Method for simultaneously detecting chlorogenic acid, ferulic acid, dracorhodin, cinnamaldehyde, ligustilide and osthole in rhizoma dioscoreae blood-activating patch

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting chlorogenic acid, ferulic acid, dracorhodin, cinnamaldehyde, ligustilide and osthole in a rhizoma dioscoreae blood-activating patch. According to the invention, a'quantitative analysis of multi-components by single marker 'method is adopted, effective components in monarch drugs of radix angelicae pubescentis and ligusticum wallichii and ministerial drugs of dragon's blood and cinnamon in the rhizoma cyperi blood-activating patch are taken as indexes, cinnamaldehyde with clear pharmacological action and high content is taken as an internal standard, and calculation is carried out through relative correction factors between the other five to-be-detected compounds and cinnamaldehyde; the content of chlorogenic acid, ferulic acid, dracorhodin, cinnamaldehyde, ligustilide and cnidium lactone in the rhizoma gastrodiae blood-activating patch is obtained by detecting the content of cinnamaldehyde in the rhizoma gastrodiae blood-activating patch, so that the content of chlorogenic acid, ferulic acid, dracorhodin, cinnamaldehyde, ligustilide and cnidium lactone in the rhizoma gastrodiae blood-activating patch can be detected at the same time only by detecting the content of cinnamaldehyde in the rhizoma gastrodiae blood-activating patch; the aim of objectively, comprehensively and accurately performing quantitative control on the rhizoma cyperi blood-activating patch is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for simultaneously detecting chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in Xiangduhuoxuetie. Background Art

[0002] Xiangdu Huoxue Patch (gel patch) is derived from the external prescription handed down by Mr. Du Qiongshu, a famous Chinese medicine orthopedic expert. The prescription consists of Duhuo, Chuanxiong, frankincense, myrrh, Sanguisorba officinalis, cinnamon, Sichuan costusroot, fennel, Chuanwu, Caowu, Sanguisorba officinalis, and borneol. Duhuo in the prescription is pungent, bitter, and slightly warm. It has the effect of dispelling rheumatism and relieving pain. It is an important medicine for treating rheumatic arthralgia. "Ming Yi Bie Lu" says: "It can treat all kinds of thieves' wind and gout in all joints. There is no long-term new one." Chuanxiong is pungent and warm. It has the effect of promoting blood circulation and qi, dispelling wind and relieving pain. "Ben Cao Hui Yan" calls it "blood qi medicine". The two are used together as the monarch. Frankincense, myrrh, Sanguisorba officinalis, and cinnamon are the ministerial drugs. Among them, frankincense and myrrh are used together. They have the effect of promoting blood circulation and relieving pain. They are mainly used to treat traumatic injuries and stasis pain. Sanguisorba officinalis enters the blood and disperses blood stasis and relieves pain. It is an important medicine for traumatology. The three medicines combined with cinnamon warm the blood vessels and increase the effect of promoting blood circulation and removing blood stasis. Sichuan costus root and fennel promote qi and relieve pain, while Sichuan aconite and kusnezoffii dispel wind and dampness, dispel cold and relieve pain. "Changsha Yaojie" says: "Aconitum, warm and dry, descends, its nature is rapid, opens the pores, and expels cold and dampness very quickly." The above four flavors are adjuvants. Borneol is pungent, bitter, slightly cold, and can pass through the twelve meridians. It is a guiding drug. The combination of these medicines can promote blood circulation and remove blood stasis, dispel wind and dampness, and warm the meridians to relieve pain for blood stasis, qi stagnation, wind, cold and dampness. A large number of studies have shown that external treatment with traditional Chinese medicine has great advantages in the treatment of soft tissue injuries. The transdermal absorption of drugs through the epidermal route can effectively avoid the first-pass effect, and it is easy to use and has a definite effect. It is favored by the majority of people, so Xiangdu Huoxue Patch has a broad application prospect.

[0003] The detection method for the plaster for treating soft tissue injury in the related art is mainly the external standard method. For example, the related art discloses a method for detecting the content of osthole and ligustilide in the bitong patch by high performance liquid chromatography, in which the mobile phase used is acetonitrile-water and the chromatographic column is Shiseido C 18 MG chromatographic column. However, the above detection method only determined the content of the active ingredient in one of the main drugs in Bitongtie, namely, Angelica dahurica, and could not achieve the simultaneous detection of multiple ingredients in the plaster. Summary of the invention

[0004] The object of the present invention is to provide a method for simultaneously detecting chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in Xiangduhuoxue patch. The method provided by the present invention realizes the simultaneous detection of the contents of chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in Xiangduhuoxue patch, and objectively, comprehensively and accurately controls the quality of Xiangduhuoxue patch.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for simultaneously detecting chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in Xiangdu Huoxue Patch, comprising the following steps:

[0007] dissolving the first cinnamaldehyde standard sample in methanol to obtain a cinnamaldehyde standard sample solution;

[0008] The cinnamaldehyde standard sample solution is subjected to a first high performance liquid chromatography test to obtain a first cinnamaldehyde standard sample peak area;

[0009] Remove the film from the Xiang Du Huo Xue patch sample to be tested, crush it, and extract it with a first phosphoric acid-methanol solution to obtain a sample solution to be tested; the mass percentage of phosphoric acid in the first phosphoric acid-methanol solution is 2-4%;

[0010] The sample solution to be tested is subjected to a second high performance liquid chromatography test to obtain a peak area of ​​the sample to be tested, and the concentration of cinnamaldehyde in the sample solution to be tested is calculated using an external standard method according to the concentration of the cinnamaldehyde standard sample solution, the peak area of ​​the first cinnamaldehyde standard sample and the peak area of ​​the sample to be tested;

[0011] According to the peak area of ​​the first cinnamaldehyde standard sample, the peak area of ​​the sample to be tested, the concentration of cinnamaldehyde in the sample solution to be tested and the relative correction factor, the concentrations of chlorogenic acid, ferulic acid, dracotin, ligustilide and osthole in the sample solution to be tested are calculated respectively; the relative correction factors are the chlorogenic acid correction factor, the ferulic acid correction factor, the dracotin correction factor, the osthole correction factor and the ligustilide correction factor respectively;

[0012] The conditions of the first HPLC detection and the second HPLC detection independently include: the chromatographic column is C 18Chromatographic column; mobile phase system comprises mobile phase A and mobile phase B, wherein mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; elution mode is gradient elution, and the procedure of the gradient elution is as follows: 0min, the volume fraction of the mobile phase A is 95%; 0-5min, the volume fraction of the mobile phase A is reduced from 95% to 90%; 5-15min, the volume fraction of the mobile phase A is 90%; 15-17min, the volume fraction of the mobile phase A is reduced from 90% to 82%; 17-30min, the volume fraction of the mobile phase A is 82%; 30-31min, the volume fraction of the mobile phase A is reduced from 82% to 75% ; 31-41min, the volume fraction of the mobile phase A is reduced from 75% to 70%; 41-45min, the volume fraction of the mobile phase A is 70%; 45-50min, the volume fraction of the mobile phase A is reduced from 70% to 65%; 50-55min, the volume fraction of the mobile phase A is reduced from 65% to 52%; 55-85min, the volume fraction of the mobile phase A is 52%; 85-86min, the volume fraction of the mobile phase A is reduced from 52% to 0%; 86-90min, the volume fraction of the mobile phase A is 0%; 90-91min, the volume fraction of the mobile phase A is increased from 0% to 95%; 91-95min, the volume fraction of the mobile phase A is 95%;

[0013] The Angelica Angelicae Pubescentis blood patch comprises a backing material, a coating, a Chinese medicine composition and a matrix; in terms of weight, the matrix comprises 10-45 parts of a skeleton material, 1-3 parts of a cross-linking agent, 30-70 parts of a moisturizer, 1-3 parts of a pH adjuster, 15-35 parts of a viscosity enhancer and 50-120 parts of purified water; in terms of weight, the Chinese medicine composition comprises 5-24 parts of Angelicae Pubescentis, 5-24 parts of Ligusticum chuanxiong, 5-24 parts of Radix Aucklandiae, 4-14 parts of Fennel, 4-14 parts of Cinnamon bark, 4-14 parts of Frankincense, 4-14 parts of Myrrh, 1-12 parts of processed Aconiti Kusnezoffii, 1-12 parts of processed Aconiti Kusnezoffii, 1-12 parts of Sanguisorba officinalis and 1-8 parts of Borneol.

