CN110108814B

CN110108814B - Detection method of spray for tendon and bone injuries

Info

Publication number: CN110108814B
Application number: CN201910434923.0A
Authority: CN
Inventors: 程吉祥; 刘青; 罗天军; 薛静; 伍朝平; 杨玉兰
Original assignee: Guizhou Remote Pharmaceutical Co ltd
Current assignee: Guizhou Remote Pharmaceutical Co ltd
Priority date: 2019-05-23
Filing date: 2019-05-23
Publication date: 2022-07-05
Anticipated expiration: 2039-05-23
Also published as: CN110108814A

Abstract

The invention relates to a detection method for a muscle and bone injury spray, which is characterized in that icariin in a product is qualitatively identified, high performance liquid chromatography is used for measuring the content of icariin in the product, gas chromatography is used for measuring the content of menthol in the product, and high performance liquid chromatography is used for measuring the content of ellagic acid in the product; compared with the existing standard, the invention adds the qualitative and quantitative detection method for epimedium herb and the quantitative detection method for menthol and red shank powder, thereby further effectively controlling the quality of the product; the invention is examined by methodology, the instrument precision and linear relation are good, and the specificity is strong; good stability and repeatability, etc.

Description

Detection method of spray for tendon and bone injuries

Technical Field

The invention relates to the field of medicine invention, in particular to a detection method of a spray for tendon and bone injuries.

Background

Soft tissue injury refers to pathological damage of tissues such as skin, subcutaneous superficial and deep fascia, muscles, tendons, tendon sheaths, ligaments, joint capsules, synovial capsules, intervertebral discs, peripheral nerve vessels and the like of a human body caused by various acute trauma or chronic strain and pathological conditions of the disease, and is called soft tissue injury, and clinical manifestations of the soft tissue injury are as follows: pain, swelling, deformity, dysfunction.

The muscle and bone injury spray is developed and produced by Guizhou remote pharmacy limited company, has the effects of promoting blood circulation to remove blood stasis and relieving swelling and pain, and is mainly used for soft tissue injury.

The quantitative index in the existing quality standard is paeoniflorin in red paeony root, the thin-layer chromatography is used for identifying the erythroptera stricta, qualitative and quantitative detection is not carried out on epimedium, and quantitative detection is also not carried out on menthol and the erythroptera stricta, so that the standard is relatively simple, and the product quality cannot be effectively detected.

The company has made a lot of studies on the detection method of the tendon and bone injury spray, such as: a content determination method for simultaneously determining the active ingredients of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in a tendon and bone injury spray is developed, and the method has the advantages of convenience in operation, strong specificity, good separation degree, high stability, high efficiency and capability of simultaneously determining the content of 7 active ingredients in the tendon and bone injury spray, but has the following problems: firstly, epimedium in the product is not qualitatively identified and menthol is not quantitatively detected, so that the quality condition of the epimedium and the content condition of the menthol in the product cannot be comprehensively mastered; secondly, the main component of epimedium in the product is icariin, the main component of the polygonum runcinatum is ellagic acid, 7 components in the product are simultaneously measured by the existing detection method, a targeted content detection method is not established for the icariin in the epimedium and the ellagic acid in the polygonum runcinatum, and the detection time is long, and the separation degree and the stability need to be further improved.

Therefore, the method aims to solve the problem that the quality condition of the epimedium herb and the content condition of the menthol in the product cannot be comprehensively mastered in the prior art, further improve the separation degree and stability of the content detection method, shorten the detection time and save the detection cost, thereby comprehensively and effectively detecting the quality condition of the product and controlling the quality of the product.

Disclosure of Invention

The invention aims to provide a detection method of a spray for tendon and bone injuries.

The technical scheme of the invention is as follows:

the invention relates to a detection method for tendon and bone injury spray, which comprises the following steps of crushing the six raw materials except menthol into coarse powder, mixing, placing the coarse powder into a container, adding 55% ethanol, sealing, soaking for 10 days, taking leachate, standing and filtering, wherein the six raw materials comprise 100g of shin-sha powder, 30g of red peony root, 50g of epimedium herb, 10g of earthworm, 2g of prepared kusnezoff monkshood root and 20g of menthol. Dissolving menthol in ethanol, adding the dissolved menthol into the filtrate, adding ethanol to adjust the ethanol content to 45-55% to 1000ml, and uniformly mixing to obtain the menthol crystal. The detection method comprises the following steps: qualitatively identifying icariin in the product, measuring icariin content in the product by high performance liquid chromatography, measuring menthol content in the product by gas chromatography, and measuring ellagic acid content in the product by high performance liquid chromatography.

The method for qualitatively identifying the icariin in the product comprises the following steps: taking the tendon and bone injury spray solution as a test solution, taking 0.3-0.7G of epimedium control medicinal material, adding 8-12 ml of ethanol, soaking for 25-35 min, filtering, evaporating the filtrate, dissolving the residue with 1ml of ethanol, centrifuging, taking the supernatant as a control solution, taking a proper amount of icariin control, adding methanol to prepare a solution containing 0.5mg of icariin per 1ml, taking the solution as a control solution, performing a thin-layer chromatography test, sucking 2-3 mu l of each of the three solutions, respectively dropping the three solutions on the same silica gel G thin-layer plate, and respectively dropping chloroform-methanol-water lower-layer solution with the ratio of 13.5:5: 2-13.5: 6:2 or the lower-layer solution with the ratio of 10: 1: 1: 1, developing with ethyl acetate-butanone-formic acid-water as developing agent, taking out, air drying, spraying aluminum trichloride test solution, air drying, and inspecting under 365nm ultraviolet lamp to show fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference material and the reference solution.

Preferably, in the method for detecting the bone and muscle injury spray, the method for qualitatively identifying icariin in the product is as follows: taking the tendon and bone injury spray solution as a test solution, taking 0.5G of epimedium control medicinal material, adding 10ml of ethanol, soaking for 30min, filtering, evaporating the filtrate, dissolving the residue with 1ml of ethanol, centrifuging, taking the supernatant as a control solution, taking a proper amount of icariin control, adding methanol to prepare a solution containing 0.5mg of icariin per 1ml as a control solution, performing a thin-layer chromatography test, sucking 2-3 mu l of each of the three solutions, respectively dropping the three solutions on a same silica gel G thin-layer plate, taking a chloroform-methanol-water lower-layer solution with the ratio of 13.5:5.5:2 as a developing agent, spreading, taking out, drying in the air, spraying an aluminum trichloride test solution, drying in the air, placing under a 365nm ultraviolet lamp for inspection, and displaying fluorescent spots with the same color in positions corresponding to the control medicinal material and the control solution in the chromatogram of the test solution.

