CN104007205B - A kind of detection method for the treatment of the pharmaceutical preparation of diabete - Google Patents
A kind of detection method for the treatment of the pharmaceutical preparation of diabete Download PDFInfo
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Abstract
The invention belongs to Chinese medicine preparation quality testing field, be specifically related to a kind of detection method for the treatment of the pharmaceutical preparation of diabete, the bulk drug of this pharmaceutical preparation consists of: silkworm excrement 6000-8000 weight portion and Radix Glycyrrhizae 100-200 weight portion; The content that this detection method measures 1-DNJ by HPLC method detects described pharmaceutical preparation.Detection method of the present invention is quick, comprehensive, with strong points, is conducive to controling effectively to the quality of this medicine, contributes to the safety and stability improving this drug use.
Description
Technical field
The invention belongs to Chinese medicine preparation detection field, be specifically related to a kind of detection method for the treatment of the pharmaceutical preparation of diabete.
Background technology
At present, in global range, the patient of diabete (diabetes) is about more than 200,000,000, and wherein the patient of 85% is old-aged diabetic, and the incidence of disease has the trend day by day increased; China is one of country that diabetic's number is in the world maximum, is only second to the U.S..The health even life of the mankind 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 primarily of silkworm excrement and Radix Glycyrrhizae composition, silkworm excrement in prescription has dispels rheumatism, to invigorate blood circulation effect of analgesic therapy, this pharmaceutical composition has good hypoglycemic effect, has good curative effect to treatment diabetes.Also disclose in the document, by methods such as TLC method detection cupreol, HPLC method detection ammonium glycyrrhizinate and HPLC method detection Pipecolic Acids, quality control is carried out to this pharmaceutical composition.
But, along with the further investigation to this medicine, clinical test results shows, the alkaloid that the active principle of these medicine composite for curing diabetes mainly contains in silkworm excrement, there is the effect significantly suppressing a-glucosidase activity, and described alkaloidal main active is 1-DNJ.But existing quality control standard and detection method are all only measure for the Pipecolic Acid wherein contained, the index not only detected is comparatively single, nor is beneficial to and control effectively to the quality of this medicine on the whole.Therefore, a kind of quality testing of aforementioned pharmaceutical compositions that can be quick, comprehensive, with strong points is set up and the method for quality control has great importance.
Summary of the invention
For this reason, to be solved by this invention be the detection method of the pharmaceutical composition of existing treatment diabete exist Testing index single, be unfavorable for problem that the quality of this medicine is control effectively, thus propose a kind of can fast, comprehensively, targetedly to the method that the pharmaceutical composition of described treatment diabetes detects.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions:
A kind of detection method for the treatment of the pharmaceutical preparation 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 makes the reference substance storing solution of every mL containing 0.1-0.5mg; Precision measures reference substance storing solution described in 1mL, and precision adds the NaHCO that mass concentration is 1-3%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 1-4mL of solution 1-4mL and 1-3mg/mL, mixing also 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, adding with concentrated hydrochloric acid adjust pH is the water 30-60mL of 3-4, stirs, ultrasonic process 20-30min, and centrifuging and taking supernatant; It is that the water of 3-4 washs in right amount that residue adds pH value, centrifugal merging supernatant, and is concentrated into 10-20mL; Concentrate is placed in 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, be concentrated near dry, the 10mL that adds water subsequently dissolves, ultrasonic process 1-5 minute, and add water and be settled to 20-30mL, obtain test sample storing solution; Precision measures test sample storing solution described in 1mL, adds the NaHCO that mass concentration is 1-3% successively
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1-3mg/mL, mixing also carries out water-bath 40min in 30 DEG C, is settled to 20-30mL, as need testing solution subsequently with the acetonitrile of 30-60%;
According to high performance liquid chromatography, take octadecylsilane chemically bonded silica as filling agent, with second eyeball be mobile phase A, phosphoric acid solution with 0.2% carries out gradient elution according to following program for Mobile phase B: 0-8min, A:B are for 25%:75%; 8-40min, A:B are 25%:75% → 50%:50%; 40-45min, A:B are 50%:50%; 45-46min, A:B are 50%:50% → 25%:75%; 46-75min, A:B are 25%:75%; Coutroi velocity 1.0mL/min, column temperature 25 DEG C, determined wavelength 263nm, number of theoretical plate calculates should be not less than 6000 by 1-DNJ peak;
The described reference substance solution of accurate absorption and each 20 μ L of need testing solution, injection liquid chromatography, measures.