[0014] Preferably, the method for obtaining the relative correction factor comprises the following steps:

[0015] Dissolving a chlorogenic acid standard sample, a ferulic acid standard sample, a dracotoxin standard sample, an osthole standard sample, a ligustilide standard sample, and a second cinnamaldehyde standard sample in a second phosphoric acid-methanol solution to obtain a mixed standard sample solution;

[0016] The mixed standard sample solution is subjected to a third high performance liquid chromatography test to obtain a mixed standard sample peak area;

[0017] Using cinnamaldehyde as an internal standard, the relative correction factor is calculated by the internal standard method according to the peak area of ​​the mixed standard sample and the concentration of the mixed standard sample solution;

[0018] The mixed standard sample peak area includes the chlorogenic acid standard sample peak area, the ferulic acid standard sample peak area, the dracotoxin standard sample peak area, the osthole standard sample peak area, the ligustilide standard sample peak area and the second cinnamaldehyde standard sample peak area;

[0019] The conditions for the third high performance liquid chromatography detection include: the chromatographic column is C 18 Chromatographic column; mobile phase system comprises mobile phase A and mobile phase B, wherein mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; elution mode is gradient elution, and the procedure of the gradient elution is as follows: 0min, the volume fraction of the mobile phase A is 95%; 0-5min, the volume fraction of the mobile phase A is reduced from 95% to 90%; 5-15min, the volume fraction of the mobile phase A is 90%; 15-17min, the volume fraction of the mobile phase A is reduced from 90% to 82%; 17-30min, the volume fraction of the mobile phase A is 82%; 30-31min, the volume fraction of the mobile phase A is reduced from 82% to 75% ; 31-41min, the volume fraction of the mobile phase A is reduced from 75% to 70%; 41-45min, the volume fraction of the mobile phase A is 70%; 45-50min, the volume fraction of the mobile phase A is reduced from 70% to 65%; 50-55min, the volume fraction of the mobile phase A is reduced from 65% to 52%; 55-85min, the volume fraction of the mobile phase A is 52%; 85-86min, the volume fraction of the mobile phase A is reduced from 52% to 0%; 86-90min, the volume fraction of the mobile phase A is 0%; 90-91min, the volume fraction of the mobile phase A is increased from 0% to 95%; 91-95min, the volume fraction of the mobile phase A is 95%.

[0020] Preferably, the concentration of the second cinnamaldehyde standard sample in the mixed standard sample solution is 80-1890 μg / mL, the concentration of the chlorogenic acid standard sample is 38-349 μg / mL, the concentration of the ferulic acid standard sample is 81-734 μg / mL, the concentration of the dracotoxin standard sample is 36-324 μg / mL, the concentration of the ligustilide standard sample is 90-1524 μg / mL, and the concentration of the osthole standard sample is 28-252 μg / mL.

[0021] Preferably, the conditions for the third HPLC detection also include: column temperature of 25-35°C; flow rate of mobile phase of 0.8-1.2 mL / min; injection volume of 4-12 μL; detection wavelength of 319-324 nm.

[0022] Preferably, the calculation formula of the relative correction factor is: i / r =A i C r / A r C i ;

[0023] Among them, f i / r is the relative correction factor, A i is the peak area of ​​the second cinnamaldehyde standard sample; C i A is the concentration of the second cinnamaldehyde standard sample in the mixed standard sample solution; r is the peak area of ​​the standard sample to be calibrated; C r is the concentration of the standard sample to be calibrated in the mixed standard sample solution;

[0024] The standard samples to be calibrated are respectively a chlorogenic acid standard sample, a ferulic acid standard sample, a dracotoxin standard sample, an osthole standard sample and a ligustilide standard sample.

[0025] Preferably, the concentration of the first cinnamaldehyde standard sample in the cinnamaldehyde standard sample solution is 80 to 1890 μg / mL.

[0026] Preferably, the conditions of the first HPLC detection and the second HPLC detection independently include: column temperature of 25-35°C; flow rate of mobile phase of 0.8-1.2 mL / min; injection volume of 4-12 μL; detection wavelength of 319-324 nm.

[0027] Preferably, the solid-to-liquid ratio of the Xiangduhuoxue patch sample to be tested and the first phosphoric acid-methanol solution is 1 g: 3.56-5 mL.

[0028] Preferably, the extraction comprises ultrasonic extraction; the extraction temperature is 20 to 60° C., and the extraction time is 20 to 40 minutes.

[0029] Preferably, the calculation formula for the concentration of chlorogenic acid, ferulic acid, dracotoxin, ligustilide and osthole in the sample solution to be tested is: x =A x C ii f i / r / A ii ;

[0030] Among them, C x A is the concentration of chlorogenic acid, ferulic acid, dracotoxin, osthole or ligustilide in the sample solution to be tested;x is the peak area of ​​the sample to be tested; C ii is the concentration of the first cinnamaldehyde standard sample in the cinnamaldehyde standard sample solution; A ii is the peak area of ​​the first cinnamaldehyde standard sample; f i / r is the relative correction factor.

[0031] The invention provides a method for simultaneously detecting chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in a Xiangduhuoxue patch. The method comprises the following steps: dissolving a first cinnamaldehyde standard sample in methanol to obtain a cinnamaldehyde standard sample solution; removing the coating from a Xiangduhuoxue patch sample to be tested, crushing it, and extracting it with a first phosphoric acid-methanol solution to obtain a sample solution to be tested; performing a first high performance liquid chromatography detection on the cinnamaldehyde standard sample solution to obtain a first cinnamaldehyde standard sample peak area; performing a second high performance liquid chromatography detection on the sample solution to be tested to obtain a sample peak area, and calculating the concentration of cinnamaldehyde in the sample solution to be tested by an external standard method according to the concentration of the cinnamaldehyde standard sample solution, the first cinnamaldehyde standard sample peak area and the sample peak area; and respectively calculating the concentrations of chlorogenic acid, ferulic acid, dracoside, ligustilide and osthole in the sample solution to be tested according to the first cinnamaldehyde standard sample peak area, the sample peak area, the concentration of cinnamaldehyde in the sample solution to be tested and a relative correction factor. The present invention adopts a "one test, multiple evaluations" method, taking the active ingredients of the main medicines Angelica dahurica and Ligusticum chuanxiong, and the auxiliary medicines Dragon's blood and cinnamon in the Xiangdu Huoxue Patch as indicators, and taking cinnamaldehyde, which has a clear pharmacological effect and a large content, as an internal standard, and calculating the relative correction factors between the other five test compounds (chlorogenic acid, ferulic acid, dragon's blood, ligustilide and osthole) and cinnamaldehyde to obtain the contents of chlorogenic acid, ferulic acid, dragon's blood, cinnamaldehyde, ligustilide and osthole in the Xiangdu Huoxue Patch. The method provided by the present invention realizes the simultaneous detection of the contents of chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in the Xiangdu Huoxue Patch, and objectively, comprehensively and accurately controls the quality of the Xiangdu Huoxue Patch; moreover, the method provided by the present invention only uses a cinnamaldehyde standard sample as a single external standard sample, which is simple to operate and has lower cost than the traditional external standard method, and can accurately calculate the contents of the remaining five test compounds on the basis of reducing the use of standard samples, has good reproducibility and stability, and the obtained chromatographic peak has a good peak shape, which provides strong data support for the establishment of the quality standard of the Xiangdu Huoxue Patch.