The method for detecting the bone and muscle injury spray comprises the following specific steps of measuring the content of icariin in a product by using a high performance liquid chromatography:

the content of herba Epimedii is determined by high performance liquid chromatography

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water in a ratio of 23:77 is taken as a mobile phase; the detection wavelength is 270nm, and the number of theoretical plates is not less than 10000 calculated according to icariin;

preparing reference solution by accurately weighing icariin reference, adding methanol to obtain solution containing 0.1mg per 1ml, and shaking;

preparing a test solution, uniformly mixing 5 bottles of solution of a tendon and bone injury spray, precisely weighing 10ml, drying by distillation in a water bath, ultrasonically dissolving 20ml of residue in 20ml of water for several times, completely transferring the residue to a separating funnel, adding ethyl acetate, extracting for 2-4 times, extracting for 15-25 ml each time, combining ethyl acetate solutions, drying by distillation, and adding methanol into the residue to fix the volume to 10ml to obtain the finished product;

the determination method comprises precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

Preferably, the method for detecting the bone and muscle injury spray comprises the following specific steps of measuring the content of icariin in a product by using a high performance liquid chromatography:

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water in a ratio of 23:77 is taken as a mobile phase; the detection wavelength is 270nm, and the number of theoretical plates is not less than 1000 calculated according to icariin;

preparing a test solution, uniformly mixing 5 bottles of solution of the tendon and bone injury spray, precisely measuring 10ml, drying by steaming in a water bath, ultrasonically dissolving residues in 20ml of water for several times, completely transferring to a separating funnel, adding ethyl acetate, extracting for 3 times, extracting for 20ml each time, combining ethyl acetate solutions, drying by steaming, and adding methanol into residues to reach a constant volume of 10ml to obtain the final product;

The method for measuring the content of the menthol in the product by using the gas chromatography in the detection method of the muscle and bone injury spray is as follows:

determining menthol by gas chromatography

Chromatographic conditions and system applicability tests of the capillary column with cross-linked bonded polyethylene glycol as a stationary phase; the column temperature is 80-120 ℃; the temperature of a sample inlet is 180-220 ℃; the temperature of the detector is 220-240 ℃; split-flow sample introduction is carried out, the split-flow ratio is 20:1, and the number of theoretical plates is not lower than 15000 calculated according to the menthol crystal;

preparing reference substance solution by precisely weighing appropriate amount of Mentholum reference substance, and adding anhydrous ethanol to obtain reference substance solution containing 0.05mg of Mentholum per 1 ml;

preparing a test solution, precisely weighing 50 mu L of the tendon and bone injury spray, placing the spray in a 25ml measuring flask, adding absolute ethyl alcohol to the scale, and shaking uniformly to obtain the test solution;

the determination method comprises precisely sucking 1 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

Preferably, the method for measuring the content of menthol in the product by using the gas chromatography in the method for detecting the bone and muscle injury spray provided by the invention is as follows:

determining menthol by gas chromatography

Chromatographic conditions and system applicability tests of the capillary column with cross-linked bonded polyethylene glycol as a stationary phase; the column temperature is 100 ℃; the temperature of a sample inlet is 200 ℃; the temperature of the detector is 230 ℃; split-flow sample injection with a split-flow ratio of 20: 1. The number of theoretical plates is not less than 15000 calculated according to menthol;

The method for determining the content of ellagic acid in the product by using the high performance liquid chromatography in the method for detecting the spray for treating the tendon and bone injuries comprises the following steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking a methanol-0.5 percent phosphoric acid aqueous solution with the ratio of 42:58 as a mobile phase; the detection wavelength is 368nm, and the number of theoretical plates is not less than 4000 calculated according to ellagic acid;

preparing a reference substance solution by precisely weighing ellagic acid reference substance, adding methanol to obtain a solution containing 0.5mg per 1ml, and shaking;

preparing a test solution, uniformly mixing 5 bottles of solution of the tendon and bone injury spray, and filtering a proper amount of mixed solution to obtain the test solution;

the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

In the detection method of the muscle and bone injury spray, each 1ml of the muscle and bone injury spray contains epimedium herb and icariin (C)₃₃H₄₀O₁₅) Measured, not less than 70.0 mug; the spray for treating bone injury contains ellagic acid (C) in the form of radix Rumicis Crispi 1ml₁₄H₆O₈) Calculated, the content of the active ingredient should not be less than 0.35 mg.

The muscle and bone injury spray in the detection method of the muscle and bone injury spray contains menthol (C)₁₀H₂₀O) should be 85.0% to 115.0% of the indicated amount.

Compared with the prior art, the invention has the following advantages:

1. the quantitative index in the existing quality standard is paeoniflorin in red paeony root, the thin-layer chromatography is used for identifying the erythroptera, the qualitative and quantitative detection is not carried out on epimedium, and the quantitative detection is also not carried out on menthol and the erythroptera, so that the standard is relatively simple and the product quality cannot be effectively detected; the invention adds the qualitative and quantitative detection methods of epimedium herb and the quantitative detection methods of menthol and red shank powder, and further effectively checks and controls the quality of the product.

2. In the prior art, a content determination method for simultaneously determining the active ingredients of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray is developed, and the method has the advantages of convenient operation, strong specificity, good separation degree, high stability, high efficiency and capability of simultaneously determining the content of 7 active ingredients in the muscle and bone injury spray, but the technology has the following problems: firstly, the epimedium in the product is not qualitatively identified and the menthol is not quantitatively detected, so that the quality condition of the epimedium and the content condition of the menthol in the product cannot be comprehensively controlled; ② the main component of epimedium in the product is icariin, the main component of rupestris is ellagic acid, the existing detection method is to simultaneously determine 7 components in the product, no specific content detection method is established for icariin in epimedium and ellagic acid in rupestris, the detection time is longer, and the separation degree and stability need to be further improved.

3. The method further improves the separation degree and stability of the content detection method, shortens the detection time, saves the detection cost, comprehensively and effectively detects the quality condition of the product and controls the quality of the product.

4. The invention screens the optimal developing agent for the verification of a newly-added epimedium thin-layer identification method, and the reproducibility of the chromatographic effect is good through the verification of silicon thin-layer plates of different manufacturers.

5. Through the verification of an HPLC (high performance liquid chromatography) content determination method of icariin in the newly added epimedium herb, through methodology investigation, the peak area RSD (n is 6) is 1.2 percent, which indicates that the precision of the instrument is good; according to linear relation test investigation, icariin is in good linear relation within the range of 0.2969-1.9793 mu g; through specificity test investigation, the epimedium negative sample does not have a chromatographic peak at the corresponding retention time of the icariin, which shows that the negative sample has no interference to detection and has good specificity; stability tests show that the test solution is stable within 24 hours; through a repeatability test, the content of RSD (n is 6) is 2.83%, which indicates that the repeatability is good; by the accuracy test (sample recovery test), the average recovery rate was 99.80%, and RSD (n ═ 6) was 2.03%; through a durability test, the content of epimedium herb is 1.21 percent of RSD (n is 3) and the average content is 94.0 mu g/ml which are measured on the same Waters 2996 liquid chromatograph by using 3 chromatographic columns with different brands; adopting Phenomenex Luna C₁₈The RSD (n ═ 3) of the epimedium content was 1.27%, and the average content was 98.3 μ g/ml, as determined by chromatography on 3 different brands of hplc.

6. Through the verification of a newly added method for measuring the GC content of the menthol, the RSD of the peak area is 2.30% (n is 6) through precision test investigation, which indicates that the precision of the instrument is good; through a specificity test, a chromatographic peak which is consistent with the retention time of a reference substance is not detected in the negative solution, so that the negative solution is proved to be interference-free and has good specificity; through a stability test, the peak area RSD of the menthol is 3.77% (n is 7), which indicates that the test solution has better stability within 24 h; through a linear relation test, the linear equation is that y is 85795380.690x +86003.117, and r is 0.9995(n is 7), which indicates that the linear relation is good; through a repeatability test, the average content of the menthol is 100.63%, and the RSD is 3.25% (n is 6), which indicates that the method has good repeatability; the average recovery of menthol was 100.42% and RSD was 2.41% (n ═ 6) in the sample recovery test; the RSD of the menthol content was 2.02% (n ═ 3) by the durability test, indicating that the method was well durable.

