CN109765320B - Content determination method for tendon and bone injury spraying agent - Google Patents

Content determination method for tendon and bone injury spraying agent Download PDF

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CN109765320B
CN109765320B CN201910183127.4A CN201910183127A CN109765320B CN 109765320 B CN109765320 B CN 109765320B CN 201910183127 A CN201910183127 A CN 201910183127A CN 109765320 B CN109765320 B CN 109765320B
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epimedin
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methanol
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dissolving
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CN109765320A (en
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伍庆
程吉祥
刘青
唐勇
薛静
罗天军
伍朝平
王娇
李佳蔚
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Guizhou Remote Pharmaceutical Co ltd
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Abstract

The invention relates to a content determination method for a tendon and bone injury spray, in particular to a content determination method for 7 components such as active ingredients paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C, baohuoside I and the like in the tendon and bone injury spray.

Description

Content determination method for tendon and bone injury spraying agent
Technical Field
The invention relates to the field of medicine invention, in particular to a content determination method for a tendon and bone injury spraying agent.
Background
The muscle and bone injury spray is a traditional Chinese medicine compound preparation prepared by extracting red shin powder, red peony root, epimedium, earthworm, prepared kusnezoff monkshood root and menthol, has the effects of activating blood circulation to dissipate blood stasis and relieving swelling and pain, is used for treating soft tissue injury, and comprises the following effective components: paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C, baohuoside I and the like, but the key active ingredients in the muscle and bone injury spray are not a scientific detection method, so that the comprehensive quality detection is realized.
The quantitative index in the existing quality standard of the tendon and bone injury spray is paeoniflorin in red paeony root, the dosage of the red paeony root in a prescription is small, and the quality standard of the Chi-shank powder is only identified in a thin-layer chromatography mode, so that the standard is too simple and the quality of a product is difficult to realize effective control.
The existing literature is allowed to be ultrafinely developed, the quality research and quality standard of a Miao medicine ' tendon and bone injury spray ' are improved [ D ], Guiyang medical college, 2015, and a method for simultaneously measuring 4 components of 3,3' -dimethyl ellagic acid, 3',4' -trimethyl ellagic acid, paeoniflorin and icariin in the tendon and bone injury spray by using HPLC is disclosed, the method simultaneously measures the content of 4 components in a traditional Chinese medicine preparation, but the components influencing the quality of tendon and bone injury are many, only 4 components in the 4 components can not be effectively measured to detect the effective components in the product, wherein the methyl ellagic acid accounts for 2, experiments show that the effective components in epimedium are more, only the icariin is measured to obviously not effectively detect the effective components in the epimedium, the method can not scientifically measure the content of the effective components in the tendon and bone injury spray, and bone injury spray can be repeatedly measured by using different reference substances, the operation is complex and the efficiency is not high.
Therefore, in order to better and more accurately measure the content of the effective components in the muscle and bone injury spraying agent and improve and control the quality of the muscle and bone injury spraying agent, the invention provides the method which is convenient to operate, strong in specificity, good in separation degree, high in stability, high in efficiency and capable of simultaneously measuring the content of 7 effective components in the muscle and bone injury spraying agent.
Disclosure of Invention
Aiming at the existing problems, the invention aims to provide a method for simultaneously measuring the contents of active ingredients, namely paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in a muscle and bone injury spray and applying the method to the study of the stability investigation of the muscle and bone injury spray, and the method is convenient to operate, strong in specificity, good in separation degree, high in stability and high in efficiency, can simultaneously measure 7 ingredients, and realizes effective detection on the quality of a product.
The invention relates to a content determination method of a spray for tendon and bone injury, which comprises the following steps: the method comprises the following steps of simultaneously measuring the content of 7 effective components in the muscle and bone injury spray by adopting a high performance liquid chromatography, wherein the 7 effective components in the content measurement are as follows: paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I, wherein the content determination method comprises the following steps: preparing a test solution, preparing a reference solution and measuring by an HPLC method.
The preparation method of the test solution comprises the following specific steps: taking 2-10 ml of tendon and bone injury spray, precisely sucking, placing in a 100-200 ml evaporating dish, evaporating to dryness, dissolving in 2-10 ml of water, transferring the dissolved solution to a 25-50 ml test tube, adding 5-20 ml of water saturated n-butyl alcohol solution into the test tube, extracting, adding 5-20 ml of water saturated n-butyl alcohol solution for extraction, taking supernatant, placing in a 100-200 ml evaporating dish, evaporating to dryness, dissolving in methanol, fixing the volume to a 2-10 ml volumetric flask, filtering with a microporous membrane, and taking a subsequent filtrate as a sample solution.
Preferably, the preparation method of the test solution of the invention comprises the following steps: taking 6-8 ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of water for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, then adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 10ml volumetric flask, filtering with a microporous filter membrane, and taking the subsequent filtrate as a sample solution.
The preparation method of the reference substance solution comprises the following specific steps: dissolving icariin reference substances in methanol until the concentration is 0.15-0.32 mg/ml for later use; dissolving epimedin A reference substance in methanol until the concentration is 1-2 mg/ml for later use; dissolving epimedin B reference substance in methanol until the concentration is 0.8-2 mg/ml for later use; dissolving epimedin C reference substance in methanol to a concentration of 0.01-0.1 mg/ml for later use; taking baohuoside I reference substance, adding methanol to dissolve until the concentration is 0.02-0.03 mg/ml for later use; taking an ellagic acid reference substance, adding methanol to dissolve the ellagic acid reference substance until the concentration is 0.8-2 mg/ml for later use; taking a paeoniflorin reference substance, adding methanol to dissolve the paeoniflorin reference substance until the concentration is 0.8-2 mg/ml for later use; and preparing the prepared reference substance solutions of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I into 7-component mixed reference substance solutions by respectively taking 1-2 ml, 1-1.5 ml, 100-150 ul, 50-100 ul, 250-300 ul and 100-150 ul.