The detection method of the pharmaceutical preparation of the above-mentioned treatment diabete of the present invention, the described content to 1-DNJ carries out determination step and comprises:
Accurately weighed 1-DNJ reference substance 10mg, thin up makes the reference substance storing solution of every mL containing 0.2mg; Precision measures reference substance storing solution described in 1mL, and precision adds the NaHCO that mass concentration is 2%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 2mL of solution 2mL and 2mg/mL, mixing also carries out water-bath 40min in 50 DEG C, is settled to 25mL subsequently, in contrast product solution with the acetonitrile of 30%;
Get pharmaceutical preparation porphyrize to be measured, and precision takes 1g, adding with concentrated hydrochloric acid adjust pH is the water 50mL of 3-4, stirs, ultrasonic process 30min, and centrifuging and taking supernatant; It is that the water of 3-4 washs in right amount that residue adds pH value, centrifugal merging supernatant, and is concentrated into 10mL; Concentrate is placed in 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, be concentrated near dry, the 10mL that adds water subsequently dissolves, ultrasonic process 1 minute, and add water and be settled to 25mL, obtain test sample storing solution; Precision measures test sample storing solution described in 1mL, adds the NaHCO that mass concentration is 2% successively
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1mg/mL, mixing also carries out water-bath 40min in 50 DEG C, is settled to 25mL, as need testing solution subsequently with the acetonitrile of 30%;
According to high performance liquid chromatography, take octadecylsilane chemically bonded silica as filling agent, with second eyeball be mobile phase A, phosphoric acid solution with 0.2% carries out gradient elution according to following program for Mobile phase B: 0-8min, A:B are for 25%:75%; 8-40min, A:B are 25%:75% → 50%:50%; 40-45min, A:B are 50%:50%; 45-46min, A:B are 50%:50% → 25%:75%; 46-75min, A:B are 25%:75%; Coutroi velocity 1.0mL/min, column temperature 25 DEG C, determined wavelength 263nm, number of theoretical plate calculates should be not less than 6000 by 1-DNJ peak;
The described reference substance solution of accurate absorption and each 20 μ L of need testing solution, 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 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; Rinse resin by the 2M NaOH solution of 5 times of resin volumes, be washed to neutrality; Rinse resin with the 2M hydrochloric acid solution of 5 times of resin volumes, be washed to neutrality; Rinse resin by the 2M NaOH solution of 5 times of resin volumes, be washed to neutrality; Rinse resin with the 2M hydrochloric acid solution 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 process 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, the above-mentioned two kinds of need testing solutions of accurate absorption and reference substance solution each 1-3 μ L, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride of 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 measures the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 5-15mg is that the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 50-70:30-50:0.5-1.5 dissolves and is diluted to 50mL with volume ratio, product solution in contrast;
Get pharmaceutical preparation 1-3 gram to be measured, add methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution that volume ratio is 50-70:30-50:0.5-1.5, ultrasonic process 30 minutes, continue to add described mixed solution after letting cool and be settled to 25mL, centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid of 50-70:30-50:0.5-1.5 be mobile phase, determined wavelength is 250nm; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;
The described reference substance solution of accurate absorption and need testing solution each 5-15 μ L respectively, injection liquid chromatography, measures;
C, HPLC method measures 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 that mass concentration is 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, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, in contrast product solution;
Accurately weighed pharmaceutical preparation 0.3-0.8g to be measured, add water after jolting, 20-40 minute is heated in 60-90 DEG C of water-bath, add water after letting cool and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 1-3mL, and add that mass concentration is 0.8% successively 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, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution of 15-25:0.1-1:75-85 be mobile phase, determined wavelength is 390nm; Number of theoretical plate calculates should be not less than 2500 by Pipecolic Acid peak;
Accurate absorption reference substance solution 4-6 μ L and need testing solution 5-15 μ L 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, the above-mentioned two kinds of need testing solutions of accurate absorption and each 2 μ L of reference substance solution, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride of 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 measures the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 10mg is that the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 60:40:1 dissolves and is diluted to 50mL with volume ratio, product solution in contrast;
Get pharmaceutical preparation to be measured 2.0 grams, add methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution that volume ratio is 60:40:1, ultrasonic process 30 minutes, continue to add described mixed solution after letting cool and be settled to 25mL, centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid of 60:40:1 be mobile phase, determined wavelength is 250nm; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;
The described reference substance solution of accurate absorption and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures;
C, HPLC method measures 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 that mass concentration is 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, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, in contrast product solution;
Accurately weighed pharmaceutical preparation 0.4g to be measured, add water after jolting, in 60 DEG C of water-baths heat 30 minutes, add water after letting cool and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 2mL, and add the sodium bicarbonate solution 2mL that mass concentration is DNF acetonitrile solution 2mL and 0.5mol/L of 0.8% successively, in 60 DEG C of water-baths 1 hour, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution of 21:0.5:79 be mobile phase, determined wavelength is 390nm; Number of theoretical plate calculates should be not less than 2500 by Pipecolic Acid peak;
Accurate absorption reference substance solution 5 μ L and need testing solution 10 μ L 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 content disclosed in Chinese patent CN101496829A.
