CN105527362A

CN105527362A - Analysis method of fingerprint spectrum of amino acids in granulation-promoting ointment

Info

Publication number: CN105527362A
Application number: CN201510966611.6A
Authority: CN
Inventors: 周永梅; 房德敏; 冯鑫; 王巨存; 徐卫国; 邢国胜
Original assignee: TIANJIN HOSPITAL TIANJIN CITY
Current assignee: TIANJIN HOSPITAL TIANJIN CITY
Priority date: 2015-12-17
Filing date: 2015-12-17
Publication date: 2016-04-27

Abstract

The invention provides an analysis method of a fingerprint spectrum of amino acids in a granulation-promoting ointment. The method comprises the steps: determining the amino acids in the granulation-promoting ointment by high performance liquid chromatography, taking the granulation-promoting ointment, heating in water bath to make the granulation-promoting ointment melted, adding hot water, fully stirring, allowing to stand, cooling, then placing in a refrigerator, freezing, after solidification, taking an aqueous-layer frozen layer, melting, filtering, washing the filter residue with anhydrous ethanol, and drying; taking the dried material, adding a 6 mol/L hydrochloric acid solution, carrying out hydrolysis for 12 h at the temperature of 110 DEG C, placing to room temperature, filtering, repeatedly swashing the residue with purified water, making the volume constant, and shaking evenly; and measuring to take the solution, heating to dry the solvent by evaporation, dissolving the residue with a 0.1 mol/L hydrochloric acid solution, making the volume constant to obtain a residue solution, carrying out pre-column derivatization on the residue solution, and then carrying out gradient elution. With use of the high performance liquid chromatography, a granulation-promoting ointment amino acid HPLC fingerprint spectrum shared pattern is established; the precision, stability and repeatability of the method are in accordance with requirements of traditional Chinese medicine fingerprint spectrum related technical parameters.

Description

The analytical approach of amino acid finger-print in a kind of myogenic cream

Technical field

The present invention relates to technical field of traditional Chinese medicines, more specifically to the analytical approach of amino acid finger-print in a kind of myogenic cream.

Background technology

Treatment by Chinese herbs disease has the history of several thousand, along with the development of science and technology, Chinese medicine is also faced with modernization, go on world market, this just requires the control must carrying out traditional Chinese medicine quality, and the quality control of the component content of Chinese medicine is then most critical, especially for the Chinese medicine preparation of compound, want the quality comprehensively controlling Chinese medicine, the Accurate Measurement of the content of each composition must be carried out.

Myogenic cream comes from Zhang Shanlei " ulcer section outline ", it is TCM surgery common medicine, by more than elephant hide, calamine, tortoise plastron, blood, the medicinal material such as Radix Angelicae Sinensis, dried rhizome of rehmannia to boil through sesame oil high temperature and forms, there is myogenic, sore effect, be used for the treatment of the caused chronic wound such as wound concurrent infection, pressure ulcer, diabetes, Venous Ulcers, burn and scald evident in efficacy.Trophic factor plays an important role in wound healing.Effective energy support can be repaired wound for body and be provided sufficient energy and nutriment, reduces the degraded of body oneself protein, strengthens immunity of organisms, promote the functional union of the surface of a wound.Energy, carbohydrates, protein, fat, vitamin and mineral metabolism all affect the process of wound healing.Protein affects one of most important factor of wound healing.Potein deficiency is by the obstacle of aspects such as causing that capillary formation, fibroblast proliferation, proteoglycan synthesis, collage synthesis and wound are reinvented.The shortage of protein also can have influence on immune system, shows weakening and infection chance increase of leukocytic phagocytosis.Amino acid abundant in myogenic cream plays an important role in wound healing.Due to formulation reason, said preparation quality control lacks effective standard always, determines that this standard then needs, when measuring each component content, must to remove disturbing factor, measure content accurately, accomplishes the control drug quality of imitating, ensure the safety of medicine.

Summary of the invention

Instant invention overcomes deficiency of the prior art, provide the analytical approach of amino acid finger-print in a kind of myogenic cream.

