolute ethyl alcohol 3-8mL, in 60-90 DEG C of water-bath backflow 20-40 minute, leave standstill and get supernatant, put in water-bath and steam to 0.5-2.0mL, as need testing solution;
Separately get cupreol reference substance, add absolute ethyl alcohol and make the reference substance solution containing 0.5-2.0mg in every 1mL;
Test according to thin-layered chromatography, accurate above-mentioned two kinds of need testing solutions and the each 1-3 μ of the reference substance solution L of drawing, put respectively on same silica gel g thin-layer plate, methenyl choloride taking volume ratio as 18-19.8:0.2-2.0: acetone is developping agent, launch, take out, dry, spray, with 5-15% phosphorus molybdenum acid solution, is differentiated in 100-110 DEG C of colour developing;
B, HPLC method are measured the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 5-15mg, the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution taking volume ratio as 50-70:30-50:0.5-1.5 dissolves and is diluted to 50mL, in contrast product solution;
Get pharmaceutical preparation 1-3 gram to be measured, adding volume ratio is methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 50-70:30-50:0.5-1.5, and ultrasonic processing 30 minutes, continues to add described mixed solution to be settled to 25mL after letting cool, centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid taking volume ratio as 50-70:30-50:0.5-1.5 is mobile phase, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 2000 by ammonium glycyrrhetate peak;
Accurate described reference substance solution and the each 5-15 μ of the need testing solution L of drawing respectively, injection liquid chromatography, measures;
C, HPLC method are measured the content of Pipecolic Acid
Accurately weighed Pipecolic Acid reference substance 3-8mg, thin up is settled to 50mL; Precision measures 0.5-2.0mL solution, add water successively 0.5-2.0mL, add mass concentration be 0.8% 2, the sodium bicarbonate solution 1-3mL of 4-dinitrofluorobenzene acetonitrile solution 1-3mL and 0.5mol/L, in 60-90 DEG C of water-bath 0.5-2.0 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, product solution in contrast;
Accurately weighed pharmaceutical preparation 0.3-0.8g to be measured, add water after jolting, in 60-90 DEG C of water-bath, heat 20-40 minute, after letting cool, add water and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 1-3mL, and add successively mass concentration be 0.8% 2, the sodium bicarbonate solution 1-3mL of 4-dinitrofluorobenzene acetonitrile solution 1-3mL and 0.5mol/L, in 60-90 DEG C of water-bath 0.5-2.0 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution taking volume ratio as 15-25:0.1-1:75-85 is mobile phase, and detection wavelength is 390nm; Number of theoretical plate calculates and should be not less than 2500 by Pipecolic Acid peak;
Accurate reference substance solution 4-6 μ L and the need testing solution 5-15 μ L of drawing respectively, injection liquid chromatography, measures.
6. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 5, is characterized in that, the method also comprises at least one in the step of following discriminating and/or assay:
A, TLC differentiate
Get described pharmaceutical preparation 0.5g to be measured, add absolute ethyl alcohol 5mL, reflux 30 minutes in 60 DEG C of water-baths, leave standstill and get supernatant, put in water-bath and steam to 1mL, as need testing solution;
Separately get cupreol reference substance, add absolute ethyl alcohol and make the reference substance solution containing 1mg in every 1mL;
Test according to thin-layered chromatography, accurate above-mentioned two kinds of need testing solutions and the each 2 μ L of reference substance solution of drawing, put respectively on same silica gel g thin-layer plate, methenyl choloride taking volume ratio as 19.5:0.5: acetone is developping agent, launch, take out, dry, spray, with 10% phosphorus molybdenum acid solution, is differentiated in 105 DEG C of colour developings;
B, HPLC method are measured the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 10mg, the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution taking volume ratio as 60:40:1 dissolves and is diluted to 50mL, in contrast product solution;
Get 2.0 grams of pharmaceutical preparations to be measured, adding volume ratio is methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 60:40:1, and ultrasonic processing 30 minutes, continues to add described mixed solution to be settled to 25mL after letting cool, and centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid taking volume ratio as 60:40:1 is mobile phase, and detection wavelength is 250nm; Number of theoretical plate calculates and should be not less than 2000 by ammonium glycyrrhetate peak;
Accurate described reference substance solution and the each 10 μ L of need testing solution of drawing respectively, injection liquid chromatography, measures;
C, HPLC method are measured the content of Pipecolic Acid
Accurately weighed Pipecolic Acid reference substance 5mg, thin up is settled to 50mL; Precision measures 1mL solution, add water successively 1mL, add mass concentration be 0.8% 2, the sodium bicarbonate solution 2mL of 4-dinitrofluorobenzene acetonitrile solution 2mL and 0.5mol/L, in 60 DEG C of water-baths 1 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, product solution in contrast;
Accurately weighed pharmaceutical preparation 0.4g to be measured, add water after jolting, in 60 DEG C of water-baths, heat 30 minutes, after letting cool, add water and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 2mL, and to add successively mass concentration be 0.8% DNF acetonitrile solution 2mL and the sodium bicarbonate solution 2mL of 0.5mol/L, in 60 DEG C of water-baths 1 hour, let cool the rear phosphate buffer with pH7.0,0.2mol/L and be settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, taking octadecylsilane chemically bonded silica as filling agent; Acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution taking volume ratio as 21:0.5:79 is mobile phase, and detection wavelength is 390nm; Number of theoretical plate calculates and should be not less than 2500 by Pipecolic Acid peak;
Accurate reference substance solution 5 μ L and the need testing solution 10 μ L of drawing respectively, injection liquid chromatography, measures.