Preferably, the preparation method of the reference solution of the present invention comprises the following specific steps: dissolving icariin control in methanol to concentration of 0.236 mg/ml; dissolving epimedin A control in methanol to concentration of 1.5 mg/ml; dissolving epimedin B reference substance in methanol to concentration of 1.2 mg/ml; dissolving epimedin C control in methanol to concentration of 0.05 mg/ml; dissolving baohuoside I control with methanol to concentration of 0.026 mg/ml; dissolving ellagic acid control in methanol to concentration of 1.2 mg/ml; dissolving penoniflorin control in methanol to concentration of 1.43 mg/ml;
the prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I are respectively prepared into 7-component mixed control solution by taking 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul and 140ul to 5 ml.
The chromatographic conditions measured by the HPLC method are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6 x 150mm,5 um; the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, and the mobile phase D is acetonitrile; the flow rate is 0.85-1 ml/min, the detection wavelength is 0-23 min, 230nm, 23-54 min, 254nm, 54-137 min, 270nm, the column temperature is 35 ℃, and gradient elution is carried out.
The gradient elution method comprises the following steps:
Time mobile phase B Mobile phase C Mobile phase D Flow rate of flow
0~16min 92%~90% —— 8%~10% 1ml/min
16~20min 90% —— 10% 1ml/min
20~22min 90%~88% —— 10%~12% 1ml/min
22~33min 88%~83% —— 12%~17% 1ml/min
33min~50min 83%~79% —— 17%~21% 1ml/min
50~51min 79%~77.5% —— 21%~22.5% 1~0.85ml/min
51~80min 77.5% —— 22.5% 0.85ml/min
80~80.01min 77.5%~67% 0~11% 22.5%~22% 0.85~1ml/min
80.01~97min 67%~65% 11%~0% 22%~35% 1ml/min
97~107min 65% —— 35% 1ml/min
107~117min 65%~50% —— 35%~50% 1ml/min
117~137min 50%~35% —— 50~65% 1ml/min
Preferably, the gradient elution method of the present invention is:
Time mobile phase Mobile phase C Mobile phase D Flow rate of flow
0-14min 92%-90% —— 8%-10% 1ml/min
14-17.5min 90% —— 10% 1ml/min
17.5-22min 90%-88% —— 10%-12% 1ml/min
22-33min 88%-83% —— 12%-17% 1ml/min
33-50min 83%-79% —— 17%-21% 1ml/min
50-51min 79%-77.5% —— 21%-22.5% 1-0.85ml/min
51-80min 77.5% —— 22.5% 0.85ml/min
80-80.01min 77.5%-67% 0-11% 22.5%-22% 0.85-1ml/min
80.01-97min 67%-65% 11%-0% 22%-35% 1ml/min
97-107min 65% —— 35% 1ml/min
107-117min 65%-50% —— 35%-50% 1ml/min
117-137min 50%-35% —— 50-65% 1ml/min
The HPLC method comprises the following steps: respectively and precisely absorbing 5-10 uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the test solution.
Through actual measurement, the content of paeoniflorin in the tendon and bone injury spray is 2000-2500 ug/ml; the content of the ellagic acid is 400-500 ug/ml; the content of the epimedin A is 20-30 ug/ml; the content of the epimedin B is 30-40 ug/ml; the content of the epimedin C is 20-30 ug/ml; the content of icariin is 100-150 ug/ml; the content of baohuoside I is 10-15 ug/ml.
Through actual measurement, preferably, the content of paeoniflorin in the muscle and bone injury spray is 2028 ug/ml; the content of ellagic acid is 476 ug/ml; the content of epimedin A is 25 ug/ml; the content of epimedin B is 38 ug/ml; the content of epimedin C is 23 ug/ml; the icariin content is 148 ug/ml; the baohuoside I content is 11 ug/ml.
The method for simultaneously measuring the content of 7 effective components in the muscle and bone injury spray is applied to the stability research and investigation of the muscle and bone injury spray.
Compared with the prior art, the invention has the following advantages:
1. compared with the technology disclosed by the 'muscle and bone injury spray' with the 'super-flying' Miao medicine, quality standard improvement [ D ]. Guiyang medical college, 2015, the content of 4 ingredients in a traditional Chinese medicine preparation is simultaneously determined by a comparison document, while the content of 7 active ingredients is simultaneously determined by the invention, so that the defect that 7 active ingredients in a product cannot be effectively and simultaneously detected in the prior art is overcome.
2. The invention establishes a content determination method for simultaneously determining 7 effective components in the tendon and bone injury spray for the first time, realizes the simultaneous determination of 7 components, effectively detects the contents of 7 most representative effective components of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in three main medicinal materials in the tendon and bone injury spray, and provides a rapid and efficient determination scheme for the establishment of a liquid-phase fingerprint library of the tendon and bone injury spray.
3. Provides a quantitative investigation method for the stability of effective components.
4. The content detection method disclosed by the invention has the advantages of stable detection baseline, high separation degree, high detection accuracy and good stability.