The bulk drug of pharmaceutical preparation of the present invention consists of: silkworm excrement 7150 weight portion and Radix Glycyrrhizae 137.5 weight portion; Or silkworm excrement 6150 weight portion and Radix Glycyrrhizae 187.5 weight portion; Or silkworm excrement 7850 weight portion and Radix Glycyrrhizae 117.5 weight portion.
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 getting selected weight portion adds 2-6 times amount 50-70% ethanol, steeped overnight, slowly be heated to boil, reflux 1-3 time, each 0.5-1.5 hour, filter, merging filtrate, reclaim ethanol, being concentrated into relative density is 0.95-1.10, adds 1 times of water gaging and continues to be heated to boil, cooling, placement is spent the night, get supernatant, be concentrated into the thick paste A that relative density is 1.00-1.15, for subsequent use; The Radix Glycyrrhizae of separately getting selected weight portion adds 8-15 times amount soak by water 1-3 time, each 1-3 hour, merging filtrate, and 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 customary adjuvant, conveniently technique and make clinical acceptable formulation.
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 getting selected weight portion adds 4 times amount 60% ethanol, steeped overnight, slowly be heated to boil, backflow twice, each 1 hour, filter, merging filtrate, reclaim ethanol, being concentrated into relative density is 1.01-1.06, adds 1 times of water gaging and continues 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 amount soak by water 2 times, each 2 hours, merging filtrate, and placing spends the night makes precipitation, and getting supernatant concentration to relative density is 1.04-1.15 thick paste, for subsequent use; Merge above-mentioned two thick pastes, add customary adjuvant, conveniently technique and make clinical acceptable formulation.
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, measures the content of 1-DNJ by HPLC method.This detection method is quick, comprehensive, with strong points, is conducive to controling effectively to the quality of this medicine, contributes to the safety and stability improving this drug use.
Accompanying drawing explanation
In order to make content of the present invention be 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 scarce silkworm excrement in the embodiment of the present invention 2;
Fig. 3 is the capsule sample chromatogram that 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.
2 tastes more than [method for making], get silkworm excrement and add 4 times amount 60% ethanol, steeped overnight, slowly be heated to boil, backflow twice, each 1 hour, filter, merging filtrate, reclaim ethanol, being concentrated into relative density is 1.01-1.06 (55 DEG C), adds 1 times of water gaging and continues 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 (55 DEG C of survey), for subsequent use.Another extracting Radix Glycyrrhizae 10 times amount soak by water 2 times, each 2 hours, merging filtrate, placing spends the night made precipitation, and getting supernatant concentration to relative density is 1.04-1.15 (55 DEG C of surveys) thick paste, for subsequent use.Merge two thick pastes, add customary adjuvant according to a conventional method and make 1000, to obtain final product.
Embodiment 2HPLC method measures 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 1-DNJ reference substance and be about 10mg, 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, in 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, and jolting 10 seconds, 50 DEG C of water-bath 40min, are settled to 25mL with 30% acetonitrile, obtain final product.
2.1.2 the preparation of need testing solution
The pre-treatment of D001-CC type Zeo-karb: in turn with 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; Rinse resin by the 2M NaOH solution of 5 times of resin volumes, be washed to neutrality; Rinse resin with the 2M hydrochloric acid solution of 5 times of resin volumes, be washed to neutrality; Rinse resin by the 2M NaOH solution 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.
Example 1 prepares the content porphyrize of the capsule of gained, gets about 1g, accurately weighed, put in 100mL beaker, adding with concentrated hydrochloric acid adjust pH is the water 50mL of 3-4, stirs, ultrasonic process (power 300W, frequency 50KHz) 30min, centrifugal 10min (4000 turns/min), divide and get supernatant, it is that the water of 3-4 washs in right amount that residue adds pH value, centrifugal 10min (4000 turns/min), merges supernatant, is concentrated into about 10mL.Put processed D001-CC type cation exchange resin column (resin volume 50mL, footpath (2.5cm) height ratio: 1:8, H
+), 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 dry, add water 10mL, ultrasonic process (power 300W, frequency 50KHz) 1 minute, transfer in 25mL volumetric flask, add water to scale, shake up.Precision measures 1mL, in 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, and jolting 10 seconds, 50 DEG C of water-bath 40min, are settled to 25mL with 30% acetonitrile, obtain final product.
2.1.3 the preparation of negative sample solution
By the prescription extracting Radix Glycyrrhizae medicinal material of embodiment 1, by preparation method's preparation of need testing solution listed by 2.1 containing the negative sample solution of silkworm excrement.