7. according to the detection method of the pharmaceutical preparation of the arbitrary described treatment diabete of claim 1-6, it is characterized in that, the bulk drug of described pharmaceutical preparation consists of: silkworm excrement 7150 weight portions and Radix Glycyrrhizae 137.5 weight portions; Or silkworm excrement 6150 weight portions and Radix Glycyrrhizae 187.5 weight portions; Or silkworm excrement 7850 weight portions and Radix Glycyrrhizae 117.5 weight portions.
8. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 7, it is characterized in that, described pharmaceutical preparation is prepared by the following method: the silkworm excrement of getting selected weight portion adds 2-6 doubly to measure 50-70% ethanol, dipping spends the night, slowly be heated to boil, backflow 1-3 time, each 0.5-1.5 hour, filter, merging filtrate, reclaims ethanol, and being concentrated into relative density is 0.95-1.10, add 1 times of water gaging to continue to be heated to boil, cooling, placement is spent the night, and gets supernatant, be concentrated into relative density and be the thick paste A of 1.00-1.15, for subsequent use; The Radix Glycyrrhizae of separately getting selected weight portion adds 8-15 times of water gaging and decocts 1-3 time, each 1-3 hour, and merging filtrate, placing spends the night makes precipitation, gets the thick paste B that supernatant concentration to relative density is 1.00-1.15, for subsequent use; Merge above-mentioned thick paste A and B, add conventional auxiliary material, make clinical acceptable formulation according to common process.
9. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 8, it is characterized in that, described pharmaceutical preparation is prepared by the following method: the silkworm excrement of getting selected weight portion adds 4 times of amount 60% ethanol, dipping spends the night, slowly be heated to boil, reflux twice, each 1 hour, filter, merging filtrate, reclaims ethanol, and being concentrated into relative density is 1.01-1.06, add 1 times of water gaging to continue to be heated to boil, cooling, placement is spent the night, and gets supernatant, be concentrated into the thick paste that relative density is 1.06-1.15, for subsequent use; The Radix Glycyrrhizae of separately getting selected weight portion adds 10 times of water gagings and decocts 2 times, and each 2 hours, merging filtrate, placing spends the night makes precipitation, gets supernatant concentration to relative density and is 1.04-1.15 thick paste, for subsequent use; Merge above-mentioned two thick pastes, add conventional auxiliary material, make clinical acceptable formulation according to common process.
10. according to the detection method of the pharmaceutical preparation of the arbitrary described treatment diabete of claim 7-9, it is characterized in that, the pharmaceutical preparation of described treatment diabete is tablet, capsule, pill, granule, honey refining pill, sustained release preparation, quick releasing formulation, controlled release preparation, oral liquid or ejection preparation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105004829A (en) * | 2015-07-21 | 2015-10-28 | 漳州片仔癀药业股份有限公司 | Jintangning capsule quality detection method |
| CN107192777A (en) * | 2017-05-24 | 2017-09-22 | 江苏耐雀生物工程技术有限公司 | The detection method of 1 DNJ content in a kind of mulberry-leaf extract |
| CN108828121A (en) * | 2018-06-14 | 2018-11-16 | 华润三九医药股份有限公司 | A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material |
| CN110279719A (en) * | 2019-06-27 | 2019-09-27 | 北京市房山区中医医院 | It treats and presses packet and its preparation and application outside the late silkworm excrement of diabetic gastroparesis |
| CN112345676A (en) * | 2020-12-07 | 2021-02-09 | 葵花药业集团湖北武当有限公司 | HPLC method for simultaneously detecting 6 active ingredients in fritillaria cirrhosa lung-heat-clearing syrup |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020655A (en) * | 2007-03-15 | 2007-08-22 | 浙江大学 | Process of extracting 1-deoxy nojirimycin |