Description of the drawings:
FIG. 1 is an HPLC chromatogram of a mixed control solution in example 1, wherein 1-7 peaks correspond to 1 and paeoniflorin, respectively; 2. ellagic acid; 3. epimedin A; 4. epimedin B; 5. epimedin C; 6. icariin; 7. baohuoside I.
FIG. 2 is an HPLC chromatogram of the sample solution of example 1, wherein 1-7 peaks correspond to 1 and paeoniflorin, respectively; 2. ellagic acid; 3. epimedin A; 4. epimedin B; 5. epimedin C; 6. icariin; 7. baohuoside I.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The apparatus referred to in the following examples comprises: agilent 1100 HPLC (including on-line degasser, quaternary pump, autosampler, DAD detector, USA), Agilent chromatography workstation (Agilent technologies, Inc. USA); CH-250 type ultrasonic cleaning machine (Beijing Innovative ultrasonic electronic research institute); XS-105 electronic balance (Mettler-Torledo, Switzerland); a pipette (range of 20-200. mu.L and 100-1000. mu.L, eppendorf, Germany). The reagent comprises: control (purchased from Sichuan Veckqi Biotech Ltd): paeoniflorin (batch No. wkq 18032104); ellagic acid (batch number: wkq 18020502); epimedin A (batch No. wkq 17040509); epimedin B (batch No. wkq 17032809); epimedin C (batch No. wkq 17040701); baohuoside I (batch No. wkq 16081303); icariin (purchased from China institute for drug and biological products) (batch number: 110737-200414).
Example 1
The contents of the effective components of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray are detected according to the following method.
Chromatographic conditions are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6 x 150mm,5um, the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, the mobile phase D is acetonitrile, the detection wavelength is 0-23 min:230nm, 23-54 min:254nm, 54-137 min:270nm, the column temperature is 35 ℃, and the gradient elution method comprises the following steps:
Time mobile phase B Mobile phase C Mobile phase D Flow rate of flow
0~14min 92%~90% —— 8%~10% 1ml/min
14~17.5min 90% —— 10% 1ml/min
17.5~22min 90%~88% —— 10%~12% 1ml/min
22~33min 88%~83% —— 12%~17% 1ml/min
33~50min 83%~79% —— 17%~21% 1ml/min
50~51min 79%~77.5% —— 21%~22.5% 1~0.85ml/min
51~80min 77.5% —— 22.5% 0.85ml/min
80~80.01min 77.5%~67% 0~11% 22.5%~22% 0.85~1ml/min
80.01~97min 67%~65% 11%~0% 22%~35% 1ml/min
97~107min 65% —— 35% 1ml/min
107~117min 65%~50% —— 35%~50% 1ml/min
117~137min 50%~35% —— 50~65% 1ml/min
Preparation of a test solution: taking 5ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of water for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 10ml volumetric flask, filtering with a microporous membrane, and taking the subsequent filtrate as a sample solution.
Preparation of control solutions: dissolving icariin control, epimedin A control, epimedin B control, epimedin C control, baohuoside I control, ellagic acid control and paeoniflorin control with methanol to obtain icariin control solution with concentration of 0.236mg/ml, epimedin A control solution with concentration of 1.5mg/ml, epimedin B control solution with concentration of 1.2mg/ml, epimedin C control solution with concentration of 0.05mg/ml, baohuoside I control solution with concentration of 0.026mg/ml, ellagic acid control solution with concentration of 1.2mg/ml and paeoniflorin control solution with concentration of 1.43mg/ml, and collecting the prepared control solutions, paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I, 1.85ml and 1.19ml respectively, 1.34ml, 110ul, 90ul, 280ul and 140ul are metered to 5ml to prepare a mixed reference solution with 7 components.
And (3) high performance liquid chromatography determination: respectively and precisely sucking 10uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
The HPLC chromatogram of the mixed control solution is shown in FIG. 1.
The HPLC chromatogram of the test solution is shown in FIG. 2.
And (3) stability investigation: the above content measurement method was applied to stability examination.
Example 2
The contents of the effective components of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray are detected according to the following method.
Chromatographic conditions are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6 x 150mm,5um, the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, the mobile phase D is acetonitrile, the detection wavelength is 0-23 min:230nm, 23-54 min:254nm, 54-137 min:270nm, the column temperature is 35 ℃, and the gradient elution method comprises the following steps:
Figure BDA0001991951650000061
Figure BDA0001991951650000071
preparation of a test solution: taking 10ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of aqueous solution for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, then adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 5ml volumetric flask, filtering with a microporous membrane, and taking the subsequent filtrate as a sample solution.
Preparation of control solutions: dissolving icariin control, epimedin A control, epimedin B control, epimedin C control, baohuoside I control, ellagic acid control and paeoniflorin control with methanol to obtain icariin control solution with concentration of 0.236mg/ml, epimedin A control solution with concentration of 1.5mg/ml, epimedin B control solution with concentration of 1.2mg/ml, epimedin C control solution with concentration of 0.05mg/ml, baohuoside I control solution with concentration of 0.026mg/ml, ellagic acid control solution with concentration of 1.2mg/ml and paeoniflorin control solution with concentration of 1.43mg/ml, and collecting the prepared control solutions, paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I, 1.85ml and 1.19ml respectively, 1.34ml, 110ul, 90ul, 280ul and 140ul are metered to 5ml to prepare a mixed reference solution with 7 components.