7. The HPLC content determination method of the ellagic acid in the newly added Ruipi stauntonii is verified, and the precision test shows that the peak area RSD (n is 6) is 0.80 percent, which indicates that the precision of the instrument is good; through a linear relation test, ellagic acid is in a good linear relation within the range of 0.4905-7.3575 mu g; through a specificity test, the negative sample of the polygonum runcinatum does not have a chromatographic peak at the corresponding retention time of the ellagic acid, which indicates that the negative sample has no interference to the detection and has good specificity; stability tests show that the test solution is stable within 48 hours; through a repeatability test, the content of RSD (n is 6) is 0.0%, which indicates that the repeatability is good; by the accuracy test (sample recovery test), the average recovery was 106.70% and RSD (n ═ 6) was 0.87%; through a durability test, the content of the erythro leg RSD (n is 3) is 2.00% and the average content is 0.50mg/ml, which are measured on the same agilent Technologies 1260 liquid chromatograph by using 3 kinds of chromatographic columns with different brands; the content of the shin-san is 2.29 percent of RSD (n is 3) and the average content is 0.50mg/ml by measuring the content by using a senkyo AQ chromatographic column through 3 high performance liquid chromatographs of different brands.

Description of the drawings:

FIG. 1: icariin content determination method in the muscle and bone injury spray detection method is icariin linear relation chart.

FIG. 2 shows HPLC chromatogram of specificity test of icariin content determination method in the detection method of tendon and bone injury spray.

FIG. 3: the icariin content determination method in the muscle and bone injury spray detection method uses different brands of chromatographic columns for durability test chromatograms (1: icariin).

FIG. 4: a durability test chromatogram (1: icariin) of instruments of different brands is used in the icariin content determination method in the muscle and bone injury spray detection method.

FIG. 5: a special test chart for the method for measuring the GC content of menthol in the method for detecting the spray for treating muscle and bone injuries is disclosed.

FIG. 6: menthol GC content determination method in the muscle and bone injury spray detection method is a menthol standard curve graph.

FIG. 7: ellagic acid content determination method in the bone and tendon injury spray detection method adopts an ellagic acid linear relation diagram.

FIG. 8: a specific test HPLC chromatogram is adopted in the method for determining the content of ellagic acid in the method for detecting the tendon and bone injury spray.

FIG. 9: the method for measuring the content of the ellagic acid in the method for detecting the tendon and bone injury spray uses chromatographic column durability test chromatograms of different brands (1: ellagic acid).

FIG. 10: the method for measuring the content of ellagic acid in the method for detecting the spray for treating the tendon and bone injury uses a durability test chromatogram map of instruments of different brands (1: ellagic acid).

FIG. 11: in the tendon and bone injury spray detection method, a thin-layer chromatogram map screened by an epimedium thin-layer identification development system [ development agent: ethyl acetate-butanone-formic acid-water (10: 1: 1: 1), Qingdao H-plate ].

FIG. 12: tendon and bone injury spray detection epimedium thin layer identification development system screening thin layer chromatogram (development agent: chloroform-methanol-water (13.5:6:2), Qingdao G board ].

FIG. 13: in the tendon and bone injury spray detection method, a thin-layer chromatography chromatogram (chloroform-methanol-water (13.5:5.5:2) and Qingdao G plate) is screened by a herba epimedii thin-layer identification and development system.

FIG. 14: in the muscle and bone injury spray detection method, epimedium thin-layer identification and development system screening thin-layer chromatography is adopted [ chloroform-methanol-water (13.5:5:2) and Qingdao G plate ].

FIG. 15: thin-layer identification and verification thin-layer chromatogram (Qingdao high-efficiency G plate) of epimedium in the muscle and bone injury spray detection method.

FIG. 16: thin-layer identification and verification thin-layer chromatogram (Qingdao common prefabricated G plate) of epimedium in the muscle and bone injury spray detection method.

FIG. 17: thin-layer identification and verification thin-layer chromatogram (high-efficiency G plate) of herba Epimedii in the tendon and bone injury spray detection method.

FIG. 18: thin-layer chromatography (common prefabricated G plate of cigarette platform) for identifying and verifying herba Epimedii in the method for detecting tendon and bone injury spray.

FIG. 19 is a schematic view of: herba Epimedii identification thin layer chromatogram in the detection method of tendon and bone injury spray (common prefabricated G plate of cigarette platform, 13 batches of samples).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

Detection method of spray for treating muscle and bone injuries

[ CHEM ] Chi shank powder 100g Red peony root 30g epimedium 50g earthworm 10g

Prepared wild aconite root 2g menthol 20g

[ PREPARATION METHOD ] pulverizing the above six ingredients except Mentholum into coarse powder, mixing, placing in a container, adding 55% ethanol, sealing, soaking for 10 days, collecting the extractive solution, standing, and filtering. Dissolving menthol in ethanol, adding the dissolved menthol into the filtrate, adding ethanol to adjust the ethanol content to 45-55% to 1000ml, and uniformly mixing to obtain the menthol crystal.

[ IDENTIFICATION ]

Taking the tendon and bone injury spray solution as a test solution, taking 0.3G of epimedium control medicinal material, adding 8ml of ethanol, soaking for 25min, filtering, evaporating filtrate to dryness, dissolving residues with 1ml of ethanol, centrifuging, taking supernatant as a control medicinal material solution, taking a proper amount of icariin control, adding methanol to prepare a solution containing 0.5mg of icariin per 1ml, taking the solution as a control solution, performing thin-layer chromatography test, sucking 2-3 mu l of the three solutions respectively, respectively dropping the three solutions on a same silica gel G thin-layer plate, taking a lower layer solution of chloroform-methanol-water (13.5:5.5:2) as a developing agent, spreading, taking out, drying, spraying an aluminum trichloride test solution, airing, placing under a 365nm ultraviolet lamp for inspection, and displaying fluorescent spots with the same color in the chromatogram of the test solution of the tendon and bone injury at positions corresponding to the chromatograms of the control medicinal material and the control.

[ MEASUREMENT OF CONTENT ]

Herba Epimedii is determined by high performance liquid chromatography (general rule 0512).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (23:77) is used as a mobile phase; the detection wavelength is 270nm, and the number of theoretical plates is not less than 10000 calculated according to icariin;

preparing a test solution, uniformly mixing 5 bottles of solution of the tendon and bone injury spray, precisely measuring 10ml, drying by steaming in a water bath, ultrasonically dissolving residues in 20ml of water for several times, completely transferring to a separating funnel, adding ethyl acetate, extracting for 2 times, extracting for 15ml each time, combining ethyl acetate solutions, drying by steaming, and adding methanol into residues to reach a constant volume of 10ml to obtain the composition;

A spray for treating injury of bones and muscles contains icariin (C) and herba Epimedii in each 1ml₃₃H₄₀O₁₅) It should not be less than 70.0 μ g.

(II) determining menthol by gas chromatography (Tong Chao 0512)

Chromatographic condition and system applicability test with cross-linked polyethylene glycol as the capillary column of the stationary phase; the column temperature is 80 ℃; the temperature of a sample inlet is 180 ℃; the temperature of the detector is 220 ℃; split-flow sample introduction is carried out, the split-flow ratio is 20:1, and the number of theoretical plates is not lower than 15000 calculated according to the menthol crystal;

Spray containing Mentholum (C) for treating muscle and bone injury₁₀H₂₀O) should be 85.0% to 115.0% of the indicated amount.