[0032] In addition, compared with the external standard method used in the related art, the "one test, multiple evaluations" method provided by the present invention is more convenient, efficient and has lower cost. The present invention limits the high performance liquid phase detection conditions (mobile phase A is phosphoric acid aqueous solution, and the detection wavelength is 319-324 nm) to obtain a better chromatogram peak shape. The gradient is used for detection to increase the separation of the compounds to be tested and facilitate detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the chromatogram of the mixed standard sample to be tested;

[0034] Figure 2 This is the chromatogram of the Xiangdu Huoxue patch sample to be tested;

[0035] Figure 3 is the chromatogram of negative sample solution 1 to be tested;

[0036] Figure 4 is the chromatogram of negative sample solution 2 to be tested;

[0037] Figure 5 is the chromatogram of negative sample solution 3 to be tested;

[0038] Figure 6 is the chromatogram of negative sample solution 4 to be tested;

[0039] Figure 1 to Figure 6 Among them, peak 1 is chlorogenic acid, peak 2 is ferulic acid, peak 3 is dracotoxin, peak 4 is cinnamaldehyde, peak 5 is osthole, and peak 6 is ligustilide. DETAILED DESCRIPTION

[0040] The present invention provides a method for simultaneously detecting chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in Xiangdu Huoxue Patch, comprising the following steps:

[0041] dissolving the first cinnamaldehyde standard sample in methanol to obtain a cinnamaldehyde standard sample solution;

[0042] The cinnamaldehyde standard sample solution is subjected to a first high performance liquid chromatography test to obtain a first cinnamaldehyde standard sample peak area;

[0043] Remove the film from the Xiang Du Huo Xue patch sample to be tested, crush it, and extract it with a first phosphoric acid-methanol solution to obtain a sample solution to be tested; the mass percentage of phosphoric acid in the first phosphoric acid-methanol solution is 2-4%;

[0044] The sample solution to be tested is subjected to a second high performance liquid chromatography test to obtain a peak area of ​​the sample to be tested, and the concentration of cinnamaldehyde in the sample solution to be tested is calculated using an external standard method according to the concentration of the cinnamaldehyde standard sample solution, the peak area of ​​the first cinnamaldehyde standard sample and the peak area of ​​the sample to be tested;

[0045] According to the peak area of ​​the first cinnamaldehyde standard sample, the peak area of ​​the sample to be tested, the concentration of cinnamaldehyde in the sample solution to be tested and the relative correction factor, the concentrations of chlorogenic acid, ferulic acid, dracotin, ligustilide and osthole in the sample solution to be tested are calculated respectively; the relative correction factors are the chlorogenic acid correction factor, the ferulic acid correction factor, the dracotin correction factor, the osthole correction factor and the ligustilide correction factor respectively;

[0046] The conditions of the first HPLC detection and the second HPLC detection independently include: the chromatographic column is C 18 Chromatographic column; mobile phase system comprises mobile phase A and mobile phase B, wherein mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; elution mode is gradient elution, and the procedure of the gradient elution is as follows: 0min, the volume fraction of the mobile phase A is 95%; 0-5min, the volume fraction of the mobile phase A is reduced from 95% to 90%; 5-15min, the volume fraction of the mobile phase A is 90%; 15-17min, the volume fraction of the mobile phase A is reduced from 90% to 82%; 17-30min, the volume fraction of the mobile phase A is 82%; 30-31min, the volume fraction of the mobile phase A is reduced from 82% to 75% ; 31-41min, the volume fraction of the mobile phase A is reduced from 75% to 70%; 41-45min, the volume fraction of the mobile phase A is 70%; 45-50min, the volume fraction of the mobile phase A is reduced from 70% to 65%; 50-55min, the volume fraction of the mobile phase A is reduced from 65% to 52%; 55-85min, the volume fraction of the mobile phase A is 52%; 85-86min, the volume fraction of the mobile phase A is reduced from 52% to 0%; 86-90min, the volume fraction of the mobile phase A is 0%; 90-91min, the volume fraction of the mobile phase A is increased from 0% to 95%; 91-95min, the volume fraction of the mobile phase A is 95%;

[0047] The Angelica Angelicae Pubescentis blood patch comprises a backing material, a coating, a Chinese medicine composition and a matrix; in terms of weight, the matrix comprises 10-45 parts of a skeleton material, 1-3 parts of a cross-linking agent, 30-70 parts of a moisturizer, 1-3 parts of a pH adjuster, 15-35 parts of a viscosity enhancer and 50-120 parts of purified water; in terms of weight, the Chinese medicine composition comprises 5-24 parts of Angelicae Pubescentis, 5-24 parts of Ligusticum chuanxiong, 5-24 parts of Radix Aucklandiae, 4-14 parts of Fennel, 4-14 parts of Cinnamon bark, 4-14 parts of Frankincense, 4-14 parts of Myrrh, 1-12 parts of processed Aconiti Kusnezoffii, 1-12 parts of processed Aconiti Kusnezoffii, 1-12 parts of Sanguisorba officinalis and 1-8 parts of Borneol.

[0048] In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.

[0049] The present invention dissolves the first cinnamaldehyde standard sample in methanol to obtain a cinnamaldehyde standard sample solution. In the present invention, the concentration of the first cinnamaldehyde standard sample in the cinnamaldehyde standard sample solution is preferably 80-1890 μg / mL, more preferably 400-1240 μg / mL, and most preferably 700-890 μg / mL. In the present invention, the preparation method of the cinnamaldehyde standard sample solution preferably includes the following steps: dissolving the first cinnamaldehyde standard sample in methanol to obtain a first cinnamaldehyde standard sample stock solution; diluting the first cinnamaldehyde standard sample stock solution with methanol to obtain a cinnamaldehyde standard sample solution. In the present invention, the concentration of the first cinnamaldehyde standard sample stock solution is preferably 0.4-9.8 mg / mL, more preferably 2-6.4 mg / mL, and most preferably 3.5-5.4 mg / mL.

[0050] After obtaining the cinnamaldehyde standard sample solution, the present invention performs a first high performance liquid chromatography detection on the cinnamaldehyde standard sample solution to obtain a first cinnamaldehyde standard sample peak area.