| CN101496829A (en) * | 2008-01-30 | 2009-08-05 | 漳州片仔癀药业股份有限公司 | A pharmaceutical composition for treating diabetes, and its preparation method |
| CN101654428A (en) * | 2009-09-11 | 2010-02-24 | 成都市金医生科技健康产业有限公司 | Method for extracting and separating 1-deoxynojirimycin with high purity from natural products |
| CN102507831A (en) * | 2011-10-13 | 2012-06-20 | 厦门市医药研究所 | Determination method of 1-deoxynojirimycin content in silkworm powder |
| CN102890125A (en) * | 2011-07-20 | 2013-01-23 | 北京北大维信生物科技有限公司 | Building method and mass detection method for fingerprint of total alkaloid components of cortex mori radicis medicinal material or cortex mori radicis extract |
| CN103274992A (en) * | 2013-06-14 | 2013-09-04 | 扬州大学 | Method for preparing high-purity 1-deoxynojirimycin with combined membrane separation and column chromatography technology |
-
2014
- 2014-06-13 CN CN201410264670.4A patent/CN104007205B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020655A (en) * | 2007-03-15 | 2007-08-22 | 浙江大学 | Process of extracting 1-deoxy nojirimycin |
| CN101496829A (en) * | 2008-01-30 | 2009-08-05 | 漳州片仔癀药业股份有限公司 | A pharmaceutical composition for treating diabetes, and its preparation method |
| CN101654428A (en) * | 2009-09-11 | 2010-02-24 | 成都市金医生科技健康产业有限公司 | Method for extracting and separating 1-deoxynojirimycin with high purity from natural products |
| CN102890125A (en) * | 2011-07-20 | 2013-01-23 | 北京北大维信生物科技有限公司 | Building method and mass detection method for fingerprint of total alkaloid components of cortex mori radicis medicinal material or cortex mori radicis extract |
| CN102507831A (en) * | 2011-10-13 | 2012-06-20 | 厦门市医药研究所 | Determination method of 1-deoxynojirimycin content in silkworm powder |
| CN103274992A (en) * | 2013-06-14 | 2013-09-04 | 扬州大学 | Method for preparing high-purity 1-deoxynojirimycin with combined membrane separation and column chromatography technology |
Non-Patent Citations (5)
| Title |
|---|
| JIN-WON KIM ET AL.: "Determination of 1-deoxynojirimycin in Morus alba L. leaves by derivatization with 9-fluorenylmethyl chloroformate followed by reversed-phase high-performance liquid chromatography", 《JOURNAL OF CHROMATOGRAPHY A》, vol. 1002, no. 12, 20 June 2003 (2003-06-20) * |
| KEFEI HU ET AL.: "Analysis of 1-deoxynojirimycin component correlation between medicinal parasitic loranthus from loranthaceae and their mulberry host trees", 《JOURNAL OF MEDICINAL PLANTS RESEARCH》, vol. 5, no. 17, 9 September 2011 (2011-09-09) * |
| 刘月琴 等: "不同生长阶段家蚕1-脱氧野尻霉素富集与食桑量的相关性研究", 《天然产物研究与开发》, vol. 25, 31 December 2013 (2013-12-31) * |
| 朱旭东 等: "不同地域和季节蚕沙中DNJ含量差异性的研究", 《天津药学》, vol. 23, no. 6, 31 December 2011 (2011-12-31) * |
| 陈岩 等: "HPLC法测定蚕桑资源中1-脱氧野尻霉素的含量", 《中国药房》, vol. 21, no. 3, 31 December 2010 (2010-12-31) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105004829A (en) * | 2015-07-21 | 2015-10-28 | 漳州片仔癀药业股份有限公司 | Jintangning capsule quality detection method |
| CN107192777A (en) * | 2017-05-24 | 2017-09-22 | 江苏耐雀生物工程技术有限公司 | The detection method of 1 DNJ content in a kind of mulberry-leaf extract |
| CN108828121A (en) * | 2018-06-14 | 2018-11-16 | 华润三九医药股份有限公司 | A kind of white detection method of content for setting the high nojirimycin of α-in medicinal material |
| CN110279719A (en) * | 2019-06-27 | 2019-09-27 | 北京市房山区中医医院 | It treats and presses packet and its preparation and application outside the late silkworm excrement of diabetic gastroparesis |
| CN110279719B (en) * | 2019-06-27 | 2021-01-15 | 北京市房山区中医医院 | Night silkworm excrement external ironing bag for treating diabetic gastroparesis and preparation and use methods thereof |
| CN112345676A (en) * | 2020-12-07 | 2021-02-09 | 葵花药业集团湖北武当有限公司 | HPLC method for simultaneously detecting 6 active ingredients in fritillaria cirrhosa lung-heat-clearing syrup |
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