Object of the present invention is achieved by following technical proposals.

The analytical approach of amino acid finger-print in a kind of myogenic cream, myogenic cream comprises the bulk drug of following composition by weight: elephant hide 15-20 part, Radix Angelicae Sinensis 10-15 part, 10-15 part more than blood, dried rhizome of rehmannia 20-30 part, tortoise plastron 20-30 part, sesame oil 400-500 part, calamine 28-32 part, plaster stone 25-30 part and beeswax 80-100 part, measure by the following method:

Step 1, the preparation of need testing solution: get myogenic cream and be placed in conical flask, heating water bath makes it melt, hot water is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing, after to be solidified, water intaking layer frozen coating, fusing, filter, after filter residue absolute ethanol washing, dry thing is obtained after drying, get dry thing and be placed in ampoule, 6mol/L hydrochloric acid solution is added in ampoule, 110 DEG C of hydrolysis, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water repeatedly to rinse, add purified water to scale, shake up, measure the solution in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain need testing solution,

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 10-15 part, 10-15 part more than blood, dried rhizome of rehmannia 20-30 part, tortoise plastron 20-30 part, sesame oil 400-500 part, calamine 28-32 part, plaster stone 25-30 part and beeswax 80-100 part, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample and be placed in conical flask, heating water bath makes it melt, hot water is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing, after to be solidified, water intaking layer frozen coating, fusing, filter, after filter residue absolute ethanol washing, dry thing is obtained after drying, get dry thing and be placed in ampoule, 6mol/L hydrochloric acid solution is added in ampoule, 110 DEG C of hydrolysis, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water repeatedly to rinse, add purified water to scale, shake up, measure the solution in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 3, the preparation of reference substance solution: take the 17 seed amino acid reference substances such as aspartic acid, glutamic acid, L-hydroxyproline, serine, glycocoll, histidine, threonine, alanine, arginine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, lysine in measuring bottle, adding 0.1mol/L hydrochloric acid solution makes reference substance solution;

Step 4, the derivatization of solution: by need testing solution, blank sample solution and reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution of 1mol/L triethylamine and the acetonitrile solution of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixes a period of time, room temperature is placed, add normal hexane wherein again, after whirlpool mixes a period of time, place, take off a layer solution, normal hexane is added again in lower floor's solution, jolting, place, pick and place and put later lower floor's solution, after micro porous filtration, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Step 5, chromatographic determination: chromatographic condition is that chromatographic column adopts length to be 250mm, internal diameter is 4.6mm, and sample introduction grain size is 5 μm, and mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, Mobile phase B is acetonitrile-water, carry out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

In described step 1, get myogenic cream 30-50g and be placed in conical flask, heating water bath makes it melt, hot water 30-50mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 2-4h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 2-4 time, dry thing is obtained after drying, get dry thing 0.1-0.5g and be placed in ampoule, 6mol/L hydrochloric acid solution 4-8mL is added in ampoule, 110 DEG C of hydrolysis 10-14h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water repeatedly to rinse 2-4 time, add purified water to scale, shake up, measure the solution 3-8mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain need testing solution.

In described step 2, according to Radix Angelicae Sinensis 10-15 part, 10-15 part more than blood, dried rhizome of rehmannia 20-30 part, tortoise plastron 20-30 part, sesame oil 400-500 part, calamine 28-32 part, plaster stone 25-30 part and beeswax 80-100 part, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 30-50g and be placed in conical flask, heating water bath makes it melt, hot water 30-50mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 2-4h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 2-4 time, dry thing is obtained after drying, get dry thing 0.1-0.5g and be placed in ampoule, 6mol/L hydrochloric acid solution 4-8mL is added in ampoule, 110 DEG C of hydrolysis 10-14h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water repeatedly to rinse 2-4 time, add purified water to scale, shake up, measure the solution 3-8mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution.