[0051] In the present invention, the conditions for the first high performance liquid chromatography detection include: the chromatographic column is C 18 Chromatographic column, the C 18 The chromatographic column preferably includes an Agilent Eclipse CDB-C 18 Chromatographic columns, Agilent ZORBAX Eclipse XDB-C 18 Chromatographic columns, Diamonsil C 18 Chromatographic column or Welchrom C 18 Chromatographic column; said C 18The specification of the chromatographic column is preferably 4.6 mm × 250 mm, 5 μm; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%, preferably 0.2-0.4%, more preferably 0.3-0.4%; the mobile phase B is acetonitrile; the elution method is gradient elution, and the procedure of the gradient elution is as follows: 0 min, the volume fraction of the mobile phase A is 95%; 0-5 min, the volume fraction of the mobile phase A is 95%; 5% to 90%; 5 to 15 minutes, the volume fraction of the mobile phase A is 90%; 15 to 17 minutes, the volume fraction of the mobile phase A is reduced from 90% to 82%; 17 to 30 minutes, the volume fraction of the mobile phase A is 82%; 30 to 31 minutes, the volume fraction of the mobile phase A is reduced from 82% to 75%; 31 to 41 minutes, the volume fraction of the mobile phase A is reduced from 75% to 70%; 41 to 45 minutes, the volume fraction of the mobile phase A is 70%; 45 to 50 minutes min, the volume fraction of mobile phase A is reduced from 70% to 65%; 50-55min, the volume fraction of mobile phase A is reduced from 65% to 52%; 55-85min, the volume fraction of mobile phase A is 52%; 85-86min, the volume fraction of mobile phase A is reduced from 52% to 0%; 86-90min, the volume fraction of mobile phase A is 0%; 90-91min, the volume fraction of mobile phase A is increased from 0% to 95%; 91-95min, the volume fraction of mobile phase A is increased from 0% to 95%. The volume fraction is 95%; the column temperature is preferably 25-35°C, more preferably 28-32°C, and most preferably 29-30°C; the flow rate of the mobile phase is preferably 0.8-1.2mL / min, more preferably 0.9-1.1mL / min, and most preferably 1-1.1mL / min; the injection volume is preferably 4-12μL, more preferably 6-11μL, and most preferably 8-10μL; the detection wavelength is preferably 319-324nm, more preferably 320-323nm, and most preferably 321-323nm.

[0052] The present invention removes the coating of the Xiangdu Huoxue patch sample to be tested, breaks it, and then uses a first phosphoric acid-methanol solution to extract it to obtain a sample liquid to be tested.

[0053] In the present invention, the Xiangdu Huoxue patch comprises a backing material, a coating, a Chinese medicine composition and a matrix.

[0054] In the present invention, the backing material preferably includes one or more of non-woven fabric, cotton fabric and paper.

[0055] In the present invention, the coating film preferably includes one or more of a plastic film, a release paper and an aluminum foil-polyethylene composite film.

[0056] In parts by mass, the matrix comprises 10 to 45 parts of a skeleton material, preferably 12 to 30 parts, and more preferably 15 parts; the skeleton material is preferably a neutralized sodium polyacrylate, more preferably sodium polyacrylate NP-700; 1 to 3 parts of a cross-linking agent, preferably 1.1 to 2 parts, and more preferably 1.2 parts; the cross-linking agent is preferably an aluminum compound, more preferably aluminum glycolate; 30 to 70 parts of a humectant, preferably 40 to 60 parts, and more preferably 50 parts; the humectant is preferably one or more of glycerol, propylene glycol, and polyethylene glycol, more preferably The invention relates to a liquid medicine comprising the following: glycerol; 1 to 3 parts of a pH regulator, preferably 1.1 to 2 parts, and more preferably 1.3 parts; the pH regulator is preferably one of tartaric acid, citric acid, lactic acid and malic acid, more preferably tartaric acid; 15 to 35 parts of a viscosity enhancer, preferably 12 to 30 parts, and more preferably 15 parts; the viscosity enhancer is preferably one or more of gelatin, gum arabic, carbomer 940, sodium carboxymethyl cellulose and hydroxypropyl methylcellulose, more preferably carbomer 940; 50 to 120 parts of purified water, preferably 55 to 100 parts, and more preferably 60 parts.

[0057] In parts by mass, the Chinese medicine composition includes 5-24 parts of Angelica dahurica, preferably 12-20 parts, and specifically preferably 14 parts; 5-24 parts of Ligusticum chuanxiong, preferably 12-20 parts, and specifically preferably 14 parts; 5-24 parts of Radix Aucklandiae lappa, preferably 12-20 parts, and specifically preferably 14 parts; 4-14 parts of fennel, preferably 6-11 parts, and specifically preferably 8 parts; 4-14 parts of cinnamon, preferably 6-11 parts, and specifically preferably 8 parts; 4-14 parts of frankincense, preferably 6-11 parts, and specifically preferably 8 parts; 4-14 parts of myrrh, preferably 6-11 parts, and specifically preferably 8 parts; 1-12 parts of processed Aconiti kusnezoffii, preferably 2-11 parts, and specifically preferably 6 parts; 1-12 parts of processed Aconiti kusnezoffii, preferably 2-11 parts, and specifically preferably 6 parts; 1-12 parts of Sanguisorba officinalis, preferably 2-11 parts, and specifically preferably 6 parts; and 1-8 parts of borneol, preferably 2-7 parts, and specifically preferably 3 parts.

[0058] In the present invention, the mass percentage of phosphoric acid in the first phosphoric acid-methanol solution is 2-4%, preferably 2.5-3.5%, more preferably 2.8-3.2%, and most preferably 2.9-3%. In the present invention, the solid-to-liquid ratio of the Xiangduhuoxue patch sample to be tested and the first phosphoric acid-methanol solution is preferably 1g:3.56-5mL, more preferably 1g:3.6-4.3mL, and most preferably 1g:3.7-4mL. In the present invention, the extraction preferably includes ultrasonic extraction; the extraction temperature is preferably 20-60°C, more preferably 30-50°C, and most preferably 40-45°C; the extraction time is preferably 20-40min, more preferably 25-35min, and most preferably 28-30min.

[0059] In the present invention, the extraction preferably includes the following steps: placing the Xiang Du Huo Xue Patch sample to be tested in a first phosphoric acid-methanol solution, weighing it, performing ultrasonic extraction, weighing it, supplementing the lost mass with the first phosphoric acid-methanol solution, filtering, and the filtrate is the sample solution to be tested.

[0060] After obtaining the sample solution to be tested, the present invention performs a second high performance liquid chromatography detection on the sample solution to be tested to obtain the peak area of ​​the sample to be tested, and according to the concentration of the cinnamaldehyde standard sample solution, the peak area of ​​the first cinnamaldehyde standard sample and the peak area of ​​the sample to be tested, the concentration of cinnamaldehyde in the sample solution to be tested is calculated by an external standard method.

[0061] In the present invention, the conditions for the second HPLC detection are the same as those for the first HPLC detection, and will not be described in detail herein.

[0062] In the present invention, the calculation formula for the concentration of cinnamaldehyde in the sample solution to be tested is preferably: y =A x C w / A w Among them, C y A is the concentration of cinnamaldehyde in the sample solution to be tested; x is the peak area of ​​the sample to be tested; C w A is the concentration of the cinnamaldehyde standard sample solution; w Peak area of ​​the first cinnamaldehyde standard sample.

[0063] After obtaining the peak area of ​​the first cinnamaldehyde standard sample, the peak area of ​​the sample to be tested, and the concentration of cinnamaldehyde in the sample solution to be tested, the present invention calculates the concentrations of chlorogenic acid, ferulic acid, dracotoxin, ligustilide and osthole in the sample solution to be tested according to the peak area of ​​the first cinnamaldehyde standard sample, the peak area of ​​the sample to be tested, the concentration of cinnamaldehyde in the sample solution to be tested and the relative correction factor.

[0064] In the present invention, the relative correction factors are chlorogenic acid correction factor, ferulic acid correction factor, dracotin correction factor, osthole correction factor and ligustilide correction factor, respectively. In the present invention, the method for obtaining the relative correction factor preferably includes the following steps: dissolving a chlorogenic acid standard sample, a ferulic acid standard sample, a dracotin standard sample, an osthole standard sample, a ligustilide standard sample and a second cinnamaldehyde standard sample in a second phosphoric acid-methanol solution to obtain a mixed standard sample solution; subjecting the mixed standard sample solution to a third high performance liquid chromatography detection to obtain a mixed standard sample peak area; using cinnamaldehyde as an internal standard, according to the mixed standard sample peak area and the concentration of the mixed standard sample solution, the relative correction factor is calculated by the internal standard method. In the present invention, the mixed standard sample peak area preferably includes the chlorogenic acid standard sample peak area, the ferulic acid standard sample peak area, the dracotin standard sample peak area, the osthole standard sample peak area, the ligustilide standard sample peak area and the second cinnamaldehyde standard sample peak area.