2.1.4 prepared by blank sample solution
According to the preparation method of need testing solution listed by 2.1, preparation is containing the blank sample solution of described test sample.
2.1.5 blank test
Be filling agent with octadecylsilane chemically bonded silica; Select Agilent Hypersil ODS-C18 chromatographic column (4.0mm × 250mm, 5 μm), diode array detector (determined 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 1-DNJ reference substance solution, negative sample solution, need testing solution and blank reagent solution respectively, inject high performance liquid chromatograph according to HPLC chromatographic condition to measure, result is shown in Fig. 1,2,3,4 respectively, result shows under this chromatographic condition, 1-DNJ peak is separated better with other impurity peaks, negative sample chromatogram, at the retention time place corresponding to reference substance chromatogram, occurs without chromatographic peak, the mensuration of licorice medicinal materials not disturbed one-DNJ in side.
2.2 system suitability
Test by under test condition listed by 2.1, 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 derivatising condition
2.3.1 2%NaHCO is investigated
3solution usage, fluorenes methoxy dicarbonyl chloride acetonitrile solution consumption are on the impact of derivative reaction
Precision measures 1-DNJ reference substance solution (0.2mg/mL) 1.0mL, each 9 parts, puts in 25mL measuring bottle respectively, and 9 increment product are divided into 3 groups, often organizes precision respectively and adds 2%NaHCO
3solution 0.5mL, 1mL, 1.5mL, jolting 10 "; at 1,2, No. 3 sample often organized successively accurate fluorenes methoxy dicarbonyl chloride acetonitrile solution (FMOC-Cl) 2mL, 4mL, 5mL adding 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 chromatographic condition listed by 2.1, 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.
Table 2 different derivatising condition 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 investigation temperature of reaction and reaction time are on the impact of derivative reaction
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, precision adds fluorenes methoxy dicarbonyl chloride acetonitrile solution (FMOC-Cl) 4mL of 2mg/mL respectively again, 9 increment product are divided into 3 groups by jolting 10 seconds, and the 3 increment product often organized are placed in 20 DEG C, 30 DEG C, 40 DEG C water-baths respectively, 1,2, No. 3 example reaction time often organized is followed successively by 20min, 30min, 40min, be settled to 25mL with 50% acetonitrile, carry out liquid phase analysis by chromatographic condition listed by 2.1 respectively, the results are shown in Table 3.Result shows employing 30 DEG C of water-bath 40min, and derivative reaction can be made 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 the amount of Extraction solvent and the selection of extraction time
Adopt 50mL water, 100mL water, ultrasonic 30min, ultrasonic 60min, backflow 60min, the capsule 's content being prepared by the lot number embodiment 1 that is 1002001 to gained extracts, and measures 1-DNJ content results in table 4.Result shows to extract sample with 50mL water, ultrasonic 30min, just can be complete by sample extraction.
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 being prepared by the lot number embodiment 1 that is 1002001 to gained extracts, and measures 1-DNJ content results in table 5.Result shows can by complete for 1-DNJ wash-out with 0.5M ammoniacal liquor 600mL 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 capsule 's content being prepared by the lot number embodiment 1 that is 1002001 to gained is investigated, and measures 1-DNJ content, the results are shown in Table 6.Result shows by the elution speed result of 2.5mL/min better.
Table 6 different elution speed 1-DNJ 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 absorption reference substance solution (I) 2mL, to in 10mL measuring bottle, be diluted with water to scale, make the reference substance solution (II) of every 1mL containing 0.084mg.
By reference substance solution preparation method listed by 2.1, obtained reference substance solution (I) #, (II) # respectively.Accurate absorption reference substance solution (II) #10 μ L, 20 μ L, reference substance solution (I) #5 μ L, 10 μ L, 15 μ L, 20 μ L, listed by same 2.1, chromatographic condition measures, and the results are shown in Table 7.With reference substance sample size (μ g) for horizontal ordinate, integrating peak areas 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 1-DNJ within the scope of 0.84 μ g ~ 8.4 μ g in good linear relationship.
Table 71-DNJ linear relationship investigation table
| NO. | Sample size (μ g) | 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
Accurate every 1mL of absorption contains the reference substance solution 1mL of 1-DNJ 0.365mg, in 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, 25mL is settled to 50% acetonitrile, accurate draw 20 μ L, repeat injection liquid chromatography 5 times continuously, measure peak area by chromatographic condition listed by 2.1, ask relative standard deviation, the results are shown in Table 8.