(III) Chi shank Scattering high performance liquid chromatography determination (Tong Chao 0512)

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; methanol-0.5% phosphoric acid water solution (42:58) is taken as a mobile phase; the detection wavelength is 368nm, and the number of theoretical plates is not less than 4000 calculated according to ellagic acid;

The spray for treating bone injury contains ellagic acid (C) in the form of radix Rumicis Crispi 1ml₁₄H₆O₈) Calculated, not less than 0.35 mg;

example 2

Detection method of spray for treating muscle and bone injuries

[ CHEM ] Chi shank powder 100g Red peony root 30g epimedium 50g earthworm 10g

Prepared wild aconite root 2g menthol 20g

[ IDENTIFICATION ]

Taking the spraying agent solution for treating the muscle and bone injuries as a test solution, taking 0.3G of epimedium herb control medicinal material, adding 8ml of ethanol, soaking for 25min, filtering, evaporating filtrate to dryness, adding 1ml of ethanol to dissolve residues, centrifuging, taking supernatant as a control medicinal material solution, taking a proper amount of icariin control material solution, adding methanol to prepare a solution containing 0.5mg of icariin per 1ml, taking the solution as a control solution, performing a thin-layer chromatography test, sucking 2-3 mu l of each of the three solutions, respectively dropping the three solutions on a same silica gel G thin-layer plate, taking a chloroform-methanol-water (13.5:5.5:2) lower-layer solution as a developing agent, developing, taking out, airing, spraying an aluminum trichloride test solution, airing, placing under a 365nm ultraviolet lamp for inspection, and displaying fluorescent spots with the same color in positions corresponding to the control medicinal material and the control material in a sample chromatogram.

[ MEASUREMENT OF CONTENT ]

preparing a test solution, uniformly mixing 5 bottles of solution of the tendon and bone injury spray, precisely measuring 10ml, drying by steaming in a water bath, ultrasonically dissolving residues in 20ml of water for several times, completely transferring to a separating funnel, adding ethyl acetate, extracting for 4 times, extracting for 25ml each time, combining ethyl acetate solutions, drying by steaming, and adding methanol into residues to reach a constant volume of 10ml to obtain the composition;

(II) determining menthol by gas chromatography (Tong Chao 0512)

Chromatographic conditions and system applicability tests of the capillary column with cross-linked bonded polyethylene glycol as a stationary phase; the column temperature is 120 ℃; the injection port temperature is 220 ℃; the temperature of the detector is 240 ℃; split-flow sample introduction is carried out, the split-flow ratio is 20:1, and the number of theoretical plates is not lower than 15000 calculated according to the menthol crystal;

The spray for treating bone injury contains ellagic acid (C) in the form of radix Rumicis Crispi 1ml₁₄H₆O₈) Calculated, the content of the active ingredient should not be less than 0.35 mg.

Example 3

Detection method of spray for treating muscle and bone injuries

[ CHEM ] Chi shank powder 100g Red peony root 30g epimedium 50g earthworm 10g

Prepared wild aconite root 2g menthol 20g

[ IDENTIFICATION ] the spray solution for treating tendon and bone injury is used as test solution, herba Epimedii control 0.5g is added with 10ml ethanol, warm-soaked for 30min, filtered, the filtrate is evaporated to dryness, the residue is dissolved with 1ml ethanol, centrifuged, and the supernatant is used as control solution. Taking appropriate amount of icariin as control, adding methanol to obtain solution containing 0.5mg per 1ml as control solution. Performing thin layer chromatography (general 0502) test, sucking 2-3 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13.5:5.5:2) lower layer solution as developing agent, taking out, air drying, spraying with aluminum trichloride solution, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference solution.

[ MEASUREMENT OF CONTENT ]

(1) Herba Epimedii is determined by high performance liquid chromatography (general rule 0512).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (23:77) is used as a mobile phase; the detection wavelength was 270 nm. The number of theoretical plates is not less than 10000 calculated by icariin.

Preparation of control solution icariin control, precisely weighing, adding methanol to obtain solution containing 0.1mg per 1ml, and shaking.

Preparation of test solution the muscle and bone injury spray is prepared by mixing 5 bottles of solution, precisely measuring 10ml, drying by evaporation in a water bath, ultrasonically dissolving residues with 20ml of water by several times, transferring all the residues to a separating funnel, adding ethyl acetate for extraction for 3 times, extracting 20ml each time, combining ethyl acetate solutions, drying by evaporation, and adding methanol into residues to a constant volume of 10 ml.

(2) Menthol was measured by gas chromatography (general rule 0521).

Chromatographic conditions and system applicability tests are carried out on a capillary column taking cross-linked bonded polyethylene glycol as a stationary phase; the column temperature is 100 ℃; the temperature of a sample inlet is 200 ℃; the temperature of the detector is 230 ℃; split-flow sample injection with a split-flow ratio of 20: 1. The number of theoretical plates should not be less than 15000 calculated as menthol.

Preparation of reference solution A proper amount of Mentholum reference is precisely weighed, and anhydrous ethanol is added to make reference solution containing 0.05mg of Mentholum per 1 ml.

Preparation of test solution A precise amount of spray for treating injury of muscle and bone (50 μ L) is prepared by placing into 25ml measuring flask, adding anhydrous ethanol to scale, and shaking.

(3) The shin powder is measured by high performance liquid chromatography (general rule 0512).

Chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; methanol-0.5% phosphoric acid water solution (42:58) is taken as a mobile phase; the detection wavelength was 368 nm. The number of theoretical plates should not be less than 4000, calculated as ellagic acid.

Preparation of control solution ellagic acid control is precisely weighed, added with methanol to obtain solution containing 0.5mg per 1ml, and shaken to obtain the final product.

Preparation of test solution the muscle and bone injury spray is prepared by mixing 5 bottles of solution, taking appropriate amount of mixed solution and filtering.

The spray for treating bone injury contains ellagic acid (C) in 1ml₁₄H₆O₈) Calculated, the content of the active ingredient should not be less than 0.35 mg.

Example 4

Detection method of spray for treating muscle and bone injuries

[ CHEM ] Chi shank powder 100g Red peony root 30g epimedium 50g earthworm 10g

Prepared wild aconite root 2g menthol 20g

[ IDENTIFICATION ] the spray solution for treating tendon and bone injury is used as test solution, herba Epimedii control 0.5g is added with 10ml ethanol, warm-soaked for 30min, filtered, the filtrate is evaporated to dryness, the residue is dissolved with 1ml ethanol, centrifuged, and the supernatant is used as control solution. Taking appropriate amount of icariin as control, adding methanol to obtain solution containing 0.5mg per 1ml as control solution. Performing thin layer chromatography (general rule 0502) test, sucking 2-3 μ l of the above three solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13.5:5:2) lower layer solution as developing agent, taking out, air drying, spraying with aluminum trichloride test solution, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference solution.

[ MEASUREMENT OF CONTENT ]

Preparation of control solution icariin control was precisely weighed, added with methanol to make a solution containing 0.1mg per 1ml, and shaken well to obtain.

(2) Menthol was measured by gas chromatography (general rule 0521).

Preparation of control solution ellagic acid control was precisely weighed, added with methanol to make into solution containing 0.5mg per 1ml, and shaken well to obtain.