[0065] In the present invention, the concentration of the second cinnamaldehyde standard sample in the mixed standard sample solution is preferably 80-1890 μg / mL, more preferably 400-890 μg / mL, and most preferably 750-890 μg / mL; the concentration of the chlorogenic acid standard sample is preferably 38-349 μg / mL, more preferably 100-300 μg / mL, and most preferably 194-252 μg / mL; the concentration of the ferulic acid standard sample is preferably 81-734 μg / mL, more preferably 100-600 μg / mL, and most preferably 300-40 8μg / mL; the concentration of the dracotoxin standard sample is preferably 36-324μg / mL, more preferably 100-300μg / mL, and most preferably 180-250μg / mL; the concentration of the ligustilide standard sample is preferably 90-1524μg / mL, more preferably 500-1500μg / mL, and most preferably 900-1200μg / mL; the concentration of the osthole standard sample is preferably 28-252μg / mL, more preferably 100-200μg / mL, and most preferably 143-180μg / mL.

[0066] In the present invention, a chlorogenic acid standard sample, a ferulic acid standard sample, a dracotin standard sample, an osthole standard sample, a ligustilide standard sample and a second cinnamaldehyde standard sample are dissolved in a second phosphoric acid-methanol solution, preferably comprising the following steps: mixing the chlorogenic acid standard sample with methanol to obtain a chlorogenic acid standard sample stock solution; mixing the ferulic acid standard sample with methanol to obtain a ferulic acid standard sample stock solution; mixing the second cinnamaldehyde standard sample with methanol to obtain a second cinnamaldehyde standard sample stock solution; mixing the osthole standard sample with methanol to obtain an osthole standard sample stock solution; mixing the ligustilide standard sample with methanol to obtain a ligustilide standard sample stock solution; mixing the dracotin standard sample with the third phosphoric acid-methanol solution to obtain a dracotin standard sample stock solution; mixing the chlorogenic acid standard sample stock solution, the ferulic acid standard sample stock solution, the second cinnamaldehyde standard sample stock solution, the osthole standard sample stock solution, the ligustilide standard sample stock solution, the dracotin standard sample stock solution and the fourth phosphoric acid-methanol solution to obtain a mixed standard sample solution.

[0067] In the present invention, the concentration of the chlorogenic acid standard sample stock solution is preferably 0.194-1.746 mg / mL, more preferably 0.5-1.5 mg / mL, and most preferably 0.97-1 mg / mL; the concentration of the ferulic acid standard sample stock solution is preferably 0.408-3.672 mg / mL, more preferably 1-3 mg / mL, and most preferably 2.04-2.5 mg / mL; the concentration of the second cinnamaldehyde standard sample stock solution is preferably 0.4-5.8 mg / mL, more preferably 2-5.4 mg / mL, and most preferably 3.8-5.4 mg / mL. L; the concentration of the osthole standard sample stock solution is preferably 0.14-1.26 mg / mL, more preferably 0.5-1 mg / mL, and most preferably 0.7-0.8 mg / mL; the concentration of the ligustilide standard sample stock solution is preferably 0.743-5.287 mg / mL, more preferably 2.15-4.5 mg / mL, and most preferably 3.0-3.9 mg / mL; the concentration of the dracotoxin standard sample stock solution is preferably 0.18-1.62 mg / mL, more preferably 0.5-1.5 mg / mL, and most preferably 0.9-1 mg / mL. In the present invention, the concentration of the third phosphoric acid-methanol solution is preferably 2-4%, more preferably 2.5-3.5%, and most preferably 2.9-3%; the concentration of the fourth phosphoric acid-methanol solution is preferably 2-4%, more preferably 2.5-3.5%, and most preferably 2.9-3%.

[0068] In the present invention, the conditions for the third HPLC detection are the same as those for the first HPLC detection, and will not be described in detail herein.

[0069] In the present invention, the calculation formula of the relative correction factor is preferably: i / r =A i C r / A r C i ; Among them, f i / r is the relative correction factor, A i is the peak area of ​​the second cinnamaldehyde standard sample; C i A is the concentration of the second cinnamaldehyde standard sample in the mixed standard sample solution; r is the peak area of ​​the standard sample to be calibrated; C r is the concentration of the standard sample to be calibrated in the mixed standard sample solution; the standard samples to be calibrated are preferably chlorogenic acid standard sample, ferulic acid standard sample, dracotoxin standard sample, osthole standard sample and ligustilide standard sample.

[0070] In the present invention, the calculation formula for the concentration of chlorogenic acid, ferulic acid, dracotoxin, ligustilide and osthole in the sample solution to be tested is preferably: x =A x C ii f i / r / A ii Among them, C x A is the concentration of chlorogenic acid, ferulic acid, dracotoxin, osthole or ligustilide in the sample solution to be tested; x is the peak area of ​​the sample to be tested; C ii is the concentration of the first cinnamaldehyde standard sample in the cinnamaldehyde standard sample solution; A ii is the peak area of ​​the first cinnamaldehyde standard sample; f i / r is the relative correction factor.

[0071] To further illustrate the present invention, a method for simultaneously detecting chlorogenic acid, ferulic acid, dracotoxin, cinnamaldehyde, ligustilide and osthole in the Xiangdu Huoxue Patch provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0072] In all embodiments of the present invention, the instruments used are: the chromatograph is a high performance liquid chromatograph and an Agilent 1200 high performance liquid chromatograph produced by Shimadzu Corporation of Japan; the chromatographic column is an Agilent Eclipse XDB8-C 18 Chromatographic columns, DiamonsilC 18 Chromatographic columns and Welchrom C 18 Chromatographic column; YH-C20002 electronic analytical balance (Wuxin Scale); JY5002 electronic analytical balance (LIANG PING); KQ-300B ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.).

[0073] The reagents used were: chlorogenic acid (110753-202119), ferulic acid (110773-201915), dracotoxin (110811-202108), cinnamaldehyde (110710-202223), and osthole (110822-202111) purchased from China Food and Drug Inspection Institutes; ligustilide (RFS-G01001909024) purchased from Chengdu Ruifensi Biotechnology Co., Ltd.; methanol (analytical grade); ethanol (analytical grade); acetonitrile (chromatographic grade); phosphoric acid (chromatographic grade); ultrapure water.

[0074] The conditions for HPLC detection are as follows: The chromatographic column used in Example 1 is Agilent Eclipse CDB-C 18 Chromatographic column; the chromatographic column used in Example 2 is Agilent Eclipse XDB-C 18 Chromatographic columns, Diamonsil C 18 Chromatographic columns and Welchrom C 18 Chromatographic column; the specifications of the chromatographic column are 4.6mm×250mm, 5μm; the mobile phase system includes mobile phase A and mobile phase B, the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.3; the elution method is gradient elution, and the procedure of the gradient elution is as follows: 0min, the volume fraction of the mobile phase A is 95%; 0-5min, the volume fraction of the mobile phase A is reduced from 95% to 90%; 5-15min, the volume fraction of the mobile phase A is 90%; 15-17min, the volume fraction of the mobile phase A is reduced from 90% to 82%; 17-30min, the volume fraction of the mobile phase A is 82%; 30-31min, the volume fraction of the mobile phase A is reduced from 82% to 75%; 31-41min in, the volume fraction of the mobile phase A is reduced from 75% to 70%; 41-45min, the volume fraction of the mobile phase A is 70%; 45-50min, the volume fraction of the mobile phase A is reduced from 70% to 65%; 50-55min, the volume fraction of the mobile phase A is reduced from 65% to 52%; 55-85min, the volume fraction of the mobile phase A is 52%; 85-86min, the volume fraction of the mobile phase A is reduced from 52% to 0%; 86-90min, the volume fraction of the mobile phase A is 0%; 90-91min, the volume fraction of the mobile phase A is increased from 0% to 95%; 91-95min, the volume fraction of the mobile phase A is 95%; the flow rate of the mobile phase is 1mL / min; the injection volume is 10μL; the detection wavelength is 321nm.