Table 81-DNJ Precision test result
2.7 stability test
Get lot number be 1002001 embodiment 1 prepare the capsule sample solution of gained, 0 hour, 1 hour, 6 hours, 14 hours, 20 hours, 24 hours sample introductions after the production respectively, analyze by chromatographic condition listed by 2.1, 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 is placed 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 content assaying method listed by 2.1, 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 of getting 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, by need testing solution preparation method and 2.1 listed by chromatographic condition measure, calculate the recovery, the results are shown in Table 11.Show that the recovery of this law conforms with the regulations, method is feasible.
Table 111-DNJ determination of recovery rates result
2.10 sample size measures
Respectively the capsule sample content assaying method that following 20 batches of embodiments 1 prepare gained is measured, the results are shown in Table 12.
Table 1220 batch 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 recorded by following 6 batches of silkworm excrements, prepares the assay result of the capsule of gained in conjunction with 20 batches of embodiments 1, intend regulation this product every containing silkworm excrement with 1-DNJ (C
6h
13nO
4) meter, must not 0.50mg be less than.
Embodiment 3HPLC method measures the content of 1-DNJ
Accurately weighed 1-DNJ reference substance 5mg, thin up makes the reference substance storing solution of every mL containing 0.1mg; Precision measures reference substance storing solution described in 1mL, and precision adds the NaHCO that mass concentration is 1%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 1mL of solution 1mL and 1mg/mL, mixing also carries out water-bath 20min in 30 DEG C, is settled to 20mL subsequently, in contrast product solution with the acetonitrile of 30%.
Example 1 prepares the content porphyrize of the capsule of gained, and precision takes 0.5g, add with concentrated hydrochloric acid adjust pH be 3 water 30mL, stir, ultrasonic process 20min, and centrifuging and taking supernatant; Residue add pH value be 3 water wash in right amount, centrifugal merging supernatant, and be concentrated into 10mL; Concentrate is placed in 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, be concentrated near dry, the 10mL that adds water subsequently dissolves, ultrasonic process 1 minute, and add water and be settled to 20mL, obtain test sample storing solution; Precision measures test sample storing solution described in 1mL, adds the NaHCO that mass concentration is 1% successively
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1mg/mL, mixing also carries out water-bath 40min in 30 DEG C, is settled to 20mL, as need testing solution subsequently with the acetonitrile of 30%.
According to high performance liquid chromatography, take octadecylsilane chemically bonded silica as filling agent, with second eyeball be mobile phase A, phosphoric acid solution with 0.2% carries out gradient elution according to following program for Mobile phase B: 0-8min, A:B are for 25%:75%; 8-40min, A:B are 25%:75% → 50%:50%; 40-45min, A:B are 50%:50%; 45-46min, A:B are 50%:50% → 25%:75%; 46-75min, A:B are 25%:75%; Coutroi velocity 1.0mL/min, column temperature 25 DEG C, determined wavelength 263nm, number of theoretical plate calculates should be not less than 6000 by 1-DNJ peak.
The described reference substance solution of accurate absorption and each 20 μ L of need testing solution, injection liquid chromatography, measures.
Embodiment 4HPLC method measures the content of 1-DNJ
Accurately weighed 1-DNJ reference substance 15mg, thin up makes the reference substance storing solution of every mL containing 0.5mg; Precision measures reference substance storing solution described in 1mL, and precision adds the NaHCO that mass concentration is 3%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 4mL and 3mg/mL, mixing also carries out water-bath 40min in 60 DEG C, is settled to 30mL subsequently, in contrast product solution with the acetonitrile of 60%.
Example 1 prepares the content porphyrize of the capsule of gained, and precision takes 2.0g, add with concentrated hydrochloric acid adjust pH be 4 water 60mL, stir, ultrasonic process 30min, and centrifuging and taking supernatant; Residue add pH value be 4 water wash in right amount, centrifugal merging supernatant, and be concentrated into 20mL; Concentrate is placed in 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, be concentrated near dry, the 10mL that adds water subsequently dissolves, ultrasonic process 5 minutes, and add water and be settled to 30mL, obtain test sample storing solution; Precision measures test sample storing solution described in 1mL, adds the NaHCO that mass concentration is 3% successively
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 3mg/mL, mixing also carries out water-bath 40min in 30 DEG C, is settled to 30mL, as need testing solution subsequently with the acetonitrile of 60%.
According to high performance liquid chromatography, take octadecylsilane chemically bonded silica as filling agent, with second eyeball be mobile phase A, phosphoric acid solution with 0.2% carries out gradient elution according to following program for Mobile phase B: 0-8min, A:B are for 25%:75%; 8-40min, A:B are 25%:75% → 50%:50%; 40-45min, A:B are 50%:50%; 45-46min, A:B are 50%:50% → 25%:75%; 46-75min, A:B are 25%:75%; Coutroi velocity 1.0mL/min, column temperature 25 DEG C, determined wavelength 263nm, number of theoretical plate calculates should be not less than 6000 by 1-DNJ peak.