In described step 3, take aspartic acid, glutamic acid, L-hydroxyproline, serine, glycocoll, histidine, threonine, alanine, arginine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, lysine etc. 17 seed amino acid reference substance is in measuring bottle, add 0.1mol/L hydrochloric acid solution and make every 1mL respectively containing aspartic acid 43 μ g, glutamic acid 122 μ g, L-hydroxyproline 84 μ g, serine 44 μ g, glycocoll 210 μ g, histidine 29 μ g, threonine 101 μ g, alanine 56 μ g, arginine 75 μ g, proline 103 μ g, tyrosine 66 μ g, valine 78 μ g, methionine 60 μ g, isoleucine 43 μ g, leucine 77 μ g, phenylalanine 91 μ g, the reference substance solution of lysine 105 μ g.

In described step 4, by need testing solution, blank sample solution and each 5-15mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 0.5-1.5mL of 1mol/L triethylamine and the acetonitrile solution 0.5-1.5mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 3-7min, room temperature places 40-80min, add normal hexane 3-5mL wherein again, after whirlpool mixing 3-7min, place 5-15min, take off a layer solution, normal hexane 3-5mL is added again in lower floor's solution, jolting, place 5-15min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.3-0.5 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization.

In described step 5, chromatographic condition is HypersilODS-2C ₁₈chromatographic column, chromatographic column adopts length to be 250mm, internal diameter is 4.6mm, sample introduction grain size is 5 μm, mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 6.0-7.0, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is (1-5): (95-100), Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is (3-5): (0.5-2), carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

Beneficial effect of the present invention is: adopt high performance liquid chromatography, set up the amino acid whose HPLC finger-print of myogenic cream, and 10 batches of myogenic creams are studied, establish myogenic cream amino acid HPLC finger-print sharing model, its precision, stability, repeatability meet the requirement of traditional Chinese medicine fingerprint associated technical parameters.Point out 18 total peaks simultaneously, and amino acid Qualitive test has been carried out to characteristic peak each in finger-print, identify 17 aminoacid ingredients altogether.Carry out similarity evaluation to 10 batches of myogenic cream amino acid, result is all greater than 0.9, and show that myogenic cream aminoacid ingredient is stablized, the method provides foundation for the quality control of myogenic cream.

Accompanying drawing explanation

Fig. 1 is through the need testing solution of derivatization, high-efficient liquid phase chromatogram through the blank sample solution of derivatization and the reference substance solution through derivatization in the present invention, wherein A is the blank sample solution through derivatization, B is the reference substance solution through derivatization, and C is the need testing solution through derivatization;

Fig. 2 be in the present invention in each embodiment through the high-efficient liquid phase chromatogram of the need testing solution of derivatization, wherein, 1 is the high-efficient liquid phase chromatogram of embodiment 1, 2 is the high-efficient liquid phase chromatogram of embodiment 2, 3 is the high-efficient liquid phase chromatogram of embodiment 3, 4 is the high-efficient liquid phase chromatogram of embodiment 4, 5 is the high-efficient liquid phase chromatogram of embodiment 5, 6 is the high-efficient liquid phase chromatogram of embodiment 6, 7 is the high-efficient liquid phase chromatogram of embodiment 7, 8 is the high-efficient liquid phase chromatogram of embodiment 8, 9 is the high-efficient liquid phase chromatogram of embodiment 9, 10 is the high-efficient liquid phase chromatogram of embodiment 10, 11 is contrast collection of illustrative plates,

Fig. 3 is the myogenic cream amino acid finger-print of gained of the present invention.

Embodiment

Below by specific embodiment, technical scheme of the present invention is further described.

The instrument adopted in experimentation and reagent, as follows:

Embodiment 1

The analytical approach of amino acid finger-print in a kind of myogenic cream, myogenic cream comprises the bulk drug of following composition by weight: elephant hide 15 parts, Radix Angelicae Sinensis 10 parts, blood mores than 10 parts, 20 parts, the dried rhizome of rehmannia, tortoise plastron 20 parts, 400 parts, sesame oil, calamine 28 parts, plaster stone 25 parts and 80 parts, beeswax, measure by the following method:

Step 1, the preparation of need testing solution: get myogenic cream 30g and be placed in conical flask, heating water bath makes it melt, hot water 30mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 2h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 2 times, dry thing is obtained after drying, get dry thing 0.1g and be placed in ampoule, 6mol/L hydrochloric acid solution 4mL is added in ampoule, 110 DEG C of hydrolysis 10h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 2 times, add purified water to scale, shake up, measure the solution 3mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain need testing solution,

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 10 parts, blood mores than 10 parts, 20 parts, the dried rhizome of rehmannia, tortoise plastron 20 parts, 400 parts, sesame oil, calamine 28 parts, plaster stone 25 parts and 80 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 30g and be placed in conical flask, heating water bath makes it melt, hot water 30mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 2h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 2 times, dry thing is obtained after drying, get dry thing 0.1g and be placed in ampoule, 6mol/L hydrochloric acid solution 4mL is added in ampoule, 110 DEG C of hydrolysis 10h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 2 times, add purified water to scale, shake up, measure the solution 3mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 3, the preparation of reference substance solution: take aspartic acid, glutamic acid, L-hydroxyproline, serine, glycocoll, histidine, threonine, alanine, arginine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, lysine etc. 17 seed amino acid reference substance is in measuring bottle, add 0.1mol/L hydrochloric acid solution and make every 1mL respectively containing aspartic acid 43 μ g, glutamic acid 122 μ g, L-hydroxyproline 84 μ g, serine 44 μ g, glycocoll 210 μ g, histidine 29 μ g, threonine 101 μ g, alanine 56 μ g, arginine 75 μ g, proline 103 μ g, tyrosine 66 μ g, valine 78 μ g, methionine 60 μ g, isoleucine 43 μ g, leucine 77 μ g, phenylalanine 91 μ g, the reference substance solution of lysine 105 μ g,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 5mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 0.5mL of 1mol/L triethylamine and the acetonitrile solution 0.5mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 3min, room temperature places 40min, add normal hexane 3mL wherein again, after whirlpool mixing 3min, place 5min, take off a layer solution, normal hexane 3mL is added again in lower floor's solution, jolting, place 5min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.3 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Step 5, chromatographic determination: chromatographic condition is HypersilODS-2C ₁₈chromatographic column (250mm × 4.6mm, 5 μm), mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, and wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 6.0, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is 1:95, Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is 3:0.5, carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

Embodiment 2

The analytical approach of amino acid finger-print in a kind of myogenic cream, myogenic cream comprises the bulk drug of following composition by weight: elephant hide 20 parts, Radix Angelicae Sinensis 15 parts, blood mores than 15 parts, 30 parts, the dried rhizome of rehmannia, tortoise plastron 30 parts, 500 parts, sesame oil, calamine 32 parts, plaster stone 30 parts and 100 parts, beeswax, measure by the following method:

Step 1, the preparation of need testing solution: get myogenic cream 50g and be placed in conical flask, heating water bath makes it melt, hot water 50mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 4h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 4 times, dry thing is obtained after drying, get dry thing 0.5g and be placed in ampoule, 6mol/L hydrochloric acid solution 8mL is added in ampoule, 110 DEG C of hydrolysis 14h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 4 times, add purified water to scale, shake up, measure the solution 8mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain need testing solution,

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 15 parts, blood mores than 15 parts, 30 parts, the dried rhizome of rehmannia, tortoise plastron 30 parts, 500 parts, sesame oil, calamine 32 parts, plaster stone 30 parts and 100 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 50g and be placed in conical flask, heating water bath makes it melt, hot water 50mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 4h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 4 times, dry thing is obtained after drying, get dry thing 0.5g and be placed in ampoule, 6mol/L hydrochloric acid solution 8mL is added in ampoule, 110 DEG C of hydrolysis 14h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 4 times, add purified water to scale, shake up, measure the solution 8mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 15mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 1.5mL of 1mol/L triethylamine and the acetonitrile solution 1.5mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 7min, room temperature places 80min, add normal hexane 5mL wherein again, after whirlpool mixing 7min, place 15min, take off a layer solution, normal hexane 5mL is added again in lower floor's solution, jolting, place 15min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.5 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Step 5, chromatographic determination: chromatographic condition is HypersilODS-2C ₁₈chromatographic column (250mm × 4.6mm, 5 μm), mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, and wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 7.0, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is 5:100, Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is 5:2, carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