And (3) high performance liquid chromatography determination: respectively and precisely sucking 10uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
And (3) stability investigation: the above content measurement method was applied to stability examination.
Example 3
The contents of the effective components of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray are detected according to the following method.
Chromatographic conditions are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6 x 150mm,5um, the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, the mobile phase D is acetonitrile, the detection wavelength is 0-23 min:230nm, 23-54 min:254nm, 54-137 min:270nm, the column temperature is 35 ℃, and the gradient elution method comprises the following steps:
Time mobile phase B Mobile phase C Mobile phase D Flow rate of flow
0~14min 92%~90% —— 8%~10% 1ml/min
14~17.5min 90% —— 10% 1ml/min
17.5~22min 90%~88% —— 10%~12% 1ml/min
22~33min 88%~83% —— 12%~17% 1ml/min
33~50min 83%~79% —— 17%~21% 1ml/min
50~51min 79%~77.5% —— 21%~22.5% 1~0.85ml/min
51~80min 77.5% —— 22.5% 0.85ml/min
80~80.01min 77.5%~67% 0~11% 22.5%~22% 0.85~1ml/min
80.01~97min 67%~65% 11%~0% 22%~35% 1ml/min
97~107min 65% —— 35% 1ml/min
107~117min 65%~50% —— 35%~50% 1ml/min
117~137min 50%~35% —— 50~65% 1ml/min
Preparation of a test solution: taking 8ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of aqueous solution for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, then adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 5ml volumetric flask, filtering with a microporous membrane, and taking the subsequent filtrate as a sample solution.
Preparation of control solutions: dissolving icariin control, epimedin A control, epimedin B control, epimedin C control, baohuoside I control, ellagic acid control and paeoniflorin control with methanol to obtain icariin control solution with concentration of 0.236mg/ml, epimedin A control solution with concentration of 1.5mg/ml, epimedin B control solution with concentration of 1.2mg/ml, epimedin C control solution with concentration of 0.05mg/ml, baohuoside I control solution with concentration of 0.026mg/ml, ellagic acid control solution with concentration of 1.2mg/ml and paeoniflorin control solution with concentration of 1.43mg/ml, respectively, and keeping for use.
The prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I are respectively prepared into 7-component mixed control solution by taking 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul and 140ul to 5 ml.
And (3) high performance liquid chromatography determination: respectively and precisely sucking 10uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
And (3) stability investigation: the above content measurement method was applied to stability examination.
Example 4
The contents of the effective components of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray are detected according to the following method.
Chromatographic conditions are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6mm by 150mm and 5um, the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, the mobile phase D is acetonitrile, the detection wavelength is 0-23 min:230nm, 23-54 min:254nm, 54-137 min:270nm, and the column temperature is 35 ℃. The gradient elution method was as follows:
Time mobile phase B Mobile phase C Mobile phase D Flow rate of flow
0~14min 92%~90% —— 8%~10% 1ml/min
14~17.5min 90% —— 10% 1ml/min
17.5~22min 90%~88% —— 10%~12% 1ml/min
22~33min 88%~83% —— 12%~17% 1ml/min
33~50min 83%~79% —— 17%~21% 1ml/min
50~51min 79%~77.5% —— 21%~22.5% 1~0.85ml/min
51~80min 77.5% —— 22.5% 0.85ml/min
80~80.01min 77.5%~67% 0~11% 22.5%~22% 0.85~1ml/min
80.01~97min 67%~65% 11%~0% 22%~35% 1ml/min
97~107min 65% —— 35% 1ml/min
107~117min 65%~50% —— 35%~50% 1ml/min
117~137min 50%~35% —— 50~65% 1ml/min
Preparation of a test solution: taking 5ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of water for dissolving, transferring a dissolved solution into a 25ml test tube, adding 10ml of ethyl acetate solution into the test tube, extracting, adding 10ml of ethyl acetate solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 10ml volumetric flask, filtering with a microporous filter membrane, and taking a subsequent filtrate as a sample solution.
Preparation of control solutions: dissolving icariin control, epimedin A control, epimedin B control, epimedin C control, baohuoside I control, ellagic acid control and paeoniflorin control with methanol to obtain icariin control solution with concentration of 0.236mg/ml, epimedin A control solution with concentration of 1.5mg/ml, epimedin B control solution with concentration of 1.2mg/ml, epimedin C control solution with concentration of 0.05mg/ml, baohuoside I control solution with concentration of 0.026mg/ml, ellagic acid control solution with concentration of 1.2mg/ml and paeoniflorin control solution with concentration of 1.43mg/ml, respectively, and keeping for use.
The prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I are respectively prepared into 7-component mixed control solution by taking 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul and 140ul to 5 ml.
And (3) high performance liquid chromatography determination: precisely absorbing 5uL of each of the test solution and the reference solution, respectively, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
And (3) stability investigation: the above content measurement method was applied to stability examination.
Example 5
The contents of the effective components of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray are detected according to the following method.