The described reference substance solution of accurate absorption and each 20 μ L of need testing solution, injection liquid chromatography, measures.
Embodiment 5TLC differentiates
The content that Example 1 prepares the capsule of gained is about 0.5g, adds absolute ethyl alcohol 5mL, and 60 DEG C of water-baths reflux 30 minutes, places, gets supernatant, puts in water-bath and steams 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 (China's coastal port annex VI B), draw each 2 μ L of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with methenyl choloride: acetone (volume ratio 19.5:0.5) is developping agent, launch, take out, dry, spray, with 10% phosphorus molybdenum acid solution, is heated to spot development in 105 DEG C clear.In test sample chromatogram, on the position corresponding to reference substance chromatogram, the spot of aobvious same color.
Embodiment 6HPLC method measures the content of mono-ammonium glycyrrhizinate
Extracting Radix Glycyrrhizae acid mono-ammonium reference substance 10mg, accurately weighed, put in 50mL measuring bottle, dissolve with mobile phase and be diluted to scale, shaking up, obtain (containing mono-ammonium glycyrrhizinate 0.2mg, amount to glycyrrhizic acid is 0.1959mg to every 1mL).
Example 1 prepares the content 2.0 grams of the capsule of gained, puts in the measuring bottle of 25mL, adds flowing and to make an appointment 20mL, ultrasonic process (power 250W, frequency 50KHz) 30 minutes, take out, let cool, add mobile phase to scale, shake up, centrifugal, get supernatant, to obtain final product.
Take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid (volume ratio 60:40:1) for mobile phase; Determined wavelength is 250nm.Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak.
Accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, test sample should present the chromatographic peak identical with reference substance retention time.
Embodiment 7HPLC method measures the content of Pipecolic Acid
Get Pipecolic Acid reference substance and be about 5mg, accurately weighed, put in 50mL measuring bottle, be diluted with water to scale, shake up; Precision measures 1mL, and put in 10mL tool plug test tube, add water 1mL, 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.
The content that Example 1 prepares the capsule of gained is about 0.4g, accurately weighed, puts in 10mL tool plug test tube, add water appropriate, after shake well, heat 30 minutes in 60 DEG C of water-baths, take out, let cool, move in 10mL measuring bottle, with moisture washing container for several times, 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.
Take octadecylsilane chemically bonded silica as filling agent; With acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution (21:0.5:79) for mobile phase; Determined wavelength is 390nm.Number of theoretical plate calculates should be not less than 2500 by Pipecolic Acid peak.
Accurate absorption reference substance solution 5 μ L and need testing solution 10 μ L respectively, injection liquid chromatography, measures, to obtain final product.
The capsule that embodiment 1 prepares gained contains silkworm excrement with Pipecolic Acid (C
6h
11nO
2) meter, must not 0.20mg be less than.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not 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 exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (10)
1. treat a detection method for the pharmaceutical preparation of diabete, it 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 makes the reference substance storing solution of every mL containing 0.1-0.5mg; Precision measures reference substance storing solution described in 1mL, and precision adds the NaHCO that mass concentration is 1-3%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 1-4mL of solution 1-4mL and 1-3mg/mL, mixing also 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, adding with concentrated hydrochloric acid adjust pH is the water 30-60mL of 3-4, stirs, ultrasonic process 20-30min, and centrifuging and taking supernatant; It is that the water of 3-4 washs in right amount that residue adds pH value, centrifugal merging supernatant, and is concentrated into 10-20mL; Concentrate is placed in 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, be concentrated near dry, the 10mL that adds water subsequently dissolves, ultrasonic process 1-5 minute, and add water and be settled to 20-30mL, obtain test sample storing solution; Precision measures test sample storing solution described in 1mL, adds the NaHCO that mass concentration is 1-3% successively
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1-3mg/mL, mixing also carries out water-bath 40min in 30 DEG C, is settled to 20-30mL, as need testing solution subsequently with the acetonitrile of 30-60%;
According to high performance liquid chromatography, be filling agent with octadecylsilane chemically bonded silica, take acetonitrile as mobile phase A, phosphoric acid solution with 0.2% carries out gradient elution according to following program for Mobile phase B: 0-8min, A:B are 25%:75%; 8-40min, A:B are 25%:75% → 50%:50%; 40-45min, A:B are 50%:50%; 45-46min, A:B are 50%:50% → 25%:75%; 46-75min, A:B are 25%:75%; Coutroi velocity 1.0mL/min, column temperature 25 DEG C, determined wavelength 263nm, number of theoretical plate calculates should be not less than 6000 by 1-DNJ peak;
The described reference substance solution of accurate absorption and each 20 μ L of need testing solution, 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, describedly comprises the step that the content of 1-DNJ measures:
Accurately weighed 1-DNJ reference substance 10mg, thin up makes the reference substance storing solution of every mL containing 0.2mg; Precision measures reference substance storing solution described in 1mL, and precision adds the NaHCO that mass concentration is 2%
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 2mL of solution 2mL and 2mg/mL, mixing also carries out water-bath 40min in 50 DEG C, is settled to 25mL subsequently, in contrast product solution with the acetonitrile of 30%;
Get pharmaceutical preparation porphyrize to be measured, and precision takes 1g, adding with concentrated hydrochloric acid adjust pH is the water 50mL of 3-4, stirs, ultrasonic process 30min, and centrifuging and taking supernatant; It is that the water of 3-4 washs in right amount that residue adds pH value, centrifugal merging supernatant, and is concentrated into 10mL; Concentrate is placed in 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, be concentrated near dry, the 10mL that adds water subsequently dissolves, ultrasonic process 1 minute, and add water and be settled to 25mL, obtain test sample storing solution; Precision measures test sample storing solution described in 1mL, adds the NaHCO that mass concentration is 2% successively