Embodiment 3

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 15 parts, blood mores than 15 parts, 30 parts, the dried rhizome of rehmannia, tortoise plastron 30 parts, 500 parts, sesame oil, calamine 32 parts, plaster stone 30 parts and 100 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 30g and be placed in conical flask, heating water bath makes it melt, hot water 30mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 2h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 2 times, dry thing is obtained after drying, get dry thing 0.1g and be placed in ampoule, 6mol/L hydrochloric acid solution 4mL is added in ampoule, 110 DEG C of hydrolysis 10h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 2 times, add purified water to scale, shake up, measure the solution 3mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 9mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 1mL of 1mol/L triethylamine and the acetonitrile solution 1mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 4min, room temperature places 50min, add normal hexane 4mL wherein again, after whirlpool mixing 4min, place 9min, take off a layer solution, normal hexane 4mL is added again in lower floor's solution, jolting, place 9min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.35 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Embodiment 4

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 10 parts, blood mores than 10 parts, 20 parts, the dried rhizome of rehmannia, tortoise plastron 20 parts, 400 parts, sesame oil, calamine 28 parts, plaster stone 25 parts and 80 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 50g and be placed in conical flask, heating water bath makes it melt, hot water 50mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 4h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 4 times, dry thing is obtained after drying, get dry thing 0.5g and be placed in ampoule, 6mol/L hydrochloric acid solution 8mL is added in ampoule, 110 DEG C of hydrolysis 14h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 4 times, add purified water to scale, shake up, measure the solution 8mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 10mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 1mL of 1mol/L triethylamine and the acetonitrile solution 1mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 5min, room temperature places 60min, add normal hexane 4mL wherein again, after whirlpool mixing 5min, place 10min, take off a layer solution, normal hexane 4mL is added again in lower floor's solution, jolting, place 10min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.4 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Embodiment 5

The analytical approach of amino acid finger-print in a kind of myogenic cream, myogenic cream comprises the bulk drug of following composition by weight: elephant hide 16 parts, Radix Angelicae Sinensis 11 parts, blood mores than 11 parts, 22 parts, the dried rhizome of rehmannia, tortoise plastron 22 parts, 450 parts, sesame oil, calamine 29 parts, plaster stone 26 parts and 85 parts, beeswax, measure by the following method:

Step 1, the preparation of need testing solution: get myogenic cream 40g and be placed in conical flask, heating water bath makes it melt, hot water 40mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.25g and be placed in ampoule, 6mol/L hydrochloric acid solution 6mL is added in ampoule, 110 DEG C of hydrolysis 12h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain need testing solution,

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 11 parts, blood mores than 11 parts, 22 parts, the dried rhizome of rehmannia, tortoise plastron 22 parts, 450 parts, sesame oil, calamine 29 parts, plaster stone 26 parts and 85 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 40g and be placed in conical flask, heating water bath makes it melt, hot water 40mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.25g and be placed in ampoule, 6mol/L hydrochloric acid solution 6mL is added in ampoule, 110 DEG C of hydrolysis 12h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 10mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 0.8mL of 1mol/L triethylamine and the acetonitrile solution 0.8mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 6min, room temperature places 70min, add normal hexane 5mL wherein again, after whirlpool mixing 6min, place 12min, take off a layer solution, normal hexane 5mL is added again in lower floor's solution, jolting, place 12min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.45 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Step 5, chromatographic determination: chromatographic condition is HypersilODS-2C ₁₈chromatographic column (250mm × 4.6mm, 5 μm), mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, and wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 6.5, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is 3:97, Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is 4:1, carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

Embodiment 6

Step 1, the preparation of need testing solution: get myogenic cream 45g and be placed in conical flask, heating water bath makes it melt, hot water 45mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.3g and be placed in ampoule, 6mol/L hydrochloric acid solution 5mL is added in ampoule, 110 DEG C of hydrolysis 11h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain need testing solution,