[0075] The sample of Angelica dahurica blood patch to be tested includes a backing material, a coating, a Chinese medicine composition and a matrix; in terms of mass fractions, the matrix includes 15 parts of a skeleton material (neutralized sodium polyacrylate); 1.2 parts of a cross-linking agent (aluminum compound); 50 parts of a moisturizer (polyol compound); 1.3 parts of a pH adjuster (carboxylic acid compound); 15 parts of a viscosity enhancer (carbomer 940); 60 parts of purified water; the Chinese medicine composition includes 14 parts of Angelica dahurica; 14 parts of Ligusticum chuanxiong; 14 parts of Aucklandia lappa; 8 parts of cumin; 8 parts of cinnamon; 8 parts of frankincense; 8 parts of myrrh; 6 parts of processed Aconiti kusnezoffii; 6 parts of processed Aconiti kusnezoffii; 6 parts of Sanguisorba officinalis; and 3 parts of borneol.

[0076] Example 1

[0077] Take chlorogenic acid standard, ferulic acid standard, cinnamaldehyde standard, osthole standard and ligustilide standard, and dissolve them in methanol respectively to obtain 0.97 mg / mL chlorogenic acid standard stock solution, 2.04 mg / mL ferulic acid standard stock solution, 4.8 mg / mL cinnamaldehyde standard stock solution, 0.7 mg / mL osthole standard stock solution, and 3.9 mg / mL ligustilide standard stock solution; take dracoside standard and dissolve it in 3% mass concentration of phosphoric acid-methanol solution to obtain 0.9 mg / mL dracoside standard stock solution.

[0078] Take chlorogenic acid standard stock solution, ferulic acid standard stock solution, dracotin standard stock solution, cinnamaldehyde standard stock solution, osthole standard stock solution and ligustilide standard stock solution, dissolve them with 3% phosphoric acid-methanol solution and make up to 5 mL volumetric flask to obtain a mixed standard solution with a chlorogenic acid concentration of 194 μg / mL, a ferulic acid concentration of 408 μg / mL, a dracotin concentration of 180 μg / mL, a cinnamaldehyde concentration of 960 μg / mL, an osthole concentration of 140 μg / mL and a ligustilide concentration of 780 μg / mL (referred to as mixed standard A), and dilute mixed standard A in sequence to obtain mixed standards B, mixed standard C, mixed standard D, mixed standard E and mixed standard F, in which the concentrations of cinnamaldehyde are 480 μg / mL, 192 μg / mL, 96 μg / mL, 48 μg / mL and 9.6 μg / mL, respectively.

[0079] Take the Xiang Du Huo Xue patch sample to be tested, remove the film, cut into pieces, add a 3% phosphoric acid-methanol solution, perform ultrasonic extraction at 30°C for 30 minutes, weigh, make up the lost weight with a 3% phosphoric acid-methanol solution, filter through a microporous filter membrane, and the filtrate is the sample solution to be tested.

[0080] 10 μL of each of the mixed standard A to mixed standard F was accurately aspirated, and 10 μL of each of the 6 batches of test solutions (recorded as test A, test B, test C, test D, test E and test F in sequence) were taken, and the high performance liquid chromatography conditions provided by the present invention were used for detection. The chromatogram of the test sample liquid is shown in FIG. Figure 1 As shown. Figure 1 It can be seen that when the high performance liquid chromatography conditions provided by the present invention are used to detect the sample liquid, the peak shapes of the six compounds to be detected in the sample liquid are good and the separation degree is high.

[0081] The concentrations of the six compounds in the Xiangdu Huoxue Patch were calculated using the method provided by the present invention and the external standard method. The results are shown in Table 1.

[0082] Table 1 Determination results of the contents of 6 compounds in Xiangdu Huoxue Patch (unit: μg / mL)

[0083]

[0084] As can be seen from Table 1, there is no significant difference in the detection results between the detection method provided by the present invention and the external standard method.

[0085] Example 2

[0086] Methodological investigation

[0087] 1. Linear relationship investigation

[0088] Mixed standards A to F prepared in Example 1 were tested in turn using the HPLC conditions provided by the present invention. The concentration of the standard sample in the mixed standard solution was taken as the X-axis, and the chromatographic peak area of ​​the standard sample was taken as the Y-axis to draw a standard curve. The obtained linear regression equation and linear relationship (linear range R 2 and correlation coefficients) are shown in Table 2.

[0089] Table 2 Linear regression equation and linear relationship (linear range R 2 and correlation coefficient)

[0090]

[0091]

[0092] As shown in Table 2, when the method provided by the present invention is used for testing, chlorogenic acid, ferulic acid, dracotoxin, cinnamaldehyde, ligustilide, and osthole all have good linearity within the range.

[0093] 2. Exclusivity Verification

[0094] The remaining 9 medicinal materials (cinnamon bark, fennel, costusroot, processed aconite, processed kusnezoffii, vinegar frankincense, vinegar myrrh, dragon's blood and borneol) except angelica dahurica and chuanxiong were ground into fine powder, and a negative sample (referred to as sample 1) without chlorogenic acid, ferulic acid, ligustilide and osthole was prepared according to the prescription dosage provided by the present invention;

[0095] The remaining 10 medicinal materials (Chuanxiong, cinnamon bark, fennel, costusroot, processed Chuanwu, processed Caowu, vinegar frankincense, vinegar myrrh, dragon's blood and borneol) except for Angelica dahurica were ground into fine powder, and a negative sample (referred to as sample 2) without osthole was prepared according to the dosage of the prescription of the present invention;

[0096] The remaining 10 medicinal materials (angelica pubescens, chuanxiong, fennel, costusroot, processed aconite, processed kusnezoffii, vinegar frankincense, vinegar myrrh, dragon's blood and borneol) except cinnamon bark were ground into fine powder, and a negative sample (referred to as sample 3) without cinnamaldehyde was prepared according to the dosage of the prescription of the present invention;

[0097] The remaining 10 medicinal materials (angelica pubescens, chuanxiong, cinnamon bark, fennel, costusroot, processed aconite, processed kusnezoffii, vinegar frankincense, vinegar myrrh and borneol) except for dragon's blood were crushed into fine powder, and a negative sample (recorded as sample 4) without dragon's blood was prepared according to the dosage of the prescription of the present invention;

[0098] The above samples 1 to 4 were prepared into negative sample solutions to be tested (respectively recorded as negative sample solution 1 to be tested, negative sample solution 2 to be tested, negative sample solution 3 to be tested and negative sample solution 4 to be tested) according to the preparation method of the sample solution to be tested provided by the present invention, and a mixed standard solution was used as a control.

[0099] The negative sample solutions 1 to 4 to be tested are subjected to high performance liquid chromatography detection. Figure 3 is the chromatogram of negative sample solution 1 to be tested; Figure 4 is the chromatogram of negative sample solution 2 to be tested; Figure 5 is the chromatogram of negative sample solution 3 to be tested;

[0100] Figure 6 is the chromatogram of negative sample solution 4 to be tested. Figure 3 to Figure 6 It can be seen that the negative sample liquid 1 to be tested has no chromatographic peaks at the retention times of chlorogenic acid, ferulic acid, ligustilide and osthole, the negative sample liquid 2 to be tested has no chromatographic peak at the retention time of osthole, and the negative sample liquid 3 to be tested has no chromatographic peak at the retention time of dracotoxin; the negative sample liquid 4 to be tested has no chromatographic peak at the retention time of cinnamaldehyde, which proves that the high performance liquid chromatography detection method provided in this application has good specificity.