Chromatographic conditions are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6 x 150mm,5um, the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, the mobile phase D is acetonitrile, the detection wavelength is 0-23 min:230nm, 23-54 min:254nm, 54-137 min:270nm, the column temperature is 35 ℃, and the gradient elution method comprises the following steps:
Time mobile phase B Mobile phase C Mobile phase D Flow rate of flow
0~16min 92%~90% —— 8%~10% 1ml/min
16~20min 90% —— 10% 1ml/min
20~22min 90%~88% —— 10%~12% 1ml/min
22~33min 88%~83% —— 12%~17% 1ml/min
33min~50min 83%~79% —— 17%~21% 1ml/min
50~51min 79%~77.5% —— 21%~22.5% 1~0.85ml/min
51~80min 77.5% —— 22.5% 0.85ml/min
80~80.01min 77.5%~67% 0~11% 22.5%~22% 0.85~1ml/min
80.01~97min 67%~65% 11%~0% 22%~35% 1ml/min
97~107min 65% —— 35% 1ml/min
107~117min 65%~50% —— 35%~50% 1ml/min
117~137min 50%~35% —— 50~65% 1ml/min
Preparation of a test solution: taking 5ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of water for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 10ml volumetric flask, filtering with a microporous membrane, and taking the subsequent filtrate as a sample solution.
Preparation of control solutions: dissolving icariin control, epimedin A control, epimedin B control, epimedin C control, baohuoside I control, ellagic acid control and paeoniflorin control with methanol to obtain icariin control solution with concentration of 0.236mg/ml, epimedin A control solution with concentration of 1.5mg/ml, epimedin B control solution with concentration of 1.2mg/ml, epimedin C control solution with concentration of 0.05mg/ml, baohuoside I control solution with concentration of 0.026mg/ml, ellagic acid control solution with concentration of 1.2mg/ml and paeoniflorin control solution with concentration of 1.43mg/ml, respectively, and keeping for use.
The prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I are respectively prepared into 7-component mixed control solution by taking 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul and 140ul to 5 ml.
And (3) high performance liquid chromatography determination: respectively and precisely sucking 10uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
The detection results of the above examples show that when the method of the present invention is used to detect the content of the bone injury spray, the baseline is stable, and all target components in the sample, paeoniflorin, icariin, ellagic acid, epimedin a, epimedin B, epimedin C and baohuoside I, have been separated, which indicates that the method of the present invention for determining the content of the tendon and bone injury spray can effectively and simultaneously determine 7 effective components thereof, and has the advantages of high separation degree, high detection accuracy and good stability.
And (3) stability investigation: the above content measurement method was applied to stability examination.
Content detection method screening test:
to further verify the effectiveness of the present invention, the inventors performed a series of experiments to screen out the most suitable reagents, parameters, methods, etc. of the present invention, as follows:
experimental example 1 a test solution was prepared using an aqueous sodium hydroxide solution, and the separation of each component was tested.
The contents of active ingredients of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray are detected according to the following method:
chromatographic conditions are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6 x 150mm,5um, the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, the mobile phase D is acetonitrile, the detection wavelength is 0-23 min:230nm, 23-54 min:254nm, 54-137 min:270nm, the column temperature is 35 ℃, and the gradient elution method comprises the following steps:
Time mobile phase B Mobile phase C Mobile phase D Flow rate of flow
0~14min 92%~90% —— 8%~10% 1ml/min
14~17.5min 90% —— 10% 1ml/min
17.5~22min 90%~88% —— 10%~12% 1ml/min
22~33min 88%~83% —— 12%~17% 1ml/min
33~50min 83%~79% —— 17%~21% 1ml/min
50~51min 79%~77.5% —— 21%~22.5% 1~0.85ml/min
51~80min 77.5% —— 22.5% 0.85ml/min
80~80.01min 77.5%~67% 0~11% 22.5%~22% 0.85~1ml/min
80.01~97min 67%~65% 11%~0% 22%~35% 1ml/min
97~107min 65% —— 35% 1ml/min
107~117min 65%~50% —— 35%~50% 1ml/min
117~137min 50%~35% —— 50~65% 1ml/min
Preparation of a test solution: taking 5ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of sodium hydroxide aqueous solution for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 10ml volumetric flask, filtering with a microporous membrane, and taking the subsequent filtrate as a sample solution.
Preparation of control solutions: dissolving icariin control, epimedin A control, epimedin B control, epimedin C control, baohuoside I control, ellagic acid control and paeoniflorin control with methanol to obtain icariin control solution with concentration of 0.236mg/ml, epimedin A control solution with concentration of 1.5mg/ml, epimedin B control solution with concentration of 1.2mg/ml, epimedin C control solution with concentration of 0.05mg/ml, baohuoside I control solution with concentration of 0.026mg/ml, ellagic acid control solution with concentration of 1.2mg/ml and paeoniflorin control solution with concentration of 1.43mg/ml, respectively, and keeping for use.
The prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I are respectively prepared into 7-component mixed control solution by taking 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul and 140ul to 5 ml.
And (3) determination by an HPLC method: respectively and precisely sucking 10uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
The detection result shows that the detection base line is stable, ellagic acid cannot be detected, the target components in the sample are reduced, and the experimental effect of simultaneously detecting 7 effective components cannot be achieved.