Example 5

Detection method of spray for treating muscle and bone injuries

[ CHEM ] Chi shank powder 100g Red peony root 30g epimedium 50g earthworm 10g

Prepared wild aconite root 2g menthol 20g

[ IDENTIFICATION ] the spray solution for treating tendon and bone injury is used as test solution, herba Epimedii control 0.5g is added with 10ml ethanol, warm-soaked for 30min, filtered, the filtrate is evaporated to dryness, the residue is dissolved with 1ml ethanol, centrifuged, and the supernatant is used as control solution. Taking appropriate amount of icariin as control, adding methanol to obtain solution containing 0.5mg per 1ml as control solution. Performing thin layer chromatography (general rule 0502) test, sucking 2-3 μ l of the above three solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13.5:6:2) lower layer solution as developing agent, taking out, air drying, spraying with aluminum trichloride test solution, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference solution.

[ MEASUREMENT OF CONTENT ]

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (23:77) is used as a mobile phase; the detection wavelength was 270 nm. The number of theoretical plates should be not less than 10000 calculated based on icariin.

Preparation of test solution the muscle and bone injury spray is prepared by mixing 5 bottles of solution, precisely weighing 10ml, drying by distillation in water bath, ultrasonically dissolving residue with 20ml water for several times, transferring all the solution into a separating funnel, extracting with ethyl acetate for 3 times, each time 20ml, combining ethyl acetate solutions, drying by distillation, and adding methanol into residue to constant volume of 10 ml.

(2) Menthol was measured by gas chromatography (general rule 0521).

(3) The shin-sha powder is measured by high performance liquid chromatography (general rule 0512).

Example 6

Detection method of spray for treating muscle and bone injuries

[ CHEM ] Chi shank powder 100g Red peony root 30g epimedium 50g earthworm 10g

Prepared wild aconite root 2g menthol 20g

[ IDENTIFICATION ] the spray solution for treating tendon and bone injury is used as test solution, herba Epimedii control 0.5g is added with 10ml ethanol, warm-soaked for 30min, filtered, the filtrate is evaporated to dryness, the residue is dissolved with 1ml ethanol, centrifuged, and the supernatant is used as control solution. Taking appropriate amount of icariin as control, adding methanol to obtain solution containing 0.5mg per 1ml as control solution. Performing thin layer chromatography (general rule 0502) test, sucking 2-3 μ l of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-butanone-formic acid-water (10: 1: 1: 1) as developing agent, taking out, air drying, spraying with aluminum trichloride test solution, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference solution.

[ MEASUREMENT OF CONTENT ]

(2) Menthol was measured by gas chromatography (general rule 0521).

To further verify the feasibility of the assay, the inventors performed a series of experiments, as follows:

test example 1 HPLC content determination method test of icariin in product

1 Instrument and reagent

1.1 instruments

Waters 2996 liquid chromatograph, THERMO DIONEX UltMate 3000 high performance liquid chromatograph, Agilengt Technologies 1260 high performance liquid chromatograph. A chromatographic column: phenomenex Luna C₁₈(2)(250mm×4.6mm,5μm)、Welch Topsil C₁₈(250 mm. times.4.6 mm,5 μm), Agela Technologies Durashell RP (250 mm. times.4.6 mm,5 μm). Model MX5 parts per million electronic balance (Mettler Toledo).

1.2 reagent

Icariin control (batch number, purity ≧ 94.2%) was purchased from the pilot hospital. 13 batches of tendon and bone injury spray samples and 1 batch of barren epimedium herb negative samples are provided by Guizhou remote pharmaceutical Limited liability company.

Acetonitrile is chromatographically pure; the water is purified water; methanol was analytically pure.

2 chromatographic conditions

Adopting Phenomenex Luna C₁₈(2) Chromatography column (250mm × 4.6mm,5 μm), mobile phase: acetonitrile-water (23: 77); flow rate 1.0ml.min^-1Column temperature 30 ℃, detection wavelength: 270 nm.

3 preparation of the solution

3.1 control solutions

Precisely weighing icariin control 5.253mg, and adding methanol to obtain control solution of 0.1051 mg/ml. 3.2 test article solution

Precisely measuring 10ml of sample solution, evaporating to dryness in water bath, adding 20ml of water, dissolving with ultrasound, extracting with ethyl acetate for 3 times (20 ml each time), mixing ethyl acetate solutions, evaporating to dryness, and adding methanol to desired volume of 10 ml. (interference exists in direct sample injection or dilution sample injection of the muscle and bone injury spray, and the interference can be removed by adopting liquid-liquid extraction.)

3.3 negative solutions

Precisely measuring 10ml of negative sample solution lacking herba Epimedii, and preparing with the method under item 3.2.

4 methodological investigation

4.1 precision test

The control solution under the term of "3.1" was precisely aspirated, and the measurement was performed 6 times with 10. mu.l per injection, and the result showed that the peak area RSD (n ═ 6) was 1.2%, indicating that the instrument precision was good, as shown in Table 1.

Table 1 precision test results (n ═ 6)

4.2 Linear relationship test

Precisely sucking 3.1 items of

control solution solutions

3, 5, 10, 15 and 20 μ l, injecting into a liquid chromatograph, measuring peak area, taking sample amount M (μ g) as abscissa and peak area A as ordinate, and calculating regression equation to obtain y of 2223.1x +3.1278 and r of 0.9997(n of 5); the results show that icariin is in good linear relation in the range of 0.2969-1.9793 mug, as shown in table 2 and figure 1.

Table 2 results of the linear relationship test (n ═ 5)

4.3 specificity test

And respectively injecting 10 μ l of the control solution, the test solution and the negative sample (lacking herba Epimedii) solution into a liquid chromatograph, wherein the result shows that no chromatographic peak is observed in the herba Epimedii negative sample at the corresponding retention time of icariin, which indicates that the negative sample has no interference to detection, and is shown in figure 2.

4.4 stability test

The sample solution (lot No. 20170602) was precisely aspirated, 10 μ l was injected at 0h, 3h, 6h, 9h, 12h, 15h, 18h, and 24h, respectively, peak areas were recorded, and RSD (n ═ 8) was calculated to be 1.55%, indicating that the sample solution was stable within 24 h. See table 3.

Table 3 stability test results (n ═ 8)

4.5 repeatability test

6 parts of the same sample batch (batch No. 20170602) are taken, 10ml of each sample batch is prepared in parallel 6 parts according to the preparation method of the test solution, 10 mul of sample injection is carried out, the content is measured, and the result shows that the content RSD (n is 6) is 2.83 percent, which indicates that the repeatability is good. See table 4.

Table 4 results of repetitive tests (n ═ 6)

4.6 accuracy test (sample recovery test)

4.6.1 preparation of control solutions

Accurately weighing appropriate amount of icariin, adding into 10ml measuring flask, adding methanol to scale mark, and making into 0.5253mg/ml control solution.

4.6.2 sample recovery test

6 portions of a sample (batch number: 20170602) with a known content are weighed, 1ml of icariin reference substance (purity 94.2%) solution with the concentration of 0.5253mg/ml is precisely added, the sample is prepared according to the method, 10 mul of the icariin reference substance is injected, and the recovery rate is calculated and calculated, so that the average recovery rate is 99.80% and the RSD (n-6) is 2.03%. See table 5.