[0101] 3. Instrument stability test

[0102] Take 10 μL of mixed standard B and inject it 6 times continuously, and calculate the RSD of the peak areas of the 6 components. The peak area RSDs (n=6) of chlorogenic acid, ferulic acid, dracotoxin, cinnamaldehyde, ligustilide and osthole were 0.46%, 0.39%, 0.20% and 0.36%, respectively, all less than 3%, indicating that the instrument has good precision.

[0103] 4. Solution stability test

[0104] The same sample solution was taken for analysis at 0, 2, 4, 8, 12, and 24 hours, and the peak area of ​​each analyte was determined. The RSD (n=6) of the peak areas of chlorogenic acid, ferulic acid, dracotoxin, cinnamaldehyde, ligustilide, and osthole were calculated to be 0.94%, 0.96%, 0.34%, 1.27%, 0.74%, and 1.37%, respectively, all less than 3%, indicating that the sample solution was relatively stable within 24 hours.

[0105] 5. Repeatability test

[0106] Take 6 samples of the same batch of Xiangdu Huoxue patch to be tested, prepare 6 sample solutions to be tested in parallel, and use the high performance liquid chromatography conditions provided by the present invention for detection. The RSDs of the contents of chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide, and osthole were measured to be 1.45%, 1.35%, 1.50%, 2.33%, 2.89%, and 1.47%, respectively, all less than 3%, indicating that the method has good repeatability.

[0107] 6. Sample recovery test

[0108] A total of 9 portions of the test E were taken and accurately weighed, and standard samples were added at 3 levels of 80%, 100% and 120% of the known content (3 samples for each level), and the test sample solution was prepared using the method provided by the present invention. The sample injection was measured and the sample recovery rates of the 6 test compounds in the test Xiangdu Huoxue Patch sample were calculated, as shown in Tables 3 to 8, respectively.

[0109] Table 3 The test results of the recovery rate of chlorogenic acid in the samples of Xiangdu Huoxue Patch to be tested

[0110]

[0111] Table 4 Test results of recovery rate of ferulic acid in the tested Xiangdu Huoxue patch samples

[0112]

[0113]

[0114] Table 5 The test results of the recovery rate of dracoside in the samples of Xiangdu Huoxue Patch to be tested

[0115]

[0116] Table 6 Test results of recovery rate of cinnamaldehyde in the samples of Xiangdu Huoxue Patch to be tested

[0117]

[0118] Table 7 Test results of recovery rate of osthole in the samples of Xiangdu Huoxue Patch to be tested

[0119]

[0120]

[0121] Table 8 Test results of recovery rate of ligustilide in the samples of Xiangduhuoxuetie to be tested

[0122]

[0123] It can be seen from Tables 3 to 8 that the one-test-multiple-evaluation method provided by the present invention has good accuracy.

[0124] 7. Determination of relative correction factor

[0125] Because cinnamaldehyde is stable, has a good peak shape, a high content, and is cheap and easy to obtain, cinnamaldehyde was selected as the internal standard, and mixed standard solutions of different mass concentrations were accurately aspirated, and the measurement was performed under the same chromatographic conditions, and the peak area of ​​each chromatographic peak was recorded. The results are shown in Table 9.

[0126] Table 9 Correction factors for chlorogenic acid, ferulic acid, dracoside, ligustilide and osthole

[0127]

[0128] From Table 9, the correction factor of chlorogenic acid is 0.1121, the correction factor of ferulic acid is 0.0576, the correction factor of dracotoxin is 1.0297, the correction factor of ligustilide is 0.1449, and the correction factor of osthole is 0.0844.

[0129] 8. Evaluation of the durability of correction factors

[0130] 8.1. Effects of different HPLC instruments and columns on the calibration factor

[0131] The mixed standard B was precisely drawn and measured using the HPLC conditions provided by the present invention, with the only difference being that the chromatographic columns used were different HPLC instruments (Shimadzu HPLC and Agilent 1200 HPLC), different chromatographic columns (Agilent Eclipse XD8-C 18 Chromatographic columns, Diamonsil C 18 Chromatographic columns and Welchrom C 18The effects of different HPLC instruments and different chromatographic columns on the correction factor are shown in Table 10.

[0132] Table 10 Effects of different HPLC instruments and different chromatographic columns on the correction factor

[0133]

[0134] It can be seen from Table 10 that the correction factor provided by the present invention has good durability when using different instruments and different chromatographic columns.

[0135] 8.2 Effects of different injection volumes, flow rates and column temperatures on the correction factor value

[0136] The mixed standard C was precisely aspirated and measured using the HPLC conditions of the present invention, with the only difference being that different injection volumes (4, 6, 8, 10 and 12 μL), different flow rates (0.8, 0.9, 1, 1.1 and 1.2 mL / min) and different column temperatures (25, 28, 30, 32 and 35° C.) were used. The different injection volumes, different flow rates and different column temperatures are shown in Tables 11 to 13.

[0137] Table 11 Effect of different injection volumes on correction factors

[0138]

[0139] Table 12 Effect of different flow rates on correction factors

[0140]

[0141]

[0142] Table 13 Effect of different column temperatures on correction factors

[0143]

[0144] It can be seen from Tables 11 to 13 that the calibration factor has good durability under different injection volumes, different flow rates and different column temperatures.

[0145] 9. Chromatographic peak location of the component to be tested

[0146] The chromatographic peaks of the components to be measured were located using relative retention time, that is, the ratio of the retention time of each component to be measured to that of cinnamaldehyde. The reproducibility of the relative retention value on different HPLC instruments and different types of chromatographic columns was investigated, as well as the reproducibility of the relative retention value under different injection volumes, different flow rates and different column temperatures. The results are shown in Tables 14 to 17.

[0147] Table 14 Effect of different HPLC instruments on t

[0148]

[0149] Table 15 Effect of different injection volumes on t

[0150]

[0151]

[0152] Table 16 Effect of different flow rates on t

[0153]

[0154] Table 17 Effect of different column temperatures on t

[0155]

[0156] It can be seen from Tables 14 to 17 that the reproducibility of relative retention time under different HPLC instruments, different types of chromatographic columns, different injection volumes, different flow rates and different column temperatures is good.