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 11 parts, blood mores than 11 parts, 22 parts, the dried rhizome of rehmannia, tortoise plastron 22 parts, 450 parts, sesame oil, calamine 29 parts, plaster stone 26 parts and 85 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 45g and be placed in conical flask, heating water bath makes it melt, hot water 45mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.3g and be placed in ampoule, 6mol/L hydrochloric acid solution 5mL is added in ampoule, 110 DEG C of hydrolysis 11h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 10mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 1mL of 1mol/L triethylamine and the acetonitrile solution 1mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 5min, room temperature places 60min, add normal hexane 4mL wherein again, after whirlpool mixing 5min, place 10min, take off a layer solution, normal hexane 4mL is added again in lower floor's solution, jolting, place 10min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.45 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Step 5, chromatographic determination: chromatographic condition is HypersilODS-2C ₁₈chromatographic column (250mm × 4.6mm, 5 μm), mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, and wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 6.5, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is 2:97, Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is 4:1.5, carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

Embodiment 7

The analytical approach of amino acid finger-print in a kind of myogenic cream, myogenic cream comprises the bulk drug of following composition by weight: elephant hide 19 parts, Radix Angelicae Sinensis 13 parts, blood mores than 13 parts, 25 parts, the dried rhizome of rehmannia, tortoise plastron 25 parts, 450 parts, sesame oil, calamine 29 parts, plaster stone 29 parts and 88 parts, beeswax, measure by the following method:

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 13 parts, blood mores than 13 parts, 25 parts, the dried rhizome of rehmannia, tortoise plastron 25 parts, 450 parts, sesame oil, calamine 29 parts, plaster stone 29 parts and 88 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 40g and be placed in conical flask, heating water bath makes it melt, hot water 40mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.25g and be placed in ampoule, 6mol/L hydrochloric acid solution 6mL is added in ampoule, 110 DEG C of hydrolysis 12h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Embodiment 8

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 13 parts, blood mores than 13 parts, 25 parts, the dried rhizome of rehmannia, tortoise plastron 25 parts, 450 parts, sesame oil, calamine 29 parts, plaster stone 29 parts and 88 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 45g and be placed in conical flask, heating water bath makes it melt, hot water 45mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.3g and be placed in ampoule, 6mol/L hydrochloric acid solution 5mL is added in ampoule, 110 DEG C of hydrolysis 11h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 5, chromatographic determination: chromatographic condition is HypersilODS-2C ₁₈chromatographic column (250mm × 4.6mm, 5 μm), mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, and wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 7.0, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is 5:95, Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is 3:2, carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

Embodiment 9

The analytical approach of amino acid finger-print in a kind of myogenic cream, myogenic cream comprises the bulk drug of following composition by weight: elephant hide 18 parts, Radix Angelicae Sinensis 15 parts, blood mores than 13 parts, 25 parts, the dried rhizome of rehmannia, tortoise plastron 25 parts, 500 parts, sesame oil, calamine 29 parts, plaster stone 29 parts and 88 parts, beeswax, measure by the following method:

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 15 parts, blood mores than 13 parts, 25 parts, the dried rhizome of rehmannia, tortoise plastron 25 parts, 500 parts, sesame oil, calamine 29 parts, plaster stone 29 parts and 88 parts, beeswax, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 40g and be placed in conical flask, heating water bath makes it melt, hot water 40mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.25g and be placed in ampoule, 6mol/L hydrochloric acid solution 6mL is added in ampoule, 110 DEG C of hydrolysis 12h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 11mL of reference substance solution are placed in centrifuge tube, add the acetonitrile solution 1.2mL of 1mol/L triethylamine and the acetonitrile solution 1.2mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 6min, room temperature places 60min, add normal hexane 4mL wherein again, after whirlpool mixing 6min, place 10min, take off a layer solution, normal hexane 6mL is added again in lower floor's solution, jolting, place 12min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.35 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Embodiment 10