Table 5 recovery test results (n ═ 6)

4.7 durability test

4.7.1 three different brands C₁₈Column

①Phenomenex Luna C₁₈(2)(250mm×4.6mm,5μm)；

②Welch Topsil C₁₈(250mm×4.6mm,5μm)；

③Agela Technologies Durashell RP(250mm×4.6mm,5μm)。

A sample (lot No. 20170602) was taken and 1 part of the sample solution was prepared by the test solution preparation method, and the content of epimedium was 1.21% in RSD (n-3) and 94.0. mu.g/ml in average, as measured on the same Waters 2996 liquid chromatograph using the above 3 kinds of chromatographic columns of different brands. See table 6, fig. 3.

Table 6 results of durability (different columns) (n ═ 3)

4.7.2 three chromatographs of different brands

Firstly, a THERMO DIONEX UltMate 3000 high performance liquid chromatograph;

agilength Technologies 1260 high performance liquid chromatograph;

③ Waters 2996 high performance liquid chromatograph.

A sample (batch No. 20170704) was taken and 1 part was prepared by the method for preparing a test solution using Phenomenex Luna C₁₈The column chromatography was performed by 3 different brands of HPLC, and the content of epimedium herb RSD (n-3) was 1.27%, and the average content was 98.3. mu.g/ml. See table 7, fig. 4.

Table 7 durability (different instruments) test results (n ═ 3)

5 sample content determination

Precisely weighing icariin reference substances 1.998 and 2.025mg, respectively placing into 20ml volumetric flasks, and adding methanol to obtain reference substance solutions with concentrations of 0.0999 and 0.1013mg/ml, respectively.

13 samples were taken and prepared in the same manner as the test solution preparation method, and the content was calculated by the external standard one-point method, and the results are shown in Table 8.

Results of icariin assay in samples of Table 8 (n ═ 13)

The content limit of icariin is determined, and the icariin content is 29.0-151.7 mu g/ml, the average content is 88.7 mu g/ml, and the RSD is 41.3% (n is 13). The prescription amount of the epimedium is 50mg/ml, the epimedium content in the epimedium is not lower than 0.50% according to the prescription, so the average theoretical transfer rate of the icariin in the muscle and bone injury spray is 88.7/(0.5% 50) 35.5%, the difference of 13 batches of data of the icariin content in the muscle and bone injury spray is large, the lower limit is temporarily set to 70.0 mu g/ml according to 80% of the average value, and 2 batches of 13 samples do not accord with the prescription according to the limit.

6 system adaptability requirement

The minimum theoretical plate number of the icariin peak on different instruments and chromatographic columns under the content determination item is counted, see table 9, and the result shows that the theoretical plate number of the icariin peak is 13000 at the minimum, so the minimum theoretical plate number in the detection method is calculated by the icariin peak, and the formulation is not less than 10000. See table 9.

TABLE 9 minimum theoretical number of icariin peaks on different instruments

Test example 2: newly added menthol GC content determination method test

1 Instrument and reagent

1.1 Instrument

Thermo fisher DSQ gas chromatograph, agilent 6890N gas chromatograph, shimadzu 2010Plus gas chromatograph, HP-INNOWAX capillary column (30m × 0.25mm, 0.25 μm), MX5 type millionth electronic balance (Mettler Toledo).

1.2 reagent

Menthol control (batch No. 110728-200506) was purchased from the institute of food and drug testing, China; 13 samples of tendon and bone injury sprays and 1 sample of lacking menthol negative were provided by Guizhou remote pharmaceutical Limited liability company.

Absolute ethanol was analytically pure.

2 chromatographic conditions

HP-INNOWAX capillary column (30 m.times.0.25 mm, 0.25 μm). The temperature of a sample inlet is 200 ℃; FID detector temperature 230 ℃; column temperature: 100 ℃; the carrier gas is helium; the flow rate is 1.0 ml/min; split-flow sample injection with a split-flow ratio of 20: 1; the amount of sample was 1. mu.l.

3 preparation of the solution

3.1 control solutions

Taking a proper amount of menthol control, precisely weighing, and adding absolute ethanol to prepare a control solution containing 0.05mg of menthol per 1 ml.

3.2 test article solution

Precisely weighing 50uL of the tendon and bone injury spray, placing into a 25ml volumetric flask, adding absolute ethyl alcohol to the scale, and shaking up to obtain the product.

3.3 negative solutions

Precisely measuring 50uL of a menthol negative sample, putting the menthol negative sample into a 25mL measuring flask, adding absolute ethyl alcohol to the scale, and shaking up to obtain the menthol negative sample.

4 methodological investigation

4.1 precision test

The control solution was injected 6 times and the peak area of menthol was recorded, the results are shown in table 10, the RSD of the peak area was 2.30% (n ═ 6), indicating good instrument precision.

TABLE 10 precision test results (n ═ 6)

4.2 specificity test

Precisely sucking the reference solution, the test solution and the menthol negative solution by 1 μ l respectively, injecting into a gas chromatograph, measuring, and recording chromatogram, wherein as shown in FIG. 5, the result negative solution does not detect chromatographic peak consistent with the retention time of the reference, and the negative is proved to be non-interfering.

4.3 stability test

The sample solution (batch No. 20170602) was sampled and measured for 0h, 4h, 8h, 12h, 16h, 20h, and 24h, the peak area was recorded, and RSD was calculated, and the results are shown in table 11, where the RSD of the peak area of menthol was 3.77% (n ═ 7), indicating that the sample solution had good stability within 24 h.

Table 11 stability test results (n ═ 7)

4.4 Linear relationship test

Accurately weighing a proper amount of menthol control, accurately weighing, adding absolute ethyl alcohol to prepare control solution containing 0.2435mg of menthol per 1ml, diluting the control solution into a series of control solutions with different concentrations, injecting samples, recording peak areas, drawing a standard curve by taking the peak area value (A) as a vertical coordinate and the concentration (C mg/ml) as a horizontal coordinate, and showing that the linear relation is good as shown in figure 6, wherein the linear equation is y-85795380.690 x +86003.117, and r-0.9995 (n-7).

4.5 repeatability test

The same batch of samples (batch No. 20170602) was sampled to prepare 6 test solutions according to the method, and the contents were measured by sampling and calculated by the external standard one-point method and expressed as the labeled amount (20mg/ml), and the results are shown in Table 12, where the average content of menthol is 100.63% and RSD is 3.25% (n is 6), indicating that the method has good reproducibility.

Table 12 repeatability test results (n ═ 6)

4.6 sample recovery test

6 parts of the known content of the muscle and bone injury spray (batch No. 20170602) are precisely weighed, 25ul of the spray is placed into a 25ml volumetric flask, 1ml of menthol control solution with the concentration of 0.5196mg/ml is precisely added, the preparation is carried out according to the method, and the results are shown in Table 13, wherein the average recovery rate of the menthol is 100.42 percent and the RSD is 2.41 percent (n is 6).

Table 13 sample recovery test results (n ═ 6)

4.7 durability test

The same sample solution (lot No. 201710602) was taken and measured for content on an Aglent 6890N gas chromatograph, a Thermo fisher DSQ mass spectrometer (switching to FID detector), and shimadzu GC-2010Plus, respectively, and the results are shown in table 14, where RSD of menthol content was 2.02% (N ═ 3), indicating that the instrument durability of the method was good.