[0157] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting chlorogenic acid, ferulic acid, dracoside, cinnamaldehyde, ligustilide and osthole in Xiangdu Huoxue Patch, characterized in that: The following steps are involved: dissolving the first cinnamaldehyde standard sample in methanol to obtain a cinnamaldehyde standard sample solution; The cinnamaldehyde standard sample solution is subjected to a first high performance liquid chromatography test to obtain a first cinnamaldehyde standard sample peak area; Remove the film from the Xiang Du Huo Xue patch sample to be tested, crush it, and extract it with a first phosphoric acid-methanol solution to obtain a sample solution to be tested; the mass percentage of phosphoric acid in the first phosphoric acid-methanol solution is 2-4%; The sample solution to be tested is subjected to a second high performance liquid chromatography test to obtain a peak area of ​​the sample to be tested, and the concentration of cinnamaldehyde in the sample solution to be tested is calculated using an external standard method according to the concentration of the cinnamaldehyde standard sample solution, the peak area of ​​the first cinnamaldehyde standard sample and the peak area of ​​the sample to be tested; According to the peak area of ​​the first cinnamaldehyde standard sample, the peak area of ​​the sample to be tested, the concentration of cinnamaldehyde in the sample solution to be tested and the relative correction factor, the concentrations of chlorogenic acid, ferulic acid, dracotin, ligustilide and osthole in the sample solution to be tested are calculated respectively; the relative correction factors are the chlorogenic acid correction factor, the ferulic acid correction factor, the dracotin correction factor, the osthole correction factor and the ligustilide correction factor respectively; The conditions of the first HPLC detection and the second HPLC detection independently include: the chromatographic column is C 18 Chromatographic column; the mobile phase system comprises mobile phase A and mobile phase B, wherein the mobile phase A is a phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; the elution mode is gradient elution, and the procedure of the gradient elution is as follows: 0min, the volume fraction of the mobile phase A is 95%; 0-5min, the volume fraction of the mobile phase A is reduced from 95% to 90%; 5-15min, the volume fraction of the mobile phase A is 90%; 15-17min, the volume fraction of the mobile phase A is reduced from 90% to 82%; 17-30min, the volume fraction of the mobile phase A is 82%; 30-31min, the volume fraction of the mobile phase A is reduced from 82% to 75%; 31-41min, the volume fraction of the mobile phase A is reduced from 75% to 70%; 41-45min, the volume fraction of the mobile phase A is 70%; 45-50min, the volume fraction of the mobile phase A is reduced from 70% to 65%; 50-55min, the volume fraction of the mobile phase A is reduced from 65% to 52%; 55-85min, the volume fraction of the mobile phase A is 52%; 85-86min, the volume fraction of the mobile phase A is reduced from 52% to 0%; 86-90min, the volume fraction of the mobile phase A is 0%; 90-91min, the volume fraction of the mobile phase A is increased from 0% to 95%; 91-95min, the volume fraction of the mobile phase A is 95%; The Angelica Angelicae Pubescentis blood patch comprises a backing material, a coating, a Chinese medicine composition and a matrix; in terms of weight, the matrix comprises 10-45 parts of a skeleton material, 1-3 parts of a cross-linking agent, 30-70 parts of a moisturizer, 1-3 parts of a pH adjuster, 15-35 parts of a viscosity enhancer and 50-120 parts of purified water; in terms of weight, the Chinese medicine composition comprises 5-24 parts of Angelicae Pubescentis, 5-24 parts of Ligusticum chuanxiong, 5-24 parts of Radix Aucklandiae, 4-14 parts of Fennel, 4-14 parts of Cinnamon bark, 4-14 parts of Frankincense, 4-14 parts of Myrrh, 1-12 parts of processed Aconiti Kusnezoffii, 1-12 parts of processed Aconiti Kusnezoffii, 1-12 parts of Sanguisorba officinalis and 1-8 parts of Borneol.

2. The method according to claim 1, characterized in that The method for obtaining the relative correction factor comprises the following steps: Dissolving a chlorogenic acid standard sample, a ferulic acid standard sample, a dracotoxin standard sample, an osthole standard sample, a ligustilide standard sample, and a second cinnamaldehyde standard sample in a second phosphoric acid-methanol solution to obtain a mixed standard sample solution; The mixed standard sample solution is subjected to a third high performance liquid chromatography test to obtain a mixed standard sample peak area; Using cinnamaldehyde as an internal standard, the relative correction factor is calculated by the internal standard method according to the peak area of ​​the mixed standard sample and the concentration of the mixed standard sample solution; The mixed standard sample peak area includes the chlorogenic acid standard sample peak area, the ferulic acid standard sample peak area, the dracotoxin standard sample peak area, the osthole standard sample peak area, the ligustilide standard sample peak area and the second cinnamaldehyde standard sample peak area; The conditions for the third high performance liquid chromatography detection include: the chromatographic column is C 18 Chromatographic column; mobile phase system comprises mobile phase A and mobile phase B, wherein mobile phase A is phosphoric acid aqueous solution, mobile phase B is acetonitrile, and the mass percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.4%; elution mode is gradient elution, and the procedure of the gradient elution is as follows: 0min, the volume fraction of the mobile phase A is 95%; 0-5min, the volume fraction of the mobile phase A is reduced from 95% to 90%; 5-15min, the volume fraction of the mobile phase A is 90%; 15-17min, the volume fraction of the mobile phase A is reduced from 90% to 82%; 17-30min, the volume fraction of the mobile phase A is 82%; 30-31min, the volume fraction of the mobile phase A is reduced from 82% to 75% ; 31-41min, the volume fraction of the mobile phase A is reduced from 75% to 70%; 41-45min, the volume fraction of the mobile phase A is 70%; 45-50min, the volume fraction of the mobile phase A is reduced from 70% to 65%; 50-55min, the volume fraction of the mobile phase A is reduced from 65% to 52%; 55-85min, the volume fraction of the mobile phase A is 52%; 85-86min, the volume fraction of the mobile phase A is reduced from 52% to 0%; 86-90min, the volume fraction of the mobile phase A is 0%; 90-91min, the volume fraction of the mobile phase A is increased from 0% to 95%; 91-95min, the volume fraction of the mobile phase A is 95%.

3. The method according to claim 2, characterized in that The concentration of the second cinnamaldehyde standard sample in the mixed standard sample solution is 80-1890 μg / mL, the concentration of the chlorogenic acid standard sample is 38-349 μg / mL, the concentration of the ferulic acid standard sample is 81-734 μg / mL, the concentration of the dracotoxin standard sample is 36-324 μg / mL, the concentration of the ligustilide standard sample is 90-1524 μg / mL, and the concentration of the osthole standard sample is 28-252 μg / mL.

4. The method according to claim 2 or 3, characterized in that: The conditions of the third high performance liquid chromatography detection also include: column temperature of 25-35°C; flow rate of mobile phase of 0.8-1.2 mL / min; injection volume of 4-12 μL; detection wavelength of 319-324 nm.

5. The method according to claim 2, characterized in that: The calculation formula of the relative correction factor is: i / r =A i C r / A r C i ; Among them, f i / r is the relative correction factor, A i is the peak area of ​​the second cinnamaldehyde standard sample; C i A is the concentration of the second cinnamaldehyde standard sample in the mixed standard sample solution; r is the peak area of ​​the standard sample to be calibrated; C r is the concentration of the standard sample to be calibrated in the mixed standard sample solution; The standard samples to be calibrated are respectively a chlorogenic acid standard sample, a ferulic acid standard sample, a dracotoxin standard sample, an osthole standard sample and a ligustilide standard sample.

6. The method according to claim 1, characterized in that The concentration of the first cinnamaldehyde standard sample in the cinnamaldehyde standard sample solution is 80-1890 μg / mL.

7. The method according to claim 1 or 6, characterized in that: The conditions of the first HPLC detection and the second HPLC detection independently include: column temperature of 25-35°C; flow rate of mobile phase of 0.8-1.2 mL / min; injection volume of 4-12 μL; detection wavelength of 319-324 nm.

8. The method according to claim 1, characterized in that The solid-to-liquid ratio of the Xiangduhuoxue patch sample to be tested and the first phosphoric acid-methanol solution is 1 g: 3.56-5 mL.

9. The method according to claim 1 or 8, characterized in that: The extraction includes ultrasonic extraction; the extraction temperature is 20-60° C. and the extraction time is 20-40 minutes.

10. The method according to claim 1 or 8, characterized in that: The calculation formula for the concentration of chlorogenic acid, ferulic acid, dracotoxin, ligustilide and osthole in the sample solution to be tested is: x =A x C ii f i / r / A ii ; Among them, C x A is the concentration of chlorogenic acid, ferulic acid, dracotoxin, osthole or ligustilide in the sample solution to be tested; x is the peak area of ​​the sample to be tested; C ii is the concentration of the first cinnamaldehyde standard sample in the cinnamaldehyde standard sample solution; A ii is the peak area of ​​the first cinnamaldehyde standard sample; f i / r is the relative correction factor.

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