Step 2, the preparation of blank sample solution: according to Radix Angelicae Sinensis 15 parts, blood mores than 13 parts, 25 parts, the dried rhizome of rehmannia, tortoise plastron 25 parts, 500 parts, sesame oil, calamine 29 parts, plaster stone 29 parts and 88 parts, beeswax are by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 45g and be placed in conical flask, heating water bath makes it melt, hot water 45mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 3h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 3 times, dry thing is obtained after drying, get dry thing 0.3g and be placed in ampoule, 6mol/L hydrochloric acid solution 5mL is added in ampoule, 110 DEG C of hydrolysis 11h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water and repeatedly rinse 3 times, add purified water to scale, shake up, measure the solution 5mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution,

Step 4, the derivatization of solution: by need testing solution, blank sample solution and each 10mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 0.8mL of 1mol/L triethylamine and the acetonitrile solution 0.8mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 5min, room temperature places 40min, add normal hexane 4mL wherein again, after whirlpool mixing 5min, place 10min, take off a layer solution, normal hexane 4mL is added again in lower floor's solution, jolting, place 10min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.45 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization,

Step 5, chromatographic determination: chromatographic condition is HypersilODS-2C ₁₈chromatographic column (250mm × 4.6mm, 5 μm), mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, and wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 6.5, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is 2:98, Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is 4:2, carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.

Gradient elution program table in embodiment 1-10, as shown in the table:

Time (minute)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
11	93	7
			13.9	88	12
14	85	15
			29	66	34
35	20	80
			36	0	100
41	0	100
			46	100	0

In embodiment 1-10, need testing solution has the relative retention time at peak, as shown in the table:

In embodiment 1-10, need testing solution has the relative peak area at peak, as shown in the table:

The similarity of need testing solution in embodiment 1-10, as shown in the table:

Adopt high performance liquid chromatography, set up the amino acid whose HPLC finger-print of myogenic cream, and 10 batches of myogenic creams are studied, establish myogenic cream amino acid HPLC finger-print sharing model, its precision, stability, repeatability meet the requirement of traditional Chinese medicine fingerprint associated technical parameters.Point out 18 total peaks simultaneously, and amino acid Qualitive test has been carried out to characteristic peak each in finger-print, identify 17 aminoacid ingredients altogether.Carry out similarity evaluation to 10 batches of myogenic cream amino acid, result is all greater than 0.9, and show that myogenic cream aminoacid ingredient is stablized, the method provides foundation for the quality control of myogenic cream.

Above to invention has been exemplary description; should be noted that; when not departing from core of the present invention, any simple distortion, amendment or other those skilled in the art can not spend the equivalent replacement of creative work all to fall into protection scope of the present invention.

Claims

1. the analytical approach of amino acid finger-print in a myogenic cream, it is characterized in that: myogenic cream comprises the bulk drug of following composition by weight: elephant hide 15-20 part, Radix Angelicae Sinensis 10-15 part, 10-15 part more than blood, dried rhizome of rehmannia 20-30 part, tortoise plastron 20-30 part, sesame oil 400-500 part, calamine 28-32 part, plaster stone 25-30 part and beeswax 80-100 part, measure by the following method:

2. the analytical approach of amino acid finger-print in a kind of myogenic cream according to claim 1, it is characterized in that: in described step 1, get myogenic cream 30-50g and be placed in conical flask, heating water bath makes it melt, hot water 30-50mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 2-4h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 2-4 time, dry thing is obtained after drying, get dry thing 0.1-0.5g and be placed in ampoule, 6mol/L hydrochloric acid solution 4-8mL is added in ampoule, 110 DEG C of hydrolysis 10-14h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water repeatedly to rinse 2-4 time, add purified water to scale, shake up, measure the solution 3-8mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain need testing solution.