Table 14 durability test results (n ═ 3)

5 sample content determination

Taking the above samples, preparing a test solution according to the method, precisely sucking 1 μ l, injecting into a gas chromatograph, measuring, recording peak area, calculating content by external standard one-point method, and expressing with menthol labeling amount (20mg/ml), and the result is shown in Table 15.

TABLE 15 measurement results of sample content

The content of the menthol in 13 samples is 88.7-114.1% by content limit, and the menthol in the muscle and bone injury spray is directly added after being dissolved, so the limit range is 85.0-115.0% of the marked amount. All 13 samples were within the limits specified.

6 system adaptability requirement

The minimum theoretical plate number of the menthol peak on different instruments under the item of content measurement is counted, see table 16, and the result shows that the minimum theoretical plate number of the menthol is 17214, so the minimum theoretical plate number in the detection method is calculated by the menthol peak, and the set value cannot be lower than 15000.

TABLE 16 minimum theoretical number of menthol peaks on different instruments

Test example 3 HPLC content determination method of ellagic acid in newly added Rutosporus trifoliatus

1 Instrument and reagent

1.1 instruments

Waters 2996 liquid chromatograph, THERMO DIONEX UltMate 3000 high performance liquid chromatograph, Agilengt Technologies 1260 high performance liquid chromatograph. And (3) chromatographic column: zishengtang AQ (250mm × 4.6mm,5 μm), Phenomenex Luna C₁₈(2)(250mm×4.6mm,5μm)、Welch Topsil C₁₈(250 mm. times.4.6 mm,5 μm). Model MX5 parts per million electronic balance (Mettler Toledo).

Model KH-600DE ultrasonic device (grahamia kuntze).

1.2 reagent

Ellagic acid control (batch number, purity ≧ 89.2%) was purchased from the pilot hospital. 13 samples of sinew bone injury sprays and 1 sample of absent-red shank negative samples were provided by Guizhou remote pharmaceutical Limited liability company.

The methanol is chromatographically pure; phosphoric acid is chromatographically pure; the water is purified water.

2 chromatographic conditions

Using a senescon AQ chromatography column (250mm × 4.6mm,5 μm), mobile phase: methanol-0.5% phosphoric acid aqueous solution (42: 58); flow rate 1.0ml.min^-1Column temperature 30 ℃, detection wavelength: 368 nm.

3 preparation of the solution

3.1 control solutions

Precisely weighing ellagic acid control substances 4.905mg and 5.075mg, and adding methanol to obtain control substance solutions 0.4905mg/ml and 0.5057mg/ml respectively.

3.2 test article solution

And (4) taking a proper amount of sample solution, and filtering to obtain the product.

3.3 negative solutions

And (4) taking a proper amount of the negative sample solution lacking the Ruditapes indica and filtering to obtain the composition.

4 methodological investigation

4.1 precision test

The control solution under the term of "3.1" was precisely aspirated, and the measurement was performed 6 times with 10. mu.l per injection, and the result showed that the peak area RSD (n ═ 6) was 0.80%, indicating that the instrument precision was good.

Table 17 precision test results (n ═ 6)

4.2 Linear relationship test

Precisely sucking 1, 3, 5, 7, 10 and 15 mu l of reference solution under item 3.1, injecting into a liquid chromatograph, measuring peak area, taking sample amount M (mu g) as abscissa and peak area A as ordinate, and calculating regression equation to obtain y which is 1.9309 x-22.769; the result shows that the ellagic acid has a good linear relation in the range of 0.4905-7.3575 mu g. See table 18, fig. 7.

Table 18 results of linear relationship test (n ═ 6)

4.3 specificity test

And (3) respectively injecting 10 mu l of the reference substance solution, the test sample solution and the negative sample (lacking ellagic acid) into a liquid chromatograph, wherein the result shows that the negative sample of the Rumex lepigone does not have a chromatographic peak at the corresponding retention time of the ellagic acid, which indicates that the negative sample does not interfere with the detection. See fig. 8.

4.4 stability test

The sample solution (batch No. 20170602) was aspirated, 10. mu.l was injected at 0h, 4h, 6h, 14h, 26h, 32h, and 48h, respectively, peak areas were recorded, and RSD (n ═ 7) was calculated to be 1.81%, indicating that the sample solution was stable within 48 h. See table 19.

Table 19 stability test results (n ═ 7)

4.5 repeatability test

6 portions of the same batch sample (batch number: 20170602) are taken, 6 portions are prepared in parallel according to the preparation method of the test solution, 10 mul is injected, the content is measured, and the result that the content RSD (n is 6) is 0.0 percent shows that the repeatability is good. See table 20.

Table 20 repeatability test results (n ═ 6)

4.6 accuracy test (sample recovery test)

4.6.1 preparation of control solutions

Precisely weighing appropriate amount of ellagic acid, adding into 10ml measuring flask, adding methanol to scale, and making into 0.4905mg/ml control solution.

4.6.2 sample recovery test

6 parts of a sample (batch number: 20170602) with a known content is weighed, 10ml of each part is accurately added with 10ml of ellagic acid control (purity 89.2%) solution with the concentration of 0.4905mg/ml, the mixture is mixed evenly, 10 mul of sample injection is carried out, the recovery rate is calculated, the average recovery rate is 106.70%, and the RSD (n-6) is 0.87%. See table 21.

Table 21 recovery test results (n ═ 6)

4.7 durability test

4.7.1 three different brands C₁₈Column

Sennanth AQ (250mm × 4.6mm,5 μm);

②Welch Topsil C₁₈(250mm×4.6mm,5μm)；

③Phenomenex Luna C₁₈(2)Agela Technologies Durashell RP(250mm×4.6mm,5μm)。

a sample (lot No. 20170602) was taken and 1 part was prepared according to the test sample solution preparation method, and the content of Rutosphaea was 2.00% in RSD (n ═ 3) and 0.50mg/ml as measured on the same Agilengt Technologies 1260 liquid chromatograph using the above-mentioned 3 kinds of columns of different brands. See table 22, fig. 9.

TABLE 22 durability (different columns) test results (n ═ 3)

4.7.2 three chromatographs of different brands

Firstly, a THERMO DIONEX UltMate 3000 high performance liquid chromatograph;

agilength Technologies 1260 high performance liquid chromatograph;

③ Waters 2996 high performance liquid chromatograph.

A sample (lot No. 20170704) was taken and 1 part was prepared according to the test sample solution preparation method, and measured by using shiitang AQ column chromatography on 3 different brands of high performance liquid chromatographs, as a result, RSD (n ═ 3) of the content of the blackleg was 2.29%, and the average content was 0.50 mg/ml. See table 23, fig. 10.

Table 23 durability (different instruments) test results (n ═ 3)

5 sample content determination

Precisely weighing ellagic acid reference substances 4.905 and 5.057mg, placing into 10ml volumetric flasks, respectively, and adding methanol to obtain reference substance solutions with concentrations of 0.4905 and 0.5057mg/ml, respectively.

13 samples were taken and prepared in the same manner as the test solution preparation method, and the content was calculated by the external standard one-point method, and the results are shown in Table 24.

The content limit defines the ellagic acid content result as 0.42-1.03 ml, the average is 0.64mg/ml, and the RSD is 25.17% (n is 13). Position of Chi shankThe formula amount is 0.1g/ml, and the content (calculated by dry product) of the ellagic acid in the ranunculus japonicus medicinal material (corresponding to the preparation batch 20170707) provided by the factory is 6.5mg/g, so the average actual transfer rate of the ellagic acid in the tendon and bone injury spray is 0.38/(6.5 × 0.1) 58.4%. The limit of the ellagic acid content in the medicinal material of the Rupitis lepigone is temporarily fixed to be 0.6 percent, and the limit of the ellagic acid content of the spray for treating the muscle and bone injuries is 6.0mg/g 58.4 percent and 0.1g/ml is 0.35 mg/ml. The tested results show that every 1ml of the spray for treating temporary bone and muscle injuries contains the erythro leg powder and the ellagic acid (C)₁₄H₆O₈) Calculated, the content of the active ingredient should not be less than 0.35 mg. See tables 24, 25.

Table 24 measurement of ellagic acid in the sample (n ═ 13)

TABLE 25 determination of ellagic acid content in Rugosa Gilg medicinal materials

6 System Adaptation requirements

The lowest theoretical plate number of the ellagic acid peak on different instruments and chromatographic columns under the item of statistical content measurement is shown in table 26, and the result shows that the lowest theoretical plate number of the ellagic acid is 4703, so that the lowest theoretical plate number in the detection method is calculated by using the ellagic acid peak, and the formulation is not less than 4000. See table 26.

TABLE 26 lowest theoretical plate number of ellagic acid peaks on different instruments

Experimental example 4 verifies the identification method of the newly added epimedium herb thin layer.

The expansion system is optimized and screened, the screened expansion system is verified, a standard method is formulated, and 13 batches of samples are detected according to the method.

The icariin reference substance, the physique injury spraying agent sample and the barren epimedium negative sample information, and under the draft instruction item for measuring the content of physique injury spraying agent epimedium, the reagents are all analytically pure.

1 optimization and screening of unfolding systems

The developing agent (ethyl acetate-butanone-formic acid-water (10: 1: 1: 1) (Qingdao H plate) is shown in figure 11.

Developing agent chloroform-methanol-water (13.5:6:2) (Qingdao G plate), and the development figure is shown in figure 12.

Developing agent (chloroform-methanol-water (13.5:5.5:2) (Qingdao G plate), and the development is shown in figure 13.

Developing agent (chloroform-methanol-water (13.5:5:2) (Qingdao G plate), and the development is shown in FIG. 14.

The chromatogram effect shows that the developing agent has the best effect, and finally the developing agent is determined to be: chloroform-methanol-water (13.5:5.5:2) lower layer solution, see FIG. 13. The second is developing agent I, developer II and developer IV.

2 verification of silicon thin-layer plates of different manufacturers

The chloroform-methanol-water (13.5:5.5:2) lower layer solution is used as a developing agent for development, and a Qingdao high-efficiency G plate, a Qingdao common prefabricated G plate, a cigarette bench high-efficiency G plate and a cigarette bench common prefabricated G plate are used for verification, the results are shown in figures 15-18, and the reproducibility of the chromatographic effect is good.

3 sample detection

And (4) inspecting according to a detection method, and detecting epimedium control medicinal materials and icariin in 13 batches of samples. See fig. 19.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A detection method for spray for muscle and bone injury, the spray for muscle and bone injury is made up of radix Paeoniae Rubra 100g, radix Paeoniae Rubra 30g, herba Epimedii 50g, earthworm 10g, radix Aconiti Kusnezoffii Preparata 2g, menthol 20g, the above six medicines, except menthol, pulverize to coarse powder, mix, put into container, add 55% ethanol, seal, soak for 10 days, take the leach solution, left standstill, filter, menthol adds ethanol to dissolve, add to filtrate, add ethanol to adjust the alcoholic content to 45% -55% to 1000ml, mix, get final product; the method is characterized in that: the detection method comprises the following steps: qualitatively identifying icariin in the product, measuring icariin content in the product by high performance liquid chromatography, measuring menthol content in the product by gas chromatography, and measuring ellagic acid content in the product by high performance liquid chromatography;

the method for qualitatively identifying icariin comprises the following steps: taking the tendon and bone injury spray solution as a test solution, taking 0.5G of epimedium herb control medicinal material, adding 10ml of ethanol, soaking for 30min, filtering, evaporating filtrate to dryness, dissolving residues with 1ml of ethanol, centrifuging, taking supernatant as a control medicinal material solution, taking a proper amount of icariin control, adding methanol to prepare a solution containing 0.5mg of icariin per 1ml, taking the solution as a control solution, performing a thin-layer chromatography test, sucking 2-3 mu l of each of the three solutions, respectively dropping the three solutions on a same silica gel G thin-layer plate, taking a chloroform-methanol-water lower-layer solution with a ratio of 13.5:5.5:2 as a developing agent, spreading, taking out, drying, spraying an aluminum trichloride test solution, drying, and placing under a 365nm ultraviolet lamp for inspection, wherein in a sample chromatogram, fluorescent spots with the same color are displayed at positions corresponding to the control medicinal material and the control substance chromatogram;

the content of icariin in the product is measured by high performance liquid chromatography, and the specific method comprises the following steps:

The chromatographic conditions and the system applicability test use octadecylsilane chemically bonded silica as a filler, and the specification of a chromatographic column is as follows: 250mm × 4.6mm,5 μm; acetonitrile-water in a ratio of 23:77 is taken as a mobile phase; the flow rate is 1ml/min, and the column temperature is 30 ℃; the detection wavelength is 270nm, and the number of theoretical plates is not less than 10000 calculated according to icariin;

the determination method comprises precisely sucking 5 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;

the method for measuring the content of menthol in the product by using the gas chromatography comprises the following specific steps:

determining menthol by gas chromatography

Chromatographic conditions and system applicability tests of the capillary column with cross-linked bonded polyethylene glycol as a stationary phase; the specification of the capillary column is 30m multiplied by 0.25mm and 0.25 mu m, and the injection port temperature is 200 ℃; FID detector temperature 230 ℃; the column temperature is 100 ℃; the carrier gas is helium; the flow rate is 1.0 ml/min; split-flow sample injection is carried out, and the split-flow ratio is 20: 1; the sample amount is 1 μ l, and the number of theoretical plates is not less than 15000 calculated according to menthol;

the determination method comprises precisely sucking 1 μ l of reference solution and test solution respectively, injecting into liquid chromatograph, and determining;

the method for determining ellagic acid content in the product by high performance liquid chromatography comprises the following steps:

the method for measuring the red shank by high performance liquid chromatography comprises the following steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; the specification of a chromatographic column is 250mm multiplied by 4.6mm,5 mu m, and a methanol-0.5 percent phosphoric acid aqueous solution with the proportion of 42:58 is taken as a mobile phase; flow rate 1.0ml/min, column temperature 30 ℃, detection wavelength: 368nm, and the number of theoretical plates is not less than 4000 calculated according to ellagic acid;

2. The method for detecting a bone injury spray according to claim 1, wherein the method comprises the following steps: a spray for treating injury of bones and muscles contains epimedium and icariin C in each 1ml₃₃H₄₀O₁₅Measured, not less than 70.0 mug; the spray for treating bone injury contains ellagic acid C in the form of radix Rumicis Crispi in 1ml₁₄H₆O₈Calculated, the content of the active ingredient should not be less than 0.35 mg.

3. The method for detecting a tendon and bone injury spray as claimed in claim 1, wherein the method comprises the steps of: spray containing Mentholum C for treating muscle and bone injury₁₀H₂₀O should be 85.0% to 115.0% of the labeled amount.