3the fluorenes methoxy dicarbonyl chloride acetonitrile solution 4mL of solution 1mL and 1mg/mL, mixing also carries out water-bath 40min in 50 DEG C, is settled to 25mL, as need testing solution subsequently with the acetonitrile of 30%;
According to high performance liquid chromatography, be filling agent with octadecylsilane chemically bonded silica, take acetonitrile as mobile phase A, phosphoric acid solution with 0.2% carries out gradient elution according to following program for Mobile phase B: 0-8min, A:B are 25%:75%; 8-40min, A:B are 25%:75% → 50%:50%; 40-45min, A:B are 50%:50%; 45-46min, A:B are 50%:50% → 25%:75%; 46-75min, A:B are 25%:75%; Coutroi velocity 1.0mL/min, column temperature 25 DEG C, determined wavelength 263nm, number of theoretical plate calculates should be not less than 6000 by 1-DNJ peak;
The described reference substance solution of accurate absorption and each 20 μ L of need testing solution, injection liquid chromatography, measures.
3. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 1, 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 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; Rinse resin by the 2M NaOH solution of 5 times of resin volumes, be washed to neutrality; Rinse resin with the 2M hydrochloric acid solution of 5 times of resin volumes, be washed to neutrality; Rinse resin by the 2M NaOH solution of 5 times of resin volumes, be washed to neutrality; Rinse resin with the 2M hydrochloric acid solution 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, the above-mentioned two kinds of need testing solutions of accurate absorption and reference substance solution each 1-3 μ L, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride of 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 measures the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 5-15mg is that the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 50-70:30-50:0.5-1.5 dissolves and is diluted to 50mL with volume ratio, product solution in contrast;
Get pharmaceutical preparation 1-3 gram to be measured, add methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution that volume ratio is 50-70:30-50:0.5-1.5, ultrasonic process 30 minutes, continue to add described mixed solution after letting cool and be settled to 25mL, centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid of 50-70:30-50:0.5-1.5 be mobile phase, determined wavelength is 250nm; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;
The described reference substance solution of accurate absorption and need testing solution each 5-15 μ L respectively, injection liquid chromatography, measures;
C, HPLC method measures 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 that mass concentration is 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, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, in contrast product solution;
Accurately weighed pharmaceutical preparation 0.3-0.8g to be measured, add water after jolting, 20-40 minute is heated in 60-90 DEG C of water-bath, add water after letting cool and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 1-3mL, and add that mass concentration is 0.8% successively 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, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution of 15-25:0.1-1:75-85 be mobile phase, determined wavelength is 390nm; Number of theoretical plate calculates should be not less than 2500 by Pipecolic Acid peak;
Accurate absorption reference substance solution 4-6 μ L and need testing solution 5-15 μ L 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, the above-mentioned two kinds of need testing solutions of accurate absorption and each 2 μ L of reference substance solution, put respectively on same silica gel g thin-layer plate, take volume ratio as the methenyl choloride of 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 measures the content of mono-ammonium glycyrrhizinate
Accurately weighed mono-ammonium glycyrrhizinate reference substance 10mg is that the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution of 60:40:1 dissolves and is diluted to 50mL with volume ratio, product solution in contrast;
Get pharmaceutical preparation to be measured 2.0 grams, add methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid mixed solution that volume ratio is 60:40:1, ultrasonic process 30 minutes, continue to add described mixed solution after letting cool and be settled to 25mL, centrifuging and taking supernatant, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the methyl alcohol-0.2mol/L ammonium acetate-glacial acetic acid of 60:40:1 be mobile phase, determined wavelength is 250nm; Number of theoretical plate calculates should be not less than 2000 by ammonium glycyrrhetate peak;
The described reference substance solution of accurate absorption and each 10 μ L of need testing solution respectively, injection liquid chromatography, measures;
C, HPLC method measures 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 that mass concentration is 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, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, in contrast product solution;
Accurately weighed pharmaceutical preparation 0.4g to be measured, add water after jolting, in 60 DEG C of water-baths heat 30 minutes, add water after letting cool and be settled to 10mL, shake up and centrifugal after, precision measures supernatant 2mL, and add the sodium bicarbonate solution 2mL that mass concentration is DNF acetonitrile solution 2mL and 0.5mol/L of 0.8% successively, in 60 DEG C of water-baths 1 hour, the phosphate buffer letting cool rear pH7.0,0.2mol/L is settled to 10mL, as need testing solution;
According to high performance liquid chromatography test, take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the acetonitrile-dimethyl formamide-0.025mol/L sodium acetate solution of 21:0.5:79 be mobile phase, determined wavelength is 390nm; Number of theoretical plate calculates should be not less than 2500 by Pipecolic Acid peak;
Accurate absorption reference substance solution 5 μ L and need testing solution 10 μ L respectively, injection liquid chromatography, measures.