Experimental example 2 the mobile phase B was water and the separation was tested
The contents of active ingredients of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in the muscle and bone injury spray are detected according to the following method:
the chromatographic conditions are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6mm by 150mm and 5um, the mobile phase B is water, the mobile phase C is methanol, the mobile phase D is acetonitrile, the detection wavelength is 0-23 min:230nm, 23-54 min:254nm, 54-137 min:270nm, the column temperature is 35 ℃, and the gradient elution method comprises the following steps:
Time mobile phase B Mobile phase C Mobile phase D Flow rate of flow
0~14min 92%~90% —— 8%~10% 1ml/min
14~17.5min 90% —— 10% 1ml/min
17.5~22min 90%~88% —— 10%~12% 1ml/min
22~33min 88%~83% —— 12%~17% 1ml/min
33~50min 83%~79% —— 17%~21% 1ml/min
50~51min 79%~77.5% —— 21%~22.5% 1~0.85ml/min
51~80min 77.5% —— 22.5% 0.85ml/min
80~80.01min 77.5%~67% 0~11% 22.5%~22% 0.85~1ml/min
80.01~97min 67%~65% 11%~0% 22%~35% 1ml/min
97~107min 65% —— 35% 1ml/min
107~117min 65%~50% —— 35%~50% 1ml/min
117~137min 50%~35% —— 50~65% 1ml/min
Preparation of a test solution: taking 5ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of water for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 10ml volumetric flask, filtering with a microporous membrane, and taking the subsequent filtrate as a sample solution.
Preparation of control solutions: dissolving icariin control, epimedin A control, epimedin B control, epimedin C control, baohuoside I control, ellagic acid control and paeoniflorin control with methanol to obtain icariin control solution with concentration of 0.236mg/ml, epimedin A control solution with concentration of 1.5mg/ml, epimedin B control solution with concentration of 1.2mg/ml, epimedin C control solution with concentration of 0.05mg/ml, baohuoside I control solution with concentration of 0.026mg/ml, ellagic acid control solution with concentration of 1.2mg/ml and paeoniflorin control solution with concentration of 1.43mg/ml, respectively, and keeping for use.
The prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I are respectively prepared into 7-component mixed control solution by taking 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul and 140ul to 5 ml.
And (3) high performance liquid chromatography determination: respectively and precisely sucking 10uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
The detection result shows that the detection base line is stable, the separation degree of each target component in the sample is poor, and the effect is inferior to that of the embodiment.
Test example 3 determination of the content of the spray component for treating muscle and bone injuries by the prior art method
According to the detection method disclosed in the quality research and quality standard improvement of the 'Jingfei' Miao medicament, namely the 'Jingfei spray' the contents of paeoniflorin, ellagic acid, epimedin A, epimedin B, epimedin C, icariin and baohuoside I are tested.
The instrument comprises the following steps: agilent 1100 to DAD, Agilent to VWD high performance liquid chromatographs (Agilent technologies, Inc., USA), KQ to 250B ultrasonic cleaners (ultrasonic instruments, Inc., Kunshan), Nippon Shimadzu AY to 120 electronic balances, HH to 4 digital display constant temperature water bath (electric appliances, Inc., China).
Chromatographic conditions are as follows: the chromatographic column is WONDASIL C18(250X 4.6mm, 5 um); detection wavelength: 230nm, 248nm and 270nm, and the specific parameters of the wavelength conversion method are as follows:
time (minutes) Wavelength (nm)
0 248
10 230
25 248
41 270
45 248
Flow rate: 1 ml/min; column temperature: 25 ℃, sample introduction: 10 ul; the gradient elution method comprises the following steps of (1) carrying out gradient elution on a mobile phase A-methanol and a mobile phase B-0.1% phosphoric acid solution:
time (minutes) Mobile phase A Mobile phase B
0 70% 30%
20 60% 40%
30 45% 55%
50 35% 65%
60 20% 80%
Reagents and reagents: methanol (chromatographic purity), phosphoric acid, methanol (analytical alcohol), lebai's purified drinking water; spray for treating injury of bones and muscles.
Preparation of a test solution: taking 5 bottles of the same batch of muscle and bone spray, taking 50ml of each bottle, precisely weighing 5ml of the mixed test solution in an evaporating dish, evaporating to dryness, dissolving in methanol, placing in a 10ml volumetric flask, performing ultrasonic treatment for 5min, fixing the volume with methanol, shaking up, and taking the supernatant to pass through a 0.45um microporous filter membrane to obtain the product.
Preparation of control solutions: dissolving icariin reference substances in methanol until the concentration is 0.2-0.3 mg/ml for later use; dissolving epimedin A reference substance in methanol until the concentration is 1-2 mg/ml for later use; dissolving epimedin B reference substance in methanol until the concentration is 1-2 mg/ml for later use; dissolving epimedin C reference substance in methanol until the concentration is 0.02-0.1 mg/ml for later use; taking baohuoside I reference substance, adding methanol to dissolve until the concentration is 0.02-0.03 mg/ml for later use; taking an ellagic acid reference substance, adding methanol to dissolve the ellagic acid reference substance until the concentration is 1-2 mg/ml for later use; taking a paeoniflorin reference substance, adding methanol to dissolve the paeoniflorin reference substance until the concentration is 1-1.8 mg/ml for later use;
the prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I are respectively prepared into a mixed control solution of 7 components by taking 1.85ml, 1.19ml, 1.34ml, 110ul, 90ul, 280ul and 140 ul.
And (3) high performance liquid chromatography determination: respectively and precisely sucking 10uL of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product.
The detection result shows that the detection baseline is stable, only paeoniflorin and icariin are detected in the sample, other components are not obvious, and the effect is far inferior to that of the embodiment.
In summary, the experimental results of the comparative examples and the examples show that the method for measuring the content of the tendon and bone injury spray in examples 1 to 7 of the invention can detect 7 active ingredients in the tendon and bone injury spray at the same time, and is a relatively comprehensive quantitative investigation method for the stability of the active ingredients.