3. the analytical approach of amino acid finger-print in a kind of myogenic cream according to claim 1, it is characterized in that: in described step 2, according to Radix Angelicae Sinensis 10-15 part, 10-15 part more than blood, dried rhizome of rehmannia 20-30 part, tortoise plastron 20-30 part, sesame oil 400-500 part, calamine 28-32 part, plaster stone 25-30 part and beeswax 80-100 part, by calamine, gesso is broken into impalpable powder, sieving for standby, Radix Angelicae Sinensis, tortoise plastron, more than blood, the dried rhizome of rehmannia and sesame oil are put together in pot fried withered, remove slag, filter, filter after beeswax is melted completely in deep fat, lower the temperature from fire, add calamine stone and gesso, constantly be stirred to and be cooled to cream, obtain blank sample, get blank sample 30-50g and be placed in conical flask, heating water bath makes it melt, hot water 30-50mL is added in conical flask, abundant stirring, standing cooling is placed on refrigerator freezing 2-4h, after to be solidified, water intaking layer frozen coating, fusing, filter, filter residue is with after absolute ethanol washing 2-4 time, dry thing is obtained after drying, get dry thing 0.1-0.5g and be placed in ampoule, 6mol/L hydrochloric acid solution 4-8mL is added in ampoule, 110 DEG C of hydrolysis 10-14h, be placed to room temperature, be filtered in 50mL measuring bottle, filter remaining residue purified water repeatedly to rinse 2-4 time, add purified water to scale, shake up, measure the solution 3-8mL in 50mL measuring bottle, evaporate to dryness, be transferred in 10mL measuring bottle with 0.1mol/L hydrochloric acid solution, constant volume, shake up, obtain blank sample solution.

4. the analytical approach of amino acid finger-print in a kind of myogenic cream according to claim 1, it is characterized in that: in described step 3, take aspartic acid, glutamic acid, L-hydroxyproline, serine, glycocoll, histidine, threonine, alanine, arginine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, lysine etc. 17 seed amino acid reference substance is in measuring bottle, add 0.1mol/L hydrochloric acid solution and make every 1mL respectively containing aspartic acid 43 μ g, glutamic acid 122 μ g, L-hydroxyproline 84 μ g, serine 44 μ g, glycocoll 210 μ g, histidine 29 μ g, threonine 101 μ g, alanine 56 μ g, arginine 75 μ g, proline 103 μ g, tyrosine 66 μ g, valine 78 μ g, methionine 60 μ g, isoleucine 43 μ g, leucine 77 μ g, phenylalanine 91 μ g, the control sample solution of lysine 105 μ g.

5. the analytical approach of amino acid finger-print in a kind of myogenic cream according to claim 1, it is characterized in that: in described step 4, by need testing solution, blank sample solution and each 5-15mL of reference substance solution are placed in centrifuge tube respectively, add the acetonitrile solution 0.5-1.5mL of 1mol/L triethylamine and the acetonitrile solution 0.5-1.5mL of 0.1mol/L phenyl isothiocyanate (PITC) successively, after whirlpool mixing 3-7min, room temperature places 40-80min, add normal hexane 3-5mL wherein again, after whirlpool mixing 3-7min, place 5-15min, take off a layer solution, normal hexane 3-5mL is added again in lower floor's solution, jolting, place 5-15min, pick and place and put later lower floor's solution, after micro porous filtration, the aperture of the miillpore filter that micro porous filtration adopts is 0.3-0.5 μm, obtain the need testing solution through derivatization, through the blank sample solution of derivatization and the reference substance solution through derivatization.

6. the analytical approach of amino acid finger-print in a kind of myogenic cream according to claim 1, it is characterized in that: in described step 5, chromatographic condition is HypersilODS-2C ₁₈chromatographic column, chromatographic column adopts length to be 250mm, internal diameter is 4.6mm, sample introduction grain size is 5 μm, mobile phase A is acetonitrile-0.1mol/L sodium acetate solution, wherein the PH of acetonitrile-0.1mol/L sodium acetate solution is 6.0-7.0, the proportioning of acetonitrile and 0.1mol/L sodium acetate solution is (1-5): (95-100), Mobile phase B is acetonitrile-water, and wherein the proportioning of acetonitrile and water is (3-5): (0.5-2), carries out gradient elution, volumetric flow rate 1mL/min, determined wavelength 254nm, column temperature 40 DEG C, sample size 5 μ L.