7. the detection method of the pharmaceutical preparation for the treatment of diabete according to claim 6, is characterized in that, the bulk drug of described pharmaceutical preparation consists of: silkworm excrement 7150 weight portion and Radix Glycyrrhizae 137.5 weight portion; Or silkworm excrement 6150 weight portion and Radix Glycyrrhizae 187.5 weight portion; Or silkworm excrement 7850 weight portion and Radix Glycyrrhizae 117.5 weight portion.
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 getting selected weight portion adds 2-6 times amount 50-70% ethanol, steeped overnight, slowly be heated to boil, reflux 1-3 time, each 0.5-1.5 hour, filter, merging filtrate, reclaim ethanol, 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 the thick paste A that relative density is 1.00-1.15, for subsequent use; The Radix Glycyrrhizae of separately getting selected weight portion adds 8-15 times amount soak by water 1-3 time, each 1-3 hour, merging filtrate, and 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 customary adjuvant, conveniently technique and make clinical acceptable formulation.
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 getting selected weight portion adds 4 times amount 60% ethanol, steeped overnight, slowly be heated to boil, backflow twice, each 1 hour, filter, merging filtrate, reclaim ethanol, 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 amount soak by water 2 times, each 2 hours, merging filtrate, and placing spends the night makes precipitation, and getting supernatant concentration to relative density is 1.04-1.15 thick paste, for subsequent use; Merge above-mentioned two thick pastes, add customary adjuvant, conveniently technique and make clinical acceptable formulation.
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, sustained release preparation, quick releasing formulation, controlled release preparation, oral liquid or ejection preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410264670.4A CN104007205B (en) | 2014-06-13 | 2014-06-13 | A kind of detection method for the treatment of the pharmaceutical preparation of diabete |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201410264670.4A CN104007205B (en) | 2014-06-13 | 2014-06-13 | A kind of detection method for the treatment of the pharmaceutical preparation of diabete |
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| 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 |
| CN108828121B (en) * | 2018-06-14 | 2020-06-05 | 华润三九医药股份有限公司 | Method for detecting content of α -high nojirimycin in white tree medicinal material |
| 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 |
| CN112345676B (en) * | 2020-12-07 | 2022-06-07 | 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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| CN100556892C (en) * | 2007-03-15 | 2009-11-04 | 浙江大学 | A kind of method of from mulberry leaf, extracting the 1-S-GI |
| CN101496829B (en) * | 2008-01-30 | 2011-09-14 | 漳州片仔癀药业股份有限公司 | Medicament composition for treating diabetes and preparation method thereof |
| CN101654428B (en) * | 2009-09-11 | 2012-05-23 | 成都市金医生科技健康产业有限公司 | Method for extracting and separating high-purity 1-deoxynojirimycin from natural product |
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| CN102507831B (en) * | 2011-10-13 | 2014-07-16 | 厦门市医药研究所 | Determination method of 1-deoxynojirimycin content in silkworm powder |
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Address after: 363000 No. 1, Amber Road, Xiangcheng District, Zhangzhou City, Fujian Province Patentee after: ZHANGZHOU PIEN TZE HUANG PHARMACEUTICAL Co.,Ltd. Address before: 363000 1 Street, Zhangzhou, Fujian Patentee before: ZHANGZHOU PIEN TZE HUANG PHARMACEUTICAL Co.,Ltd. |