Test example 4 methodological investigation and sample content determination:
the following experiments were carried out by treating the test solution, the control solution, the chromatographic conditions, etc. in accordance with the method of example 1:
(1) and (3) linear relation investigation: precisely sucking the control solution 2,5,10,15,20 and 25uL of the sample under the item of the example 1 respectively for measurement. Performing linear regression on X by peak area Y to obtain regression equation and linear range of paeoniflorin, ellagic acid, epimedin A, epimedin B, epimedin C, icariin and baohuoside I shown in Table 1:
TABLE 1 component regression equation and Linear Range
Composition (I) Regression equation Linear range
Paeoniflorin Y=73.856X~1.3175,r=0.9998(n=6) 1.058~13.225ug
Ellagic acid Y=5387X~1149.5,r=0.9998(n=6) 0.644~8.05ug
Epimedin A Y=1548.5X+8.1607,r=0.9998(n=6) 0.068~0.85ug
Epimedin B Y=1640.7X+9.8463,r=0.9999(n=6) 0.044~0.55ug
Epimedin C Y=2006.6X+27.168,r=0.9995(n=6) 0.1~1.25ug;
Icariin Y=1499.5X~22.376,r=0.9998(n=6) 0.112~1.4ug
Baohuoside I Y=5024.7X~29.95,r=0.9999(n=6) 0.058~0.725ug
(2) And (3) precision experiment: the mixed control solutions prepared in example 1 were each precisely aspirated, and sample injection was performed 6 times, and RSD of peak area integral values of paeoniflorin, ellagic acid, epimedin a, epimedin B, epimedin C, icariin, and baohuoside I was determined as shown in table 2, indicating that the instrument precision was good.
TABLE 2 RSD of peak area integral value of each component
Composition (I) RSD of peak area integral value
Paeoniflorin 2.1%
Ellagic acid 2.9%
Epimedin A 0.8%
Epimedin B 1.6%
Epimedin C 0.7%
Icariin 0.6%
Baohuoside I 0.7%
(3) And (3) repeatability experiment: taking the same batch of samples, preparing 5 parts of the sample in parallel according to the sample preparation method in the section of example 1, measuring according to the chromatographic conditions in the section of example 1, and calculating the content. The average contents and RSD of paeoniflorin, ellagic acid, epimedin a, epimedin B, epimedin C, icariin, baohuoside I in 6 parts of test solution are shown in table 3, which indicates good repeatability.
TABLE 3 average content and RSD
Composition (I) Average content RSD
Paeoniflorin 0.93mg/ml 1.4%
Ellagic acid 0.23mg/ml 2.3%
Epimedin A 0.011mg/ml 2.8%
Epimedin B 0.019mg/ml 2.4%
Epimedin C 0.012mg/ml 2.4%
Icariin 0.075mg/ml 2.3%
Baohuoside I 0.0051mg/ml 2.3%
(4) Stability test: the same batch of samples were taken, the sample solution was prepared according to the sample preparation method of example 1, and the sample was injected 0,2,4,8,24 hours after the preparation, and RSD of peak area integral values of paeoniflorin, ellagic acid, epimedin a, epimedin B, epimedin C, icariin, and baohuoside I in the sample was determined as shown in table 4, indicating that the sample solution was stable within 24 hours.
TABLE 4 RSD of integrated value of peak area
Composition (I) RSD of peak area integral value
Paeoniflorin 2.2%
Ellagic acid 2.7%
Epimedin A 2.5%
Epimedin B 1.8%
Epimedin C 2.2%
Icariin 1.6%
Baohuoside I 0.9%
(5) Sample recovery rate experiment: 2.5ml of the sample was taken, and an appropriate amount of each of paeoniflorin, ellagic acid, epimedin a, epimedin B, epimedin C, icariin, and baohuoside I was precisely added to the sample, and a sample solution was prepared according to the sample preparation method of example 1, and the recovery rate was calculated by measuring the sample solution under the chromatographic conditions of example 1, and as a result, the recovery rates (n ═ 9) and RSD of paeoniflorin, ellagic acid, epimedin a, epimedin B, epimedin C, icariin, and baohuoside I were as shown in table 5.
Table 5 recovery rate (n ═ 9) and RSD
Composition (I) Recovery rate (n ═ 9) RSD
Paeoniflorin 95.6% 1.4%
Ellagic acid 96.0% 1.8%
Epimedin A 97.8% 2.3%
Epimedin B 94.5% 1.5%
Epimedin C 93.5% 2.7%
Icariin 96.7% 2.4%
Baohuoside I 99.6% 1.9%
(6) And (3) sample content determination: three batches of muscle and bone injury sprays were prepared into test solutions according to the method described in example 1, and the content of 7 ingredients was determined, the results are shown in table 6:
TABLE 67 ingredient content measurement results table
Figure BDA0001991951650000171
According to the experimental results, the content determination is carried out by adopting an HPLC method, so that a better peak shape is obtained, the target component and the impurities of the sample are better separated, the repeatability is good, the specificity is good, and the detection of various effective components can be carried out simultaneously.
To summarize: 1. the quantitative method established in the experiment measures a plurality of batches of samples, and the result shows that the method is simple, has good specificity and high recovery rate, is simple, convenient and feasible, and can be used as a method for measuring the content of the tendon and bone injury spray; 2. the invention establishes a content determination method for simultaneously determining 7 effective components in the tendon and bone injury spray for the first time, realizes the simultaneous determination of 7 components, effectively detects the contents of 7 most representative effective components of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I in three main medicinal materials in the tendon and bone injury spray, and provides a rapid and efficient determination scheme for the establishment of a liquid-phase fingerprint library of the tendon and bone injury spray; 3. the invention provides a quantitative investigation method for the stability of effective components; the content detection method disclosed by the invention has the advantages of stable detection baseline, high separation degree, high detection accuracy and good stability.
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 (6)

1. A content determination method for a tendon and bone injury spraying agent is characterized in that high performance liquid chromatography is adopted to simultaneously determine the content of 7 effective components in the tendon and bone injury spraying agent, wherein the 7 effective components for content determination are as follows: paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I, wherein the content determination method comprises the following steps: preparing a test solution, preparing a reference solution and measuring by an HPLC method;
the preparation method of the test solution comprises the following specific steps: taking 2-10 ml of tendon and bone injury spray, precisely sucking, placing in a 100-200 ml evaporating dish, evaporating to dryness, dissolving with 2-10 ml of water, transferring the dissolved solution into a 25-50 ml test tube, adding 5-20 ml of water saturated n-butyl alcohol solution into the test tube, extracting, adding 5-20 ml of water saturated n-butyl alcohol solution for extraction, taking supernatant, placing in a 100-200 ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 2-10 ml volumetric flask, filtering with a microporous filter membrane, and taking a subsequent filtrate as a sample solution;
the preparation method of the reference solution comprises the following specific steps: dissolving icariin reference substances in methanol until the concentration is 0.15-0.32 mg/ml for later use; dissolving epimedin A reference substance in methanol until the concentration is 1-2 mg/ml for later use; dissolving epimedin B reference substance in methanol until the concentration is 0.8-2 mg/ml for later use; dissolving epimedin C reference substance in methanol to a concentration of 0.01-0.1 mg/ml for later use; taking baohuoside I reference substance, adding methanol to dissolve until the concentration is 0.02-0.03 mg/ml for later use; taking an ellagic acid reference substance, adding methanol to dissolve the ellagic acid reference substance until the concentration is 0.8-2 mg/ml for later use; taking a paeoniflorin reference substance, adding methanol to dissolve the paeoniflorin reference substance until the concentration is 0.8-2 mg/ml for later use; preparing a mixed reference solution of 7 components from 1-2 ml, 1-1.5 ml, 100-150 mul, 50-100 mul, 250-300 mul and 100-150 mul of the prepared reference solutions of paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I respectively;
the chromatographic conditions determined by the HPLC method are as follows: agilent ZORBA S8-C18 columns; the inner diameter is 4.6 x 150mm,5 μm; the mobile phase B is 0.05mol/L potassium dihydrogen phosphate solution, the mobile phase C is methanol, and the mobile phase D is acetonitrile; the flow rate is 0.85-1 ml/min, the detection wavelength is 0-23 min, 230nm, 23-54 min, 254nm, 54-137 min, 270nm, the column temperature is 35 ℃, and gradient elution is carried out; the gradient elution method comprises the following steps:
Figure FDA0003443771780000011
Figure FDA0003443771780000021
2. the method of claim 1, wherein the sample solution is prepared by: taking 6-8 ml of tendon and bone injury spray, precisely sucking, placing in a 100ml evaporating dish, evaporating to dryness, adding 5ml of water for dissolving, transferring the dissolved solution into a 25ml test tube, adding 10ml of water saturated n-butyl alcohol solution into the test tube, extracting, then adding 10ml of water saturated n-butyl alcohol solution for extracting, taking supernatant, placing in a 100ml evaporating dish, evaporating to dryness, dissolving with methanol, fixing the volume to a 10ml volumetric flask, filtering with a microporous filter membrane, and taking the subsequent filtrate as a sample solution.
3. The method according to claim 1, wherein the method for preparing the reference solution comprises the following steps: dissolving icariin control in methanol to concentration of 0.236 mg/ml; dissolving epimedin A control in methanol to concentration of 1.5 mg/ml; dissolving epimedin B reference substance in methanol to concentration of 1.2 mg/ml; dissolving epimedin C control in methanol to concentration of 0.05 mg/ml; dissolving baohuoside I control with methanol to concentration of 0.026 mg/ml; dissolving ellagic acid control in methanol to concentration of 1.2 mg/ml; dissolving penoniflorin control in methanol to concentration of 1.43 mg/ml; the prepared control solution paeoniflorin, icariin, ellagic acid, epimedin A, epimedin B, epimedin C and baohuoside I is prepared into 7-component mixed control solution by respectively taking 1.85ml, 1.19ml, 1.34ml, 110 mu l, 90 mu l, 280 mu l and 140 mu l of constant volume to 5 ml.
4. The method according to claim 1, characterized in that the gradient elution method is:
Figure FDA0003443771780000022
Figure FDA0003443771780000031
5. the method according to claim 1, characterized in that the HPLC method is: precisely absorbing 5-10 μ L of each of the test solution and the reference solution, injecting into a high performance liquid chromatograph, measuring, and calculating to obtain the final product; the content standard is that the content of paeoniflorin in the muscle and bone injury spray is 2000-2500 mug/ml; the content of ellagic acid is 400-500 mug/ml; the content of the epimedin A is 20-30 mu g/ml; the content of the epimedin B is 30-40 mu g/ml; the content of the epimedin C is 20-30 mu g/ml; the content of icariin is 100-150 mu g/ml; the content of baohuoside I is 10-15 mu g/ml.
6. The method as claimed in claim 1, wherein the method for simultaneously measuring the content of 7 active ingredients in the tendon and bone injury spray is applied to the stability research of tendon and bone injury